Research ArticleEffect of Mahuang Gancao Ganjiang Decoction onFusion and Fission of Mitochondria and Apoptosis ofLymphocytes in Mice under Cold Stress
Longyun Chen and Huimin Chen
Hubei University of Chinese Medicine Basic Medical College Wuhan 430065 China
Correspondence should be addressed to Huimin Chen chm1215hbtcmeducn
Received 4 September 2016 Revised 23 November 2016 Accepted 21 December 2016 Published 16 January 2017
Academic Editor Luciana Dini
Copyright copy 2017 L Chen and H Chen This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
MahuangGancaoGanjiangDecoction (MGGD) can effectively alleviate the symptoms of the patients suffering fromexogenous coldstress However the curative mechanism has not been fully clarified This study was designed to investigate the effect of MGGDon the apoptosis of lymphocytes induced by cold stress in mice The model mice were randomly divided into four groups thenormal control group (no handling mice) cold stress group MGGD + cold stress group and MGGD group Lymphocytes ofthe mice were isolated from the peripheral blood Electron microscopy analysis revealed cold stress resulted in mitochondrialfragmentation Accompanied with the change of morphology of mitochondria ATP production and the activity of respiratorychain complex decreased in these cells Western blot analysis showed that these cells expressed decreased fusion-related proteinsMitofusin 1 (Mfn1)Mitofusin 2 (Mfn2) and optic atrophy protein-1 (Opa-1) and increased fission-related proteins dynamin-relatedprotein 1 (Drp1) and fission 1 (Fis-1) our results also show that decreased mitochondrial fusion induces cell apoptosis during coldstress Meanwhile we found MGGD can inhibit cell apoptosis induced by cold stress through regulating expression level of Mfn1Mfn2 Drp1 Fis-1 and Opa-1 These findings are very significant for understanding howMGGD regulates cold-stress-induced cellapoptosis
1 Introduction
The metabolism and growth of cells can be affected by coldstress significantly In previous studies it has been demon-strated that cold stress can induce apoptosis in culturedmammalian cells [1ndash3] In the life and death of mammaliancells mitochondria play very important roles [4 5] Becauseof the fusion and fission of mitochondria the population andmorphology of mitochondria are dynamic in mammaliancells Imbalance of fusion and fission is always accompaniedbymitochondrial dysfunctionThemitochondrial fusion andfission are controlled by the specific molecules Mfn1 Mfn2and Opa-1 are essential for mitochondrial fusion Deletion ofMfn1 and Mfn2 leads to severe mitochondrial fragmentationand low levels of mitochondrial fusion [6 7] Opa-1 mediatesthe fusion of mitochondrial inner membrane the decrease of
Opa-1 expression can cause cell apoptosis [6] The fission ofmitochondria is regulated by Drp1 and Fis-1 [5 8]
Currently a series of clinical trials have shown that therepresentative formula Mahuang Gancao Ganjiang Decoc-tion (MGGD) in Traditional Chinese Medicine (TCM) cansignificantly ease the symptoms and improve the quality oflife in patients suffering from exogenous cold stress [9 10]However the mechanism of the treatment effect has not beenelucidated which limits the application ofMGGD on a largerscale
Imbalance of mitochondrial fusion and fission was foundin mammalian cells under cold stress which is recognizedto be related to cell apoptosis Previous study revealed thatoverexpression of both fusion proteins Mfn1 and Mfn2 candelay apoptosis [11 12] The mechanism of apoptosis inducedby the cold stress still needs to be studied Further whether
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 5132963 10 pageshttpsdoiorg10115520175132963
2 Evidence-Based Complementary and Alternative Medicine
MGGD-formula can work on the apoptosis also needs to beexplored Hence the aims of this study were to investigate theeffect ofMGGDon cold-stress-induced apoptosis and relatedmechanism
2 Materials and Methods
21 Animals and Lymphocytes Preparation SPF grade Kun-ming mice were purchased from the experimental animalcenter of Hubei University of traditional Chinese medicineThe medical laboratory animal certificate number is SCXK(Hubei) 2008-0005 The mice were randomly divided into4 groups (a) control group the mice were kept under a12-hour lightdark cycle (lights on at 800 am) with roomtemperature (20ndash23∘C) and humidity (50 plusmn 5) (b) coldstress group the mice were put under 4∘C for 4 hours inartificial climate box with humidity (50plusmn 5) each day in aweek (c)MGGD+ cold stress group themicewere put under4∘C for 4 hours daily and then were conducted by intragastricadministration of MGGD (d) MGGD group the mice werekept under room temperature and then were conductedby intragastric administration of MGGD The capacity ofintragastric administration was 1ml100 g body weight Inour study femalemicewere excluded Allmanipulationswereperformed between 700 am and 1200 am every day tominimize the influence of circadian rhythms
After treatment all the mice in the four groups wereinjected with an appropriate amount of sodium for anaesthe-sia One eye of anesthetic mice was pulled off by tweezersand the fresh blood was collected by the 5ml EP tubewith anticoagulants Lymphocytes separation was performedusing a mouse T Cell Isolation Kit (Miltenyi) accordingto the manufacturerrsquos instructions After the separationlymphocytes were used in the following experiments
22 MGGD Composition and Administration MGGD-for-mula was composed of the following traditional Chineseherbal medicines as recorded by the ldquoFormulas of ChineseMedicinerdquo [13]Mahuang (Ephedra) 20 gGancao (Liquorice)30 g and Ganjiang (dried ginger) 30 g which were com-posed in 2 3 3 proportion All raw materials in MGGD-formula were prepared and examined according to qualitycontrol standard of Chinese Pharmacopoeia [14] It wasmanufactured by the Preparation Room for TCM of theChinese Medicine Hospital of Hubei Province The mice inthe MGGD-formula group were given intragastric admin-istration with MGGD-formula (1ml100 gd) The controlgroup and cold stress group were given distilled water
23 Assessment of Mitochondrial Morphology with ElectronMicroscopy Lymphocytes were collected and fixed with 25glutaraldehyde on ice for 2 h Then cells were treated by 2osmium tetroxide and dehydrated with sequential washeswith 50 70 90 95 and 100 ethanol Cells wereblock mounted and thinly sliced Cells of thin sections wereexamined under electron microscope
24 Determination of Intracellular ATP Content The lym-phocytes were washed with cold PBS three times and 200120583l
of the lysis buffer was added The cells were L centrifuged(12000timesg 10 minutes 4∘C) and supernatants were har-vested 100 120583l ATP working solution was added to thesupernatants in 15ml tube and the tube was incubated atroom temperature for 5minutesTheATP content in sampleswas measured with Luminometer At the same time proteinconcentration was measured via BCAmethodThen the ATPconcentration is converted into the form of 120583molg protein
25 Mitochondrial Membrane Potential Detection (JC-1) Thelymphocytes of peripheral blood were washed by ice-coldPBS three times and centrifuged at 1500 rpm for 5min 5 times105 cells were suspended in 05ml cell culture medium anddyed by 05ml JC-1 dyeing fluid Cells were incubated at 37∘Cfor 20minutes After the incubation cells were centrifuged at1200 rpm for 3min and supernatants were discarded Cellswere washed by JC-1 dyeing buffer 2 times and suspendedby 05ml JC-1 dyeing buffer The mitochondrial membranepotential of lymphocytes was detected by flow cytometry
26 Measurement of Mitochondrial Respiratory Chain Com-plex Activity Lymphocytes were homogenized with 1ml ofpotassium phosphate buffer (100mM pH 74) at 4∘C Thehomogenates were centrifuged (1500timesg 5min 4∘C) and thesupernatants were collected The content of mitochondrialprotein was measured via the bicinchoninic protein assayAll activities were determined on a spectrophotometer inpotassium phosphate buffer Complex I activity (NADH-CoQ reductase) was determined bymeasuring the disappear-ance of NADH at 340 nm in the presence of decylubiquinone[15] Complex II activity (Succinate Dehydrogenase) wasevaluated by measuring the reduction of dichloroindophenol(DCIP) at 600 nm [16]
27 Immunofluorescence Analysis Cell suspension was fixedby 4 paraformaldehyde and coated on the slides drynaturally The cells were washed three times with PBS Thencells were treated by 01 Triton X-100 for 20min andblocked with 3 BSA for 30min Specific primary Absfor Mfn1 Mfn2 or Drp1 were added and incubated for1 h at room temperature Mito-Tracker Green was used todetect the mitochondria in lymphocytes Cells were thenwashed with 1 BSA for three times followed by incubationwith goat anti-rabbit IgG fluorescent secondary antibodyfor 1 h Nucleus was stained with DAPI (410158406-diamidino-2-phenylindole) Cells were observed under the fluorescencemicroscope
28 SDS-PAGE and WB Lymphocytes were harvested andlysed with 100 120583l of lysis buffer (20mM Tris-HCl [pH80] 100mM NaCl 19 [wtvol] Triton X-100 1mM 14-dithiothreitol and 5 [volvol] glycerol) for 30 min on iceAfter centrifugation for 30min at 4∘C the supernatants werecollected The concentration of protein was determined viathe Bradford method Equal amounts of protein were sepa-rated by 10 SDS-PAGE and transferred onto a nitrocellulosemembrane (GE Healthcare) Membrane was blocked with5 nonfat milk and incubated with the primary antibodies
Evidence-Based Complementary and Alternative Medicine 3
followed by HRP-conjugated goat anti-rabbit secondary anti-bodies
29 Detection of Apoptosis The lymphocytes of mice werewashed by PBS three times and centrifuged at 1500 rpm5min Cells were suspended by binding buffer 500 120583l Thenthe mixture containing 5120583l AnnexinV-FITC and 5 120583l PI wasadded into cell suspension Cells were incubated at roomtemperature for 5sim15min in dark The number of apoptoticcells was determined by flow cytometry
