Research Article The Loss of Cellular Junctions in...

Research ArticleThe Loss of Cellular Junctions in Epithelial Lung CellsInduced by Cigarette Smoke Is Attenuated by Corilagin

Ximena M Muresan1 Franco Cervellati1 Claudia Sticozzi1

Giuseppe Belmonte1 Chung Hin Chui2 Ilaria Lampronti3 Monica Borgatti1

Roberto Gambari1 and Giuseppe Valacchi1

1Department of Life Sciences and Biotechnology University of Ferrara 44121 Ferrara Italy2Clinical Division School of Chinese Medicine Hong Kong Baptist University Kowloon Tong Hong Kong3Biotechnology Center University of Ferrara 44121 Ferrara Italy

Correspondence should be addressed to Giuseppe Valacchi giuseppevalacchiunifeit

Received 25 November 2014 Revised 2 January 2015 Accepted 21 January 2015

Academic Editor Neelam Khaper

Copyright copy 2015 Ximena M Muresan et al 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

Cigarette smoke (CS) contains over 4700 compounds many of which can affect cellular redox balance through free radicalsproduction or through themodulation of antioxidant enzymesThe respiratory tract is one of the organs directly exposed to CS andit is known that CS can damage the integrity of lung epithelium by affecting cell junctions and increasing epithelium permeabilityIn this study we have used a human lung epithelial cell line Calu-3 to evaluate the effect of CS on lung epithelial cell junctionslevels with special focus on the expression of two proteins involved in intercellular communication connexins (Cx) 40 and 43CS exposure increased Cx40 gene expression but not of Cx43 CS also induced NF120581B activation and the formation of 4HNE-Cxsadducts Since corilagin a natural polyphenol is able to inhibit NF120581B activation we have determined whether corilagin couldcounteract the effect of CS on Cxs expression Corilagin was able to diminish CS induced Cx40 gene expression 4HNE-Cx40adducts formation and NF120581B activation The results of this study demonstrated that CS induced the loss of cellular junctions inlung epithelium possibly as a consequence of Cx-4HNE adducts formation and corilagin seems to be able to abolish these CSinduced alterations

1 Introduction

Cigarette smoke (CS) is a heterogeneous mixture formed bya gaseous phase and solid particles and it contains morethan 4700 compounds including carcinogen and oxidantsubstances [1 2] Due to its ability to induce cellular oxidativestress CS causes noxious effects in a large part of humanorgans such as cardiovascular system [3] cutaneous tissues[4] retina [5] and of course lung tissues The ability of CSto induce oxidative stress has been associated with cellulardamage such as DNA mutations [6] lipid peroxidation withformation of reactive aldehydes [7] and proteins oxidation[8] In addition it is documented that CS is able to promoteinflammatory responses in cells via TNF-120572 andNF120581B signal-ing pathway [9]

The main target of CS is obviously the respiratory tractcharacterized by a pseudostratified epithelium which actsas a barrier protecting