3 Results
31 Effect of MGGD on the Mitochondrial Morphology underCold Stress Previous study has demonstrated that coldstress can affect the normal morphology of mitochondria[17] In our study we observed the cold stress causes themitochondrial fragmentation Under cold stress mitochon-drial morphology became shorter and vacuole structure(Figures 1(a) and 1(b)) We also noticed that number ofmitochondriacell increased under cold stress (Figure 1(d))These indicated that the fission of mitochondria increasedbecause of the cold stress After being treated by MGGD thedamage of mitochondria in the mice reduced during coldstress In Figure 1(c) vacuolization of mitochondria was notobserved obviously and the cristae of mitochondria werevisible in lymphocytes of the mice conducted by intragastricadministration of MGGD These results demonstrated thatMGGD-formula can reduce the effect caused by cold stresson mitochondrial morphology
32 Effects of MGGD on Cold Stress Induced Decreasing ofATP Content and Mitochondrial Membrane Potential Thechange of mitochondrial morphology is closely related toits function Mitochondrial membrane potential is an indexto evaluate the function of mitochondria According toprevious study when the mitochondrial fission increasedATP synthesis and mitochondrial membrane potential willdecrease [18ndash20] In our experiment both the ATP contentand the mitochondrial membrane potential of lymphocytesdecreased in cold stress group (Figures 2(a) and 2(b)) Asshown in Figure 2(b) themitochondrial membrane potentialof cold stress group decreased about 20 compared to that ofcontrol andMGGD group After themice suffering from coldstress were treated by MGGD the ATP content recoveredMeanwhile themitochondrialmembrane potential increasedabout 10 compared to that of cold stress group Thesedata showed that MGGD can alleviate the effect caused bycold stress on ATP content and mitochondrial membranepotential
33 MGGDCan Reduce the Effect of Cold Stress on RespiratoryChain Complex The activity of respiratory chain complex isresponsible for the mitochondrial membrane potential andATP content in lymphocytes Based on the mitochondrialrespiratory chain complex activity detection results showedthat cold stress reduced the activity of complexes I and II by42 and 61 respectively (119901 lt 005) After being treatedby MGGD the activity of complexes I and II recovered to
74 and 80 compared with the control group (119901 lt 005)(Figures 3(a) and 3(b)) These data showed that respiratorychain complex activity decreased because of the imbalance ofmitochondrial fusion and fission induced by cold stress andMGGD can reduce this kind of effect
34 Effects of MGGD on the Expression of Mfn1 Mfn2 Drp1Fis-1 and Opa-1 Until now the impairment and dysfunctionof mitochondria had been demonstrated to be related to theimbalance of its fusion and fission The fusion and fission ofmitochondria are closely related to theMfn1Mfn2Drp1 Fis-1 and Opa-1 In order to check whether the expression ofMfn1 Mfn2 and Drp1 was affected by cold stress or MGGDthe Mfn1 Mfn2 and Drp1 in lymphocytes were labeled byFITC and observed under the fluorescencemicroscope Fromthe density of the fluorescence we can see that the expressionlevel of Mfn1 and Mfn2 decreased in lymphocytes undercold stress but the expression level of Drp1 increased InMGGD-formula group we observed increasing expressionlevel of Mfn1 and Mfn2 meanwhile expression level of Drp1decreased (Figure 4(a)) We also detected the expressionlevel of Mfn1 Mfn2 and Drp1 by western blotting Theresults of western blotting are consistent with immunoflu-orescence (Figures 4(b) 4(c) and 4(d)) Western blottingalso revealed that cold stress caused the increased Fis-1protein and decreasedOpa-1 protein in lymphocytes (Figures4(e) and 4(f)) These results suggested that MGGD affectsthe mitochondrial fusion and fission through regulating theexpression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1
35 MGGD Has Effect on the Colocalization of Drp-1 andMitochondria During the fission of mitochondria Drp1was recruited by Fis-1 and translocated from cytoplasm tothe mitochondrial outer membrane Drp1 distributed at thepotential division sites of mitochondrial outer membrane inthe form of ring [8] As shown in Figure 5 Drp-1 gatheredaround the mitochondria under the cold stress HoweverDrp-1 mainly distributed in the cytoplasm in other threegroups This suggested that MGGD can reduce the fission ofmitochondria through inhibiting the translocation of Drp-1
36 MGGD Can Weaken the Cold-Stress-Induced ApoptosisThe normal function of mitochondria is very important forthe growth of cells In our study we found that cold stressaffected the normal function of mitochondria and MGGDcan alleviate this kind of effect So we want to check ifMGGDcan help the cells survive during cold stressThe lymphocytesin peripheral blood of four groups of mice were isolated andthe number of apoptotic cells was detected by flow cytometryFrom Figures 6(a) and 6(b) we can see that the cold stresscan induce the apoptosis of lymphocytes The number ofapoptotic lymphocytes decreased in MGGD-formula group(Figure 6(c)) The results of western blotting showed thatactivation of caspases 3 and 9 was induced by cold stress andMGGD can reduce this effect (Figures 6(e) and 6(f)) Thesesuggested that MGGD can alleviate the cold-stress-inducedapoptosis
4 Evidence-Based Complementary and Alternative Medicine
Mock
1120583m
(a)
Cold stress
1120583m
(b)
MGGD + cold stress
1120583m
(c)
MGGD
1120583m
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0
5
10
15
20
25
30
Num
ber o
f mito
chon
dria
cel
l
(e)
Figure 1 Morphology of mitochondria in lymphocytes Lymphocytes were isolated from the peripheral blood of mice and analyzed forthe morphology of mitochondria via electron microscopy (a) Morphology of mitochondria in lymphocytes of mice which were kept undernormal temperature (15ndash20∘C) (b) Morphology of mitochondria in lymphocytes of mice which were kept under 4∘C about 4 hours eachday (c) Mice suffering from cold stress were treated by MGGD then the morphology of mitochondria in lymphocytes was observed underelectron microscopy (d) Mice were treated by MGGD alone then the morphology of mitochondria in lymphocytes was observed (e) Thenumber of mitochondriacell was counted lowast119901 lt 005 versus control group
Evidence-Based Complementary and Alternative Medicine 5
ATP content
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0000
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
120583m
olg
pro
t
(a)
Mock Cold stress
MGGD + cold stress MGGD
R R
R R
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
(b)
Figure 2 The ATP content and mitochondrial membrane potential of lymphocytes (a) Cold stress caused the decrease of ATP contentin lymphocytes The ATP content of lymphocytes recovered after the mice treated by MGGD lowast119901 lt 005 versus control group 119901 lt 005versus cold stress group (b) Mitochondrial membrane potential of lymphocytes was measured by flow cytometry The values mentioned inthe lower right corner of each flow cytometric dot-plot indicate how much membrane potential decreased Representative dot-plots of threeindependent experiments are shown Compared with the mice kept under normal temperature the mice suffering from cold stress had lowmitochondrial membrane potential in lymphocytes MGGD can make the mitochondrial membrane potential of lymphocytes recover to acertain extent
6 Evidence-Based Complementary and Alternative Medicine
Activity of complex I
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0100
0200
0300
0400
0500
0600
0700120583
mol
NA
DH
min
(a)
Activity of complex II
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0010
0020
0030
0040
0050
0060
0070
0080
0090
120583m
ol D
CPIP
min
(b)
Figure 3 The activity of mitochondrial respiratory chain complex in lymphocytes Cold stress had effect on the activity of mitochondrialrespiratory chain complexMGGDcan reduce this kind of effect (a)The activity ofmitochondrial respiratory chain complex I (b)The activityof the mitochondrial respiratory chain complex II lowast119901 lt 005 versus control group 119901 lt 005 versus cold stress group
4 Discussion
Cold cough is one of the most common clinical diseasesIn China Mahuang Gancao Ganjiang Decoction used inthe treatment of typhoid symptoms has a long history Ourstudy indicated that MGGD can regulate the cold-stress-induced apoptosis It has been demonstrated that the coldstress induces apoptosis in mammalian cells [1 2 21] Sothe mechanisms of MGGD protecting cells from this type ofinjury in vivo need to be investigated Our data presentedhere show that the response of lymphocytes to cold stressis associated with the imbalance of mitochondrial fissionand fusion MGGD can reduce this kind of effect throughincreasing the expression level of fusion-related protein anddecreasing the expression level of fission-related protein
MGGD as a popular formula in TCM was first recordedin Synopsis Prescriptions of Golden Chamber which is acomprehensive medical book edited in Eastern Han DynastyThree Chinese herbs are included in the prescription whichare Mahuang (two Taels) Gancao (four Taels) and Ganjiang(two Taels) MGGD is a representative formula for treatingasthma and anti-cold and recognized to be an alternativetherapy for cold injury Recently a clinical study performedby Qin showed that MGGD could significantly relieve thediscomfort in cold cough patients [10] However the curativemechanism has not been clearly demonstrated
Mitochondria participate in many physiological pro-cesses The responses of mammalian cells to cold stress havebeen demonstrated to be related to the changes in mito-chondrial function [22] Mitochondrial function is closelyrelated to its morphology Morphology of mitochondrial isin the dynamic balance of fusion and fission In previousstudies it has been shown that disruption of fusion causedmitochondrial heterogeneity [6 23 24] and an increased
rate of fission caused the mitochondrial fragmentation Inour study we found that the mice response to cold stressresulted in the fragmented morphology of mitochondriain lymphocytes (Figures 1(a) and 1(b)) This indicated theincreased rate of mitochondrial fission and the changes inits function Mitochondrial dysfunction was always accom-panied with the reduction of ATP content and its membranepotential [25] So we decided to check these two parts inlymphocytes and found that both of them decreased undercold stress (Figures 2(a) and 2(b)) Our data also showedthe activity of respiratory chain complexes I and II reducedunder cold stress (Figures 3(a) and 3(b)) These findingsdemonstrated cold stress causes the disruption of normalfunction ofmitochondria in lymphocytesHowever our studyalso showed that these effects induced by cold stress werealleviated by MGGD-formula
In order to find out the mechanism about how MGGDreduced the effect of cold stress onmitochondria we detectedthe level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 in lympho-cytes of four groups of mice In mammals Mfn1 Mfn2 andOpa-1 are necessary for the mitochondrial fusion OPA1 isrequired for the fusion of mitochondrial inner membraneThe loss of Opa-1 protein leads to dispersal of mitochondrialfragments and cell apoptosis Once the Mfn1 and Mfn2were deleted it will lead to the reduction of fusion andfragmentation of mitochondria [6 21] Drp1 and Fis-1 areknown as essential protein in mitochondrial fission It ismainly in the form of poly and located in the cytoplasm[8 26 27] According to the results of immunofluorescenceand western blotting the expression level of Mfn1 Mfn2and Opa-1 reduced and the expression of Drp1 and Fis-1increased during cold stress These are the reasons why theincrease of number of mitochondriacell was observed inlymphocytes of cold stress group This phenomenon can be
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
2 Evidence-Based Complementary and Alternative Medicine
MGGD-formula can work on the apoptosis also needs to beexplored Hence the aims of this study were to investigate theeffect ofMGGDon cold-stress-induced apoptosis and relatedmechanism
2 Materials and Methods
21 Animals and Lymphocytes Preparation SPF grade Kun-ming mice were purchased from the experimental animalcenter of Hubei University of traditional Chinese medicineThe medical laboratory animal certificate number is SCXK(Hubei) 2008-0005 The mice were randomly divided into4 groups (a) control group the mice were kept under a12-hour lightdark cycle (lights on at 800 am) with roomtemperature (20ndash23∘C) and humidity (50 plusmn 5) (b) coldstress group the mice were put under 4∘C for 4 hours inartificial climate box with humidity (50plusmn 5) each day in aweek (c)MGGD+ cold stress group themicewere put under4∘C for 4 hours daily and then were conducted by intragastricadministration of MGGD (d) MGGD group the mice werekept under room temperature and then were conductedby intragastric administration of MGGD The capacity ofintragastric administration was 1ml100 g body weight Inour study femalemicewere excluded Allmanipulationswereperformed between 700 am and 1200 am every day tominimize the influence of circadian rhythms
After treatment all the mice in the four groups wereinjected with an appropriate amount of sodium for anaesthe-sia One eye of anesthetic mice was pulled off by tweezersand the fresh blood was collected by the 5ml EP tubewith anticoagulants Lymphocytes separation was performedusing a mouse T Cell Isolation Kit (Miltenyi) accordingto the manufacturerrsquos instructions After the separationlymphocytes were used in the following experiments
22 MGGD Composition and Administration MGGD-for-mula was composed of the following traditional Chineseherbal medicines as recorded by the ldquoFormulas of ChineseMedicinerdquo [13]Mahuang (Ephedra) 20 gGancao (Liquorice)30 g and Ganjiang (dried ginger) 30 g which were com-posed in 2 3 3 proportion All raw materials in MGGD-formula were prepared and examined according to qualitycontrol standard of Chinese Pharmacopoeia [14] It wasmanufactured by the Preparation Room for TCM of theChinese Medicine Hospital of Hubei Province The mice inthe MGGD-formula group were given intragastric admin-istration with MGGD-formula (1ml100 gd) The controlgroup and cold stress group were given distilled water
23 Assessment of Mitochondrial Morphology with ElectronMicroscopy Lymphocytes were collected and fixed with 25glutaraldehyde on ice for 2 h Then cells were treated by 2osmium tetroxide and dehydrated with sequential washeswith 50 70 90 95 and 100 ethanol Cells wereblock mounted and thinly sliced Cells of thin sections wereexamined under electron microscope
24 Determination of Intracellular ATP Content The lym-phocytes were washed with cold PBS three times and 200120583l
of the lysis buffer was added The cells were L centrifuged(12000timesg 10 minutes 4∘C) and supernatants were har-vested 100 120583l ATP working solution was added to thesupernatants in 15ml tube and the tube was incubated atroom temperature for 5minutesTheATP content in sampleswas measured with Luminometer At the same time proteinconcentration was measured via BCAmethodThen the ATPconcentration is converted into the form of 120583molg protein
25 Mitochondrial Membrane Potential Detection (JC-1) Thelymphocytes of peripheral blood were washed by ice-coldPBS three times and centrifuged at 1500 rpm for 5min 5 times105 cells were suspended in 05ml cell culture medium anddyed by 05ml JC-1 dyeing fluid Cells were incubated at 37∘Cfor 20minutes After the incubation cells were centrifuged at1200 rpm for 3min and supernatants were discarded Cellswere washed by JC-1 dyeing buffer 2 times and suspendedby 05ml JC-1 dyeing buffer The mitochondrial membranepotential of lymphocytes was detected by flow cytometry
26 Measurement of Mitochondrial Respiratory Chain Com-plex Activity Lymphocytes were homogenized with 1ml ofpotassium phosphate buffer (100mM pH 74) at 4∘C Thehomogenates were centrifuged (1500timesg 5min 4∘C) and thesupernatants were collected The content of mitochondrialprotein was measured via the bicinchoninic protein assayAll activities were determined on a spectrophotometer inpotassium phosphate buffer Complex I activity (NADH-CoQ reductase) was determined bymeasuring the disappear-ance of NADH at 340 nm in the presence of decylubiquinone[15] Complex II activity (Succinate Dehydrogenase) wasevaluated by measuring the reduction of dichloroindophenol(DCIP) at 600 nm [16]
27 Immunofluorescence Analysis Cell suspension was fixedby 4 paraformaldehyde and coated on the slides drynaturally The cells were washed three times with PBS Thencells were treated by 01 Triton X-100 for 20min andblocked with 3 BSA for 30min Specific primary Absfor Mfn1 Mfn2 or Drp1 were added and incubated for1 h at room temperature Mito-Tracker Green was used todetect the mitochondria in lymphocytes Cells were thenwashed with 1 BSA for three times followed by incubationwith goat anti-rabbit IgG fluorescent secondary antibodyfor 1 h Nucleus was stained with DAPI (410158406-diamidino-2-phenylindole) Cells were observed under the fluorescencemicroscope
28 SDS-PAGE and WB Lymphocytes were harvested andlysed with 100 120583l of lysis buffer (20mM Tris-HCl [pH80] 100mM NaCl 19 [wtvol] Triton X-100 1mM 14-dithiothreitol and 5 [volvol] glycerol) for 30 min on iceAfter centrifugation for 30min at 4∘C the supernatants werecollected The concentration of protein was determined viathe Bradford method Equal amounts of protein were sepa-rated by 10 SDS-PAGE and transferred onto a nitrocellulosemembrane (GE Healthcare) Membrane was blocked with5 nonfat milk and incubated with the primary antibodies
Evidence-Based Complementary and Alternative Medicine 3
followed by HRP-conjugated goat anti-rabbit secondary anti-bodies
29 Detection of Apoptosis The lymphocytes of mice werewashed by PBS three times and centrifuged at 1500 rpm5min Cells were suspended by binding buffer 500 120583l Thenthe mixture containing 5120583l AnnexinV-FITC and 5 120583l PI wasadded into cell suspension Cells were incubated at roomtemperature for 5sim15min in dark The number of apoptoticcells was determined by flow cytometry
3 Results
31 Effect of MGGD on the Mitochondrial Morphology underCold Stress Previous study has demonstrated that coldstress can affect the normal morphology of mitochondria[17] In our study we observed the cold stress causes themitochondrial fragmentation Under cold stress mitochon-drial morphology became shorter and vacuole structure(Figures 1(a) and 1(b)) We also noticed that number ofmitochondriacell increased under cold stress (Figure 1(d))These indicated that the fission of mitochondria increasedbecause of the cold stress After being treated by MGGD thedamage of mitochondria in the mice reduced during coldstress In Figure 1(c) vacuolization of mitochondria was notobserved obviously and the cristae of mitochondria werevisible in lymphocytes of the mice conducted by intragastricadministration of MGGD These results demonstrated thatMGGD-formula can reduce the effect caused by cold stresson mitochondrial morphology
32 Effects of MGGD on Cold Stress Induced Decreasing ofATP Content and Mitochondrial Membrane Potential Thechange of mitochondrial morphology is closely related toits function Mitochondrial membrane potential is an indexto evaluate the function of mitochondria According toprevious study when the mitochondrial fission increasedATP synthesis and mitochondrial membrane potential willdecrease [18ndash20] In our experiment both the ATP contentand the mitochondrial membrane potential of lymphocytesdecreased in cold stress group (Figures 2(a) and 2(b)) Asshown in Figure 2(b) themitochondrial membrane potentialof cold stress group decreased about 20 compared to that ofcontrol andMGGD group After themice suffering from coldstress were treated by MGGD the ATP content recoveredMeanwhile themitochondrialmembrane potential increasedabout 10 compared to that of cold stress group Thesedata showed that MGGD can alleviate the effect caused bycold stress on ATP content and mitochondrial membranepotential
33 MGGDCan Reduce the Effect of Cold Stress on RespiratoryChain Complex The activity of respiratory chain complex isresponsible for the mitochondrial membrane potential andATP content in lymphocytes Based on the mitochondrialrespiratory chain complex activity detection results showedthat cold stress reduced the activity of complexes I and II by42 and 61 respectively (119901 lt 005) After being treatedby MGGD the activity of complexes I and II recovered to
74 and 80 compared with the control group (119901 lt 005)(Figures 3(a) and 3(b)) These data showed that respiratorychain complex activity decreased because of the imbalance ofmitochondrial fusion and fission induced by cold stress andMGGD can reduce this kind of effect
34 Effects of MGGD on the Expression of Mfn1 Mfn2 Drp1Fis-1 and Opa-1 Until now the impairment and dysfunctionof mitochondria had been demonstrated to be related to theimbalance of its fusion and fission The fusion and fission ofmitochondria are closely related to theMfn1Mfn2Drp1 Fis-1 and Opa-1 In order to check whether the expression ofMfn1 Mfn2 and Drp1 was affected by cold stress or MGGDthe Mfn1 Mfn2 and Drp1 in lymphocytes were labeled byFITC and observed under the fluorescencemicroscope Fromthe density of the fluorescence we can see that the expressionlevel of Mfn1 and Mfn2 decreased in lymphocytes undercold stress but the expression level of Drp1 increased InMGGD-formula group we observed increasing expressionlevel of Mfn1 and Mfn2 meanwhile expression level of Drp1decreased (Figure 4(a)) We also detected the expressionlevel of Mfn1 Mfn2 and Drp1 by western blotting Theresults of western blotting are consistent with immunoflu-orescence (Figures 4(b) 4(c) and 4(d)) Western blottingalso revealed that cold stress caused the increased Fis-1protein and decreasedOpa-1 protein in lymphocytes (Figures4(e) and 4(f)) These results suggested that MGGD affectsthe mitochondrial fusion and fission through regulating theexpression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1
35 MGGD Has Effect on the Colocalization of Drp-1 andMitochondria During the fission of mitochondria Drp1was recruited by Fis-1 and translocated from cytoplasm tothe mitochondrial outer membrane Drp1 distributed at thepotential division sites of mitochondrial outer membrane inthe form of ring [8] As shown in Figure 5 Drp-1 gatheredaround the mitochondria under the cold stress HoweverDrp-1 mainly distributed in the cytoplasm in other threegroups This suggested that MGGD can reduce the fission ofmitochondria through inhibiting the translocation of Drp-1
36 MGGD Can Weaken the Cold-Stress-Induced ApoptosisThe normal function of mitochondria is very important forthe growth of cells In our study we found that cold stressaffected the normal function of mitochondria and MGGDcan alleviate this kind of effect So we want to check ifMGGDcan help the cells survive during cold stressThe lymphocytesin peripheral blood of four groups of mice were isolated andthe number of apoptotic cells was detected by flow cytometryFrom Figures 6(a) and 6(b) we can see that the cold stresscan induce the apoptosis of lymphocytes The number ofapoptotic lymphocytes decreased in MGGD-formula group(Figure 6(c)) The results of western blotting showed thatactivation of caspases 3 and 9 was induced by cold stress andMGGD can reduce this effect (Figures 6(e) and 6(f)) Thesesuggested that MGGD can alleviate the cold-stress-inducedapoptosis
4 Evidence-Based Complementary and Alternative Medicine
Mock
1120583m
(a)
Cold stress
1120583m
(b)
MGGD + cold stress
1120583m
(c)
MGGD
1120583m
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0
5
10
15
20
25
30
Num
ber o
f mito
chon
dria
cel
l
(e)
Figure 1 Morphology of mitochondria in lymphocytes Lymphocytes were isolated from the peripheral blood of mice and analyzed forthe morphology of mitochondria via electron microscopy (a) Morphology of mitochondria in lymphocytes of mice which were kept undernormal temperature (15ndash20∘C) (b) Morphology of mitochondria in lymphocytes of mice which were kept under 4∘C about 4 hours eachday (c) Mice suffering from cold stress were treated by MGGD then the morphology of mitochondria in lymphocytes was observed underelectron microscopy (d) Mice were treated by MGGD alone then the morphology of mitochondria in lymphocytes was observed (e) Thenumber of mitochondriacell was counted lowast119901 lt 005 versus control group
Evidence-Based Complementary and Alternative Medicine 5
ATP content
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0000
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
120583m
olg
pro
t
(a)
Mock Cold stress
MGGD + cold stress MGGD
R R
R R
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
(b)
Figure 2 The ATP content and mitochondrial membrane potential of lymphocytes (a) Cold stress caused the decrease of ATP contentin lymphocytes The ATP content of lymphocytes recovered after the mice treated by MGGD lowast119901 lt 005 versus control group 119901 lt 005versus cold stress group (b) Mitochondrial membrane potential of lymphocytes was measured by flow cytometry The values mentioned inthe lower right corner of each flow cytometric dot-plot indicate how much membrane potential decreased Representative dot-plots of threeindependent experiments are shown Compared with the mice kept under normal temperature the mice suffering from cold stress had lowmitochondrial membrane potential in lymphocytes MGGD can make the mitochondrial membrane potential of lymphocytes recover to acertain extent
6 Evidence-Based Complementary and Alternative Medicine
Activity of complex I
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0100
0200
0300
0400
0500
0600
0700120583
mol
NA
DH
min
(a)
Activity of complex II
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0010
0020
0030
0040
0050
0060
0070
0080
0090
120583m
ol D
CPIP
min
(b)
Figure 3 The activity of mitochondrial respiratory chain complex in lymphocytes Cold stress had effect on the activity of mitochondrialrespiratory chain complexMGGDcan reduce this kind of effect (a)The activity ofmitochondrial respiratory chain complex I (b)The activityof the mitochondrial respiratory chain complex II lowast119901 lt 005 versus control group 119901 lt 005 versus cold stress group
4 Discussion
Cold cough is one of the most common clinical diseasesIn China Mahuang Gancao Ganjiang Decoction used inthe treatment of typhoid symptoms has a long history Ourstudy indicated that MGGD can regulate the cold-stress-induced apoptosis It has been demonstrated that the coldstress induces apoptosis in mammalian cells [1 2 21] Sothe mechanisms of MGGD protecting cells from this type ofinjury in vivo need to be investigated Our data presentedhere show that the response of lymphocytes to cold stressis associated with the imbalance of mitochondrial fissionand fusion MGGD can reduce this kind of effect throughincreasing the expression level of fusion-related protein anddecreasing the expression level of fission-related protein
MGGD as a popular formula in TCM was first recordedin Synopsis Prescriptions of Golden Chamber which is acomprehensive medical book edited in Eastern Han DynastyThree Chinese herbs are included in the prescription whichare Mahuang (two Taels) Gancao (four Taels) and Ganjiang(two Taels) MGGD is a representative formula for treatingasthma and anti-cold and recognized to be an alternativetherapy for cold injury Recently a clinical study performedby Qin showed that MGGD could significantly relieve thediscomfort in cold cough patients [10] However the curativemechanism has not been clearly demonstrated
Mitochondria participate in many physiological pro-cesses The responses of mammalian cells to cold stress havebeen demonstrated to be related to the changes in mito-chondrial function [22] Mitochondrial function is closelyrelated to its morphology Morphology of mitochondrial isin the dynamic balance of fusion and fission In previousstudies it has been shown that disruption of fusion causedmitochondrial heterogeneity [6 23 24] and an increased
rate of fission caused the mitochondrial fragmentation Inour study we found that the mice response to cold stressresulted in the fragmented morphology of mitochondriain lymphocytes (Figures 1(a) and 1(b)) This indicated theincreased rate of mitochondrial fission and the changes inits function Mitochondrial dysfunction was always accom-panied with the reduction of ATP content and its membranepotential [25] So we decided to check these two parts inlymphocytes and found that both of them decreased undercold stress (Figures 2(a) and 2(b)) Our data also showedthe activity of respiratory chain complexes I and II reducedunder cold stress (Figures 3(a) and 3(b)) These findingsdemonstrated cold stress causes the disruption of normalfunction ofmitochondria in lymphocytesHowever our studyalso showed that these effects induced by cold stress werealleviated by MGGD-formula
In order to find out the mechanism about how MGGDreduced the effect of cold stress onmitochondria we detectedthe level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 in lympho-cytes of four groups of mice In mammals Mfn1 Mfn2 andOpa-1 are necessary for the mitochondrial fusion OPA1 isrequired for the fusion of mitochondrial inner membraneThe loss of Opa-1 protein leads to dispersal of mitochondrialfragments and cell apoptosis Once the Mfn1 and Mfn2were deleted it will lead to the reduction of fusion andfragmentation of mitochondria [6 21] Drp1 and Fis-1 areknown as essential protein in mitochondrial fission It ismainly in the form of poly and located in the cytoplasm[8 26 27] According to the results of immunofluorescenceand western blotting the expression level of Mfn1 Mfn2and Opa-1 reduced and the expression of Drp1 and Fis-1increased during cold stress These are the reasons why theincrease of number of mitochondriacell was observed inlymphocytes of cold stress group This phenomenon can be
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
Evidence-Based Complementary and Alternative Medicine 3
followed by HRP-conjugated goat anti-rabbit secondary anti-bodies
29 Detection of Apoptosis The lymphocytes of mice werewashed by PBS three times and centrifuged at 1500 rpm5min Cells were suspended by binding buffer 500 120583l Thenthe mixture containing 5120583l AnnexinV-FITC and 5 120583l PI wasadded into cell suspension Cells were incubated at roomtemperature for 5sim15min in dark The number of apoptoticcells was determined by flow cytometry
3 Results
31 Effect of MGGD on the Mitochondrial Morphology underCold Stress Previous study has demonstrated that coldstress can affect the normal morphology of mitochondria[17] In our study we observed the cold stress causes themitochondrial fragmentation Under cold stress mitochon-drial morphology became shorter and vacuole structure(Figures 1(a) and 1(b)) We also noticed that number ofmitochondriacell increased under cold stress (Figure 1(d))These indicated that the fission of mitochondria increasedbecause of the cold stress After being treated by MGGD thedamage of mitochondria in the mice reduced during coldstress In Figure 1(c) vacuolization of mitochondria was notobserved obviously and the cristae of mitochondria werevisible in lymphocytes of the mice conducted by intragastricadministration of MGGD These results demonstrated thatMGGD-formula can reduce the effect caused by cold stresson mitochondrial morphology
32 Effects of MGGD on Cold Stress Induced Decreasing ofATP Content and Mitochondrial Membrane Potential Thechange of mitochondrial morphology is closely related toits function Mitochondrial membrane potential is an indexto evaluate the function of mitochondria According toprevious study when the mitochondrial fission increasedATP synthesis and mitochondrial membrane potential willdecrease [18ndash20] In our experiment both the ATP contentand the mitochondrial membrane potential of lymphocytesdecreased in cold stress group (Figures 2(a) and 2(b)) Asshown in Figure 2(b) themitochondrial membrane potentialof cold stress group decreased about 20 compared to that ofcontrol andMGGD group After themice suffering from coldstress were treated by MGGD the ATP content recoveredMeanwhile themitochondrialmembrane potential increasedabout 10 compared to that of cold stress group Thesedata showed that MGGD can alleviate the effect caused bycold stress on ATP content and mitochondrial membranepotential
33 MGGDCan Reduce the Effect of Cold Stress on RespiratoryChain Complex The activity of respiratory chain complex isresponsible for the mitochondrial membrane potential andATP content in lymphocytes Based on the mitochondrialrespiratory chain complex activity detection results showedthat cold stress reduced the activity of complexes I and II by42 and 61 respectively (119901 lt 005) After being treatedby MGGD the activity of complexes I and II recovered to
74 and 80 compared with the control group (119901 lt 005)(Figures 3(a) and 3(b)) These data showed that respiratorychain complex activity decreased because of the imbalance ofmitochondrial fusion and fission induced by cold stress andMGGD can reduce this kind of effect
34 Effects of MGGD on the Expression of Mfn1 Mfn2 Drp1Fis-1 and Opa-1 Until now the impairment and dysfunctionof mitochondria had been demonstrated to be related to theimbalance of its fusion and fission The fusion and fission ofmitochondria are closely related to theMfn1Mfn2Drp1 Fis-1 and Opa-1 In order to check whether the expression ofMfn1 Mfn2 and Drp1 was affected by cold stress or MGGDthe Mfn1 Mfn2 and Drp1 in lymphocytes were labeled byFITC and observed under the fluorescencemicroscope Fromthe density of the fluorescence we can see that the expressionlevel of Mfn1 and Mfn2 decreased in lymphocytes undercold stress but the expression level of Drp1 increased InMGGD-formula group we observed increasing expressionlevel of Mfn1 and Mfn2 meanwhile expression level of Drp1decreased (Figure 4(a)) We also detected the expressionlevel of Mfn1 Mfn2 and Drp1 by western blotting Theresults of western blotting are consistent with immunoflu-orescence (Figures 4(b) 4(c) and 4(d)) Western blottingalso revealed that cold stress caused the increased Fis-1protein and decreasedOpa-1 protein in lymphocytes (Figures4(e) and 4(f)) These results suggested that MGGD affectsthe mitochondrial fusion and fission through regulating theexpression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1
35 MGGD Has Effect on the Colocalization of Drp-1 andMitochondria During the fission of mitochondria Drp1was recruited by Fis-1 and translocated from cytoplasm tothe mitochondrial outer membrane Drp1 distributed at thepotential division sites of mitochondrial outer membrane inthe form of ring [8] As shown in Figure 5 Drp-1 gatheredaround the mitochondria under the cold stress HoweverDrp-1 mainly distributed in the cytoplasm in other threegroups This suggested that MGGD can reduce the fission ofmitochondria through inhibiting the translocation of Drp-1
36 MGGD Can Weaken the Cold-Stress-Induced ApoptosisThe normal function of mitochondria is very important forthe growth of cells In our study we found that cold stressaffected the normal function of mitochondria and MGGDcan alleviate this kind of effect So we want to check ifMGGDcan help the cells survive during cold stressThe lymphocytesin peripheral blood of four groups of mice were isolated andthe number of apoptotic cells was detected by flow cytometryFrom Figures 6(a) and 6(b) we can see that the cold stresscan induce the apoptosis of lymphocytes The number ofapoptotic lymphocytes decreased in MGGD-formula group(Figure 6(c)) The results of western blotting showed thatactivation of caspases 3 and 9 was induced by cold stress andMGGD can reduce this effect (Figures 6(e) and 6(f)) Thesesuggested that MGGD can alleviate the cold-stress-inducedapoptosis
4 Evidence-Based Complementary and Alternative Medicine
Mock
1120583m
(a)
Cold stress
1120583m
(b)
MGGD + cold stress
1120583m
(c)
MGGD
1120583m
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0
5
10
15
20
25
30
Num
ber o
f mito
chon
dria
cel
l
(e)
Figure 1 Morphology of mitochondria in lymphocytes Lymphocytes were isolated from the peripheral blood of mice and analyzed forthe morphology of mitochondria via electron microscopy (a) Morphology of mitochondria in lymphocytes of mice which were kept undernormal temperature (15ndash20∘C) (b) Morphology of mitochondria in lymphocytes of mice which were kept under 4∘C about 4 hours eachday (c) Mice suffering from cold stress were treated by MGGD then the morphology of mitochondria in lymphocytes was observed underelectron microscopy (d) Mice were treated by MGGD alone then the morphology of mitochondria in lymphocytes was observed (e) Thenumber of mitochondriacell was counted lowast119901 lt 005 versus control group
Evidence-Based Complementary and Alternative Medicine 5
ATP content
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0000
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
120583m
olg
pro
t
(a)
Mock Cold stress
MGGD + cold stress MGGD
R R
R R
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
(b)
Figure 2 The ATP content and mitochondrial membrane potential of lymphocytes (a) Cold stress caused the decrease of ATP contentin lymphocytes The ATP content of lymphocytes recovered after the mice treated by MGGD lowast119901 lt 005 versus control group 119901 lt 005versus cold stress group (b) Mitochondrial membrane potential of lymphocytes was measured by flow cytometry The values mentioned inthe lower right corner of each flow cytometric dot-plot indicate how much membrane potential decreased Representative dot-plots of threeindependent experiments are shown Compared with the mice kept under normal temperature the mice suffering from cold stress had lowmitochondrial membrane potential in lymphocytes MGGD can make the mitochondrial membrane potential of lymphocytes recover to acertain extent
6 Evidence-Based Complementary and Alternative Medicine
Activity of complex I
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0100
0200
0300
0400
0500
0600
0700120583
mol
NA
DH
min
(a)
Activity of complex II
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0010
0020
0030
0040
0050
0060
0070
0080
0090
120583m
ol D
CPIP
min
(b)
Figure 3 The activity of mitochondrial respiratory chain complex in lymphocytes Cold stress had effect on the activity of mitochondrialrespiratory chain complexMGGDcan reduce this kind of effect (a)The activity ofmitochondrial respiratory chain complex I (b)The activityof the mitochondrial respiratory chain complex II lowast119901 lt 005 versus control group 119901 lt 005 versus cold stress group
4 Discussion
Cold cough is one of the most common clinical diseasesIn China Mahuang Gancao Ganjiang Decoction used inthe treatment of typhoid symptoms has a long history Ourstudy indicated that MGGD can regulate the cold-stress-induced apoptosis It has been demonstrated that the coldstress induces apoptosis in mammalian cells [1 2 21] Sothe mechanisms of MGGD protecting cells from this type ofinjury in vivo need to be investigated Our data presentedhere show that the response of lymphocytes to cold stressis associated with the imbalance of mitochondrial fissionand fusion MGGD can reduce this kind of effect throughincreasing the expression level of fusion-related protein anddecreasing the expression level of fission-related protein
MGGD as a popular formula in TCM was first recordedin Synopsis Prescriptions of Golden Chamber which is acomprehensive medical book edited in Eastern Han DynastyThree Chinese herbs are included in the prescription whichare Mahuang (two Taels) Gancao (four Taels) and Ganjiang(two Taels) MGGD is a representative formula for treatingasthma and anti-cold and recognized to be an alternativetherapy for cold injury Recently a clinical study performedby Qin showed that MGGD could significantly relieve thediscomfort in cold cough patients [10] However the curativemechanism has not been clearly demonstrated
Mitochondria participate in many physiological pro-cesses The responses of mammalian cells to cold stress havebeen demonstrated to be related to the changes in mito-chondrial function [22] Mitochondrial function is closelyrelated to its morphology Morphology of mitochondrial isin the dynamic balance of fusion and fission In previousstudies it has been shown that disruption of fusion causedmitochondrial heterogeneity [6 23 24] and an increased
rate of fission caused the mitochondrial fragmentation Inour study we found that the mice response to cold stressresulted in the fragmented morphology of mitochondriain lymphocytes (Figures 1(a) and 1(b)) This indicated theincreased rate of mitochondrial fission and the changes inits function Mitochondrial dysfunction was always accom-panied with the reduction of ATP content and its membranepotential [25] So we decided to check these two parts inlymphocytes and found that both of them decreased undercold stress (Figures 2(a) and 2(b)) Our data also showedthe activity of respiratory chain complexes I and II reducedunder cold stress (Figures 3(a) and 3(b)) These findingsdemonstrated cold stress causes the disruption of normalfunction ofmitochondria in lymphocytesHowever our studyalso showed that these effects induced by cold stress werealleviated by MGGD-formula
In order to find out the mechanism about how MGGDreduced the effect of cold stress onmitochondria we detectedthe level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 in lympho-cytes of four groups of mice In mammals Mfn1 Mfn2 andOpa-1 are necessary for the mitochondrial fusion OPA1 isrequired for the fusion of mitochondrial inner membraneThe loss of Opa-1 protein leads to dispersal of mitochondrialfragments and cell apoptosis Once the Mfn1 and Mfn2were deleted it will lead to the reduction of fusion andfragmentation of mitochondria [6 21] Drp1 and Fis-1 areknown as essential protein in mitochondrial fission It ismainly in the form of poly and located in the cytoplasm[8 26 27] According to the results of immunofluorescenceand western blotting the expression level of Mfn1 Mfn2and Opa-1 reduced and the expression of Drp1 and Fis-1increased during cold stress These are the reasons why theincrease of number of mitochondriacell was observed inlymphocytes of cold stress group This phenomenon can be
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
4 Evidence-Based Complementary and Alternative Medicine
Mock
1120583m
(a)
Cold stress
1120583m
(b)
MGGD + cold stress
1120583m
(c)
MGGD
1120583m
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0
5
10
15
20
25
30
Num
ber o
f mito
chon
dria
cel
l
(e)
Figure 1 Morphology of mitochondria in lymphocytes Lymphocytes were isolated from the peripheral blood of mice and analyzed forthe morphology of mitochondria via electron microscopy (a) Morphology of mitochondria in lymphocytes of mice which were kept undernormal temperature (15ndash20∘C) (b) Morphology of mitochondria in lymphocytes of mice which were kept under 4∘C about 4 hours eachday (c) Mice suffering from cold stress were treated by MGGD then the morphology of mitochondria in lymphocytes was observed underelectron microscopy (d) Mice were treated by MGGD alone then the morphology of mitochondria in lymphocytes was observed (e) Thenumber of mitochondriacell was counted lowast119901 lt 005 versus control group
Evidence-Based Complementary and Alternative Medicine 5
ATP content
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0000
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
120583m
olg
pro
t
(a)
Mock Cold stress
MGGD + cold stress MGGD
R R
R R
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
(b)
Figure 2 The ATP content and mitochondrial membrane potential of lymphocytes (a) Cold stress caused the decrease of ATP contentin lymphocytes The ATP content of lymphocytes recovered after the mice treated by MGGD lowast119901 lt 005 versus control group 119901 lt 005versus cold stress group (b) Mitochondrial membrane potential of lymphocytes was measured by flow cytometry The values mentioned inthe lower right corner of each flow cytometric dot-plot indicate how much membrane potential decreased Representative dot-plots of threeindependent experiments are shown Compared with the mice kept under normal temperature the mice suffering from cold stress had lowmitochondrial membrane potential in lymphocytes MGGD can make the mitochondrial membrane potential of lymphocytes recover to acertain extent
6 Evidence-Based Complementary and Alternative Medicine
Activity of complex I
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0100
0200
0300
0400
0500
0600
0700120583
mol
NA
DH
min
(a)
Activity of complex II
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0010
0020
0030
0040
0050
0060
0070
0080
0090
120583m
ol D
CPIP
min
(b)
Figure 3 The activity of mitochondrial respiratory chain complex in lymphocytes Cold stress had effect on the activity of mitochondrialrespiratory chain complexMGGDcan reduce this kind of effect (a)The activity ofmitochondrial respiratory chain complex I (b)The activityof the mitochondrial respiratory chain complex II lowast119901 lt 005 versus control group 119901 lt 005 versus cold stress group
4 Discussion
Cold cough is one of the most common clinical diseasesIn China Mahuang Gancao Ganjiang Decoction used inthe treatment of typhoid symptoms has a long history Ourstudy indicated that MGGD can regulate the cold-stress-induced apoptosis It has been demonstrated that the coldstress induces apoptosis in mammalian cells [1 2 21] Sothe mechanisms of MGGD protecting cells from this type ofinjury in vivo need to be investigated Our data presentedhere show that the response of lymphocytes to cold stressis associated with the imbalance of mitochondrial fissionand fusion MGGD can reduce this kind of effect throughincreasing the expression level of fusion-related protein anddecreasing the expression level of fission-related protein
MGGD as a popular formula in TCM was first recordedin Synopsis Prescriptions of Golden Chamber which is acomprehensive medical book edited in Eastern Han DynastyThree Chinese herbs are included in the prescription whichare Mahuang (two Taels) Gancao (four Taels) and Ganjiang(two Taels) MGGD is a representative formula for treatingasthma and anti-cold and recognized to be an alternativetherapy for cold injury Recently a clinical study performedby Qin showed that MGGD could significantly relieve thediscomfort in cold cough patients [10] However the curativemechanism has not been clearly demonstrated
Mitochondria participate in many physiological pro-cesses The responses of mammalian cells to cold stress havebeen demonstrated to be related to the changes in mito-chondrial function [22] Mitochondrial function is closelyrelated to its morphology Morphology of mitochondrial isin the dynamic balance of fusion and fission In previousstudies it has been shown that disruption of fusion causedmitochondrial heterogeneity [6 23 24] and an increased
rate of fission caused the mitochondrial fragmentation Inour study we found that the mice response to cold stressresulted in the fragmented morphology of mitochondriain lymphocytes (Figures 1(a) and 1(b)) This indicated theincreased rate of mitochondrial fission and the changes inits function Mitochondrial dysfunction was always accom-panied with the reduction of ATP content and its membranepotential [25] So we decided to check these two parts inlymphocytes and found that both of them decreased undercold stress (Figures 2(a) and 2(b)) Our data also showedthe activity of respiratory chain complexes I and II reducedunder cold stress (Figures 3(a) and 3(b)) These findingsdemonstrated cold stress causes the disruption of normalfunction ofmitochondria in lymphocytesHowever our studyalso showed that these effects induced by cold stress werealleviated by MGGD-formula
In order to find out the mechanism about how MGGDreduced the effect of cold stress onmitochondria we detectedthe level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 in lympho-cytes of four groups of mice In mammals Mfn1 Mfn2 andOpa-1 are necessary for the mitochondrial fusion OPA1 isrequired for the fusion of mitochondrial inner membraneThe loss of Opa-1 protein leads to dispersal of mitochondrialfragments and cell apoptosis Once the Mfn1 and Mfn2were deleted it will lead to the reduction of fusion andfragmentation of mitochondria [6 21] Drp1 and Fis-1 areknown as essential protein in mitochondrial fission It ismainly in the form of poly and located in the cytoplasm[8 26 27] According to the results of immunofluorescenceand western blotting the expression level of Mfn1 Mfn2and Opa-1 reduced and the expression of Drp1 and Fis-1increased during cold stress These are the reasons why theincrease of number of mitochondriacell was observed inlymphocytes of cold stress group This phenomenon can be
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
Evidence-Based Complementary and Alternative Medicine 5
ATP content
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
lowast
0000
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
120583m
olg
pro
t
(a)
Mock Cold stress
MGGD + cold stress MGGD
R R
R R
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
101 102 103 104100
G
(b)
Figure 2 The ATP content and mitochondrial membrane potential of lymphocytes (a) Cold stress caused the decrease of ATP contentin lymphocytes The ATP content of lymphocytes recovered after the mice treated by MGGD lowast119901 lt 005 versus control group 119901 lt 005versus cold stress group (b) Mitochondrial membrane potential of lymphocytes was measured by flow cytometry The values mentioned inthe lower right corner of each flow cytometric dot-plot indicate how much membrane potential decreased Representative dot-plots of threeindependent experiments are shown Compared with the mice kept under normal temperature the mice suffering from cold stress had lowmitochondrial membrane potential in lymphocytes MGGD can make the mitochondrial membrane potential of lymphocytes recover to acertain extent
6 Evidence-Based Complementary and Alternative Medicine
Activity of complex I
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0100
0200
0300
0400
0500
0600
0700120583
mol
NA
DH
min
(a)
Activity of complex II
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0010
0020
0030
0040
0050
0060
0070
0080
0090
120583m
ol D
CPIP
min
(b)
Figure 3 The activity of mitochondrial respiratory chain complex in lymphocytes Cold stress had effect on the activity of mitochondrialrespiratory chain complexMGGDcan reduce this kind of effect (a)The activity ofmitochondrial respiratory chain complex I (b)The activityof the mitochondrial respiratory chain complex II lowast119901 lt 005 versus control group 119901 lt 005 versus cold stress group
4 Discussion
Cold cough is one of the most common clinical diseasesIn China Mahuang Gancao Ganjiang Decoction used inthe treatment of typhoid symptoms has a long history Ourstudy indicated that MGGD can regulate the cold-stress-induced apoptosis It has been demonstrated that the coldstress induces apoptosis in mammalian cells [1 2 21] Sothe mechanisms of MGGD protecting cells from this type ofinjury in vivo need to be investigated Our data presentedhere show that the response of lymphocytes to cold stressis associated with the imbalance of mitochondrial fissionand fusion MGGD can reduce this kind of effect throughincreasing the expression level of fusion-related protein anddecreasing the expression level of fission-related protein
MGGD as a popular formula in TCM was first recordedin Synopsis Prescriptions of Golden Chamber which is acomprehensive medical book edited in Eastern Han DynastyThree Chinese herbs are included in the prescription whichare Mahuang (two Taels) Gancao (four Taels) and Ganjiang(two Taels) MGGD is a representative formula for treatingasthma and anti-cold and recognized to be an alternativetherapy for cold injury Recently a clinical study performedby Qin showed that MGGD could significantly relieve thediscomfort in cold cough patients [10] However the curativemechanism has not been clearly demonstrated
Mitochondria participate in many physiological pro-cesses The responses of mammalian cells to cold stress havebeen demonstrated to be related to the changes in mito-chondrial function [22] Mitochondrial function is closelyrelated to its morphology Morphology of mitochondrial isin the dynamic balance of fusion and fission In previousstudies it has been shown that disruption of fusion causedmitochondrial heterogeneity [6 23 24] and an increased
rate of fission caused the mitochondrial fragmentation Inour study we found that the mice response to cold stressresulted in the fragmented morphology of mitochondriain lymphocytes (Figures 1(a) and 1(b)) This indicated theincreased rate of mitochondrial fission and the changes inits function Mitochondrial dysfunction was always accom-panied with the reduction of ATP content and its membranepotential [25] So we decided to check these two parts inlymphocytes and found that both of them decreased undercold stress (Figures 2(a) and 2(b)) Our data also showedthe activity of respiratory chain complexes I and II reducedunder cold stress (Figures 3(a) and 3(b)) These findingsdemonstrated cold stress causes the disruption of normalfunction ofmitochondria in lymphocytesHowever our studyalso showed that these effects induced by cold stress werealleviated by MGGD-formula
In order to find out the mechanism about how MGGDreduced the effect of cold stress onmitochondria we detectedthe level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 in lympho-cytes of four groups of mice In mammals Mfn1 Mfn2 andOpa-1 are necessary for the mitochondrial fusion OPA1 isrequired for the fusion of mitochondrial inner membraneThe loss of Opa-1 protein leads to dispersal of mitochondrialfragments and cell apoptosis Once the Mfn1 and Mfn2were deleted it will lead to the reduction of fusion andfragmentation of mitochondria [6 21] Drp1 and Fis-1 areknown as essential protein in mitochondrial fission It ismainly in the form of poly and located in the cytoplasm[8 26 27] According to the results of immunofluorescenceand western blotting the expression level of Mfn1 Mfn2and Opa-1 reduced and the expression of Drp1 and Fis-1increased during cold stress These are the reasons why theincrease of number of mitochondriacell was observed inlymphocytes of cold stress group This phenomenon can be
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
6 Evidence-Based Complementary and Alternative Medicine
Activity of complex I
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0100
0200
0300
0400
0500
0600
0700120583
mol
NA
DH
min
(a)
Activity of complex II
lowast
Moc
k
Col
d str
ess
+M
GG
Dco
ld st
ress
MG
GD
0000
0010
0020
0030
0040
0050
0060
0070
0080
0090
120583m
ol D
CPIP
min
(b)
Figure 3 The activity of mitochondrial respiratory chain complex in lymphocytes Cold stress had effect on the activity of mitochondrialrespiratory chain complexMGGDcan reduce this kind of effect (a)The activity ofmitochondrial respiratory chain complex I (b)The activityof the mitochondrial respiratory chain complex II lowast119901 lt 005 versus control group 119901 lt 005 versus cold stress group
4 Discussion
Cold cough is one of the most common clinical diseasesIn China Mahuang Gancao Ganjiang Decoction used inthe treatment of typhoid symptoms has a long history Ourstudy indicated that MGGD can regulate the cold-stress-induced apoptosis It has been demonstrated that the coldstress induces apoptosis in mammalian cells [1 2 21] Sothe mechanisms of MGGD protecting cells from this type ofinjury in vivo need to be investigated Our data presentedhere show that the response of lymphocytes to cold stressis associated with the imbalance of mitochondrial fissionand fusion MGGD can reduce this kind of effect throughincreasing the expression level of fusion-related protein anddecreasing the expression level of fission-related protein
MGGD as a popular formula in TCM was first recordedin Synopsis Prescriptions of Golden Chamber which is acomprehensive medical book edited in Eastern Han DynastyThree Chinese herbs are included in the prescription whichare Mahuang (two Taels) Gancao (four Taels) and Ganjiang(two Taels) MGGD is a representative formula for treatingasthma and anti-cold and recognized to be an alternativetherapy for cold injury Recently a clinical study performedby Qin showed that MGGD could significantly relieve thediscomfort in cold cough patients [10] However the curativemechanism has not been clearly demonstrated
Mitochondria participate in many physiological pro-cesses The responses of mammalian cells to cold stress havebeen demonstrated to be related to the changes in mito-chondrial function [22] Mitochondrial function is closelyrelated to its morphology Morphology of mitochondrial isin the dynamic balance of fusion and fission In previousstudies it has been shown that disruption of fusion causedmitochondrial heterogeneity [6 23 24] and an increased
rate of fission caused the mitochondrial fragmentation Inour study we found that the mice response to cold stressresulted in the fragmented morphology of mitochondriain lymphocytes (Figures 1(a) and 1(b)) This indicated theincreased rate of mitochondrial fission and the changes inits function Mitochondrial dysfunction was always accom-panied with the reduction of ATP content and its membranepotential [25] So we decided to check these two parts inlymphocytes and found that both of them decreased undercold stress (Figures 2(a) and 2(b)) Our data also showedthe activity of respiratory chain complexes I and II reducedunder cold stress (Figures 3(a) and 3(b)) These findingsdemonstrated cold stress causes the disruption of normalfunction ofmitochondria in lymphocytesHowever our studyalso showed that these effects induced by cold stress werealleviated by MGGD-formula
In order to find out the mechanism about how MGGDreduced the effect of cold stress onmitochondria we detectedthe level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 in lympho-cytes of four groups of mice In mammals Mfn1 Mfn2 andOpa-1 are necessary for the mitochondrial fusion OPA1 isrequired for the fusion of mitochondrial inner membraneThe loss of Opa-1 protein leads to dispersal of mitochondrialfragments and cell apoptosis Once the Mfn1 and Mfn2were deleted it will lead to the reduction of fusion andfragmentation of mitochondria [6 21] Drp1 and Fis-1 areknown as essential protein in mitochondrial fission It ismainly in the form of poly and located in the cytoplasm[8 26 27] According to the results of immunofluorescenceand western blotting the expression level of Mfn1 Mfn2and Opa-1 reduced and the expression of Drp1 and Fis-1increased during cold stress These are the reasons why theincrease of number of mitochondriacell was observed inlymphocytes of cold stress group This phenomenon can be
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
Evidence-Based Complementary and Alternative Medicine 7
Mock Cold stress MGGD + cold stress MGGDM
fn1
Mfn2
Drp
1
(a)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn1
GAPDH
4321
(b)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Mfn2
GAPDH
4321
(c)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Drp1
GAPDH
4321
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Fis-1
4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
GAPDH
Opa-1
4321
(f)
Figure 4 The expression level of Mfn1 Mfn2 Drp1 Fis-1 and Opa-1 (a) Mfn1 Mfn2 and Drp1 in lymphocytes were detected by specificantibody and labeled by FITC via immunofluorescence as described in Materials and Methods The nucleus was stained with DAPI (b cand d)The expression levels of Mfn1 Mfn2 and Drp1 in lymphocytes were analyzed via western blot GAPDH was used as control (e and f)Expression of Fis-1 and Opa-1 in lymphocytes was detected via western blotting with specific antibody
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
8 Evidence-Based Complementary and Alternative Medicine
Moc
kC
old
stres
sM
GG
D+
cold
stre
ssM
GG
D
Drp-1 Mitochondria Merge
Figure 5 The colocalization of Drp-1 and mitochondria was analyzed Mitochondria were labeled by Mito-Tracker Green Drp1 inlymphocytes were detected by specific antibody and labeled by Cy3 as described in Materials and Methods
reduced byMGGD becauseMGGD can increase the amountof Mfn1 Mfn2 and Opa-1 in cells At the same time theexpression of Drp1 and Fis-1 was downregulated by MGGD(Figure 4)
It is well known that mitochondrion is an importantexecutor of apoptosis the abnormalities of its structureand function can lead to cell apoptosis Previous studyelucidated that the silencing of the Mfn2 can increase theBcl-2 expression Under normal condition upregulated Bcl-2 could redeem the loss of Mfn2 However Bcl-2 expressiondecreased in cells treated with Mfn2 siRNA could notredeem the loss of Mfn2 during cold stress and the cellapoptosis occurred [28] In our experiment we also foundcold stress can induce the apoptosis of lymphocytes (Figures6(a) and 6(b)) As mitochondria are so important to cold-stress-induced apoptosis maintaining their normal functionis critical for protection of the lymphocytes against thisprocess Fortunately MGGD was found to be able to protectlymphocytes against cold-stress-induced apoptosis at certainextent (Figure 6(c))
Lymphocytes are very important for the immuneresponse in mammals The increasing of lymphocytesapoptosis may cause disorder of immune system and can not
protect the organism from being invaded by germs or viruseseffectively during cold exposure Our findings indicatedthat MGGD may maintain the immune response throughinhibiting the lymphocytes apoptosis in mice under coldstress
In conclusion the present study has demonstrated thatMGGD was able to regulate the fusion and fission of mito-chondria and the apoptosis of lymphocytes mediated byregulating the expression level of Mfn1 Mfn2 and Drp1under cold stressOur results also suggested that thismight bethe mechanism of MGGD helping mice adapt to cold stressat least in part
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
This study was supported by grants from the Hubei ProvinceEducation Department of China (no D20152002) and HubeiProvincial Department of Education (no B2016112)
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
Evidence-Based Complementary and Alternative Medicine 9
Mock
100
101
102
103
104
PI
101 102 103 104100
FITC(a)
Cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(b)
MGGD + cold stress
100
101
102
103
104
PI
101 102 103 104100
FITC(c)
MGGD
101 102 103 104100
FITC
100
101
102
103
104
PI
(d)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 3
Cleaved caspase 3
GAPDH4321
(e)
Moc
k
Col
d str
ess
MG
GD
+co
ld st
ress
MG
GD
Procaspase 9
Cleaved caspase 9
GAPDH4321
(f)
Figure 6 The apoptosis of lymphocytes was detected by flow cytometry (a b c and d) The values mentioned in the upper right cornerand lower right corner of each flow cytometric dot-plot indicate the number apoptotic cells Representative dot-plots of three independentexperiments are shown (e and f) The activation of caspases 3 and 9 Procaspase 3 procaspase 9 cleaved caspase 3 and cleaved caspase 9protein levels were analyzed by western blotting
References
[1] T R Doeppner T Grune H De Groot and U Rauen ldquoCold-induced apoptosis of rat liver endothelial cells involvement ofthe proteasomerdquo Transplantation vol 75 no 12 pp 1946ndash19532003
[2] U Kerkweg M Jacob H De Groot H-G Mannherz and URauen ldquoCold-induced apoptosis of rat liver endothelial cellscontribution ofmitochondrial alterationsrdquoTransplantation vol76 no 3 pp 501ndash508 2003
[3] U Rauen B Polzar H Stephan H G Mannherz and H DeGroot ldquoCold-induced apoptosis in cultured hepatocytes andliver endothelial cells mediation by reactive oxygen speciesrdquoFASEB Journal vol 13 no 1 pp 155ndash168 1999
[4] A Nechushtan C L Smith Y-T Hsu and R J Youle ldquoConfor-mation of the BaxC-terminus regulates subcellular location andcell deathrdquo EMBO Journal vol 18 no 9 pp 2330ndash2341 1999
[5] H Yuan A A Gerencser G Liot et al ldquoMitochondrial fissionis an upstream and required event for bax foci formation in
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
10 Evidence-Based Complementary and Alternative Medicine
response to nitric oxide in cortical neuronsrdquo Cell Death andDifferentiation vol 14 no 3 pp 462ndash471 2007
[6] H Chen S A Detmer A J Ewald E E Griffin S E Fraserand D C Chan ldquoMitofusins Mfn1 and Mfn2 coordinatelyregulate mitochondrial fusion and are essential for embryonicdevelopmentrdquo Journal of Cell Biology vol 160 no 2 pp 189ndash200 2003
[7] S-Y Choi P Huang G M Jenkins D C Chan J Schillerand M A Frohman ldquoA common lipid links Mfn-mediatedmitochondrial fusion and SNARE-regulated exocytosisrdquoNatureCell Biology vol 8 no 11 pp 1255ndash1262 2006
[8] E Smirnova L Griparic D-L Shurland and A M Van derBliek ldquoDynamin-related proteinDrp1 is required formitochon-drial division inmammalian cellsrdquoMolecular Biology of the Cellvol 12 no 8 pp 2245ndash2256 2001
[9] H Bai ldquoLiquorice and dried ginger decoction in the treatmentof lung cold cough in 48 casesrdquo Journal of Yunnan ChineseMedicine vol 21 no 4 p 31 2000
[10] Z Qin ldquoLiquorice ginger soup treatment of cut cold coughapplied researchrdquo Journal of Practical Traditional Chinese Inter-nal Medicine vol 26 no 9 pp 24ndash25 2012
[11] R Sugioka S Shimizu and Y Tsujimoto ldquoFzo1 a proteininvolved in mitochondrial fusion inhibits apoptosisrdquoThe Jour-nal of Biological Chemistry vol 279 no 50 pp 52726ndash527342004
[12] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008
[13] Z J Deng Formulas of Chinese Medicine Chinese Press ofTraditional Chinese Medicine Beijing China 2003
[14] PRCPCosMoPHoChinese Pharmacopoeia Chemical IndustryPress 2005
[15] K A Kramer D Oglesbee S J Hartman et al ldquoAutomatedspectrophotometric analysis ofmitochondrial respiratory chaincomplex enzyme activities in cultured skin fibroblastsrdquo ClinicalChemistry vol 51 no 11 pp 2110ndash2116 2005
[16] O Miro A Barrientos J R Alonso et al ldquoEffects of generalanaesthetic procedures on mitochondrial function of humanskeletalmusclerdquoEuropean Journal of Clinical Pharmacology vol55 no 1 pp 35ndash41 1999
[17] W Zhang Y Chen Q Yang et al ldquoMitofusin-2 protects againstcold stress-induced cell injury in HEK293 cellsrdquo Biochemicaland Biophysical Research Communications vol 397 no 2 pp270ndash276 2010
[18] T Ono K Isobe K Nakada and J-I Hayashi ldquoHuman cells areprotected from mitochondrial dysfunction by complementa-tion of DNA products in fused mitochondriardquo Nature Geneticsvol 28 no 3 pp 272ndash275 2001
[19] T J Collins M J Berridge P Lipp andM D Bootman ldquoMito-chondria are morphologically and functionally heterogeneouswithin cellsrdquo EMBO Journal vol 21 no 7 pp 1616ndash1627 2002
[20] F Legros A Lombes P Frachon and M Rojo ldquoMitochondrialfusion in human cells is efficient requires the inner membranepotential and is mediated by mitofusinsrdquo Molecular Biology ofthe Cell vol 13 no 12 pp 4343ndash4354 2002
[21] M Vairetti P Griffini G Pietrocola P Richelmi and I FreitasldquoCold-induced apoptosis in isolated rat hepatocytes protectiverole of glutathionerdquo Free Radical Biology and Medicine vol 31no 8 pp 954ndash961 2001
[22] M P Mollica L Lionetti R Crescenzo et al ldquoCold exposuredifferently influences mitochondrial energy efficiency in rat
liver and skeletal musclerdquo FEBS Letters vol 579 no 9 pp 1978ndash1982 2005
[23] H Chen and D C Chan ldquoEmerging functions of mammalianmitochondrial fusion and fissionrdquo Human Molecular Geneticsvol 14 no 2 pp R283ndashR289 2005
[24] H Chen A Chomyn and D C Chan ldquoDisruption of fusionresults in mitochondrial heterogeneity and dysfunctionrdquo TheJournal of Biological Chemistry vol 280 no 28 pp 26185ndash26192 2005
[25] L P Kravchenko and B J Fuller ldquoEnergy status of isolatedhepatocytes after long-term cold storage in various types ofmediardquo Ukrainskii Biokhimicheskii Zhurnal vol 73 no 5 pp55ndash60 2001
[26] A M Labrousse M D Zappaterra D A Rube and A M Vander Bliek ldquoC elegans dynamin-related protein DRP-1 controlssevering of themitochondrial outermembranerdquoMolecular Cellvol 4 no 5 pp 815ndash826 1999
[27] H-W Shin H Takatsu H Mukai E Munekata K Murakamiand K Nakayama ldquoIntermolecular and interdomain inter-actions of a dynamin-related GTP-binding protein Dnm1pVps1p-like proteinrdquoThe Journal of Biological Chemistry vol 274no 5 pp 2780ndash2785 1999
[28] R J Youle and M Karbowski ldquoMitochondrial fission in apop-tosisrdquo Nature Reviews Molecular Cell Biology vol 6 no 8 pp657ndash663 2005
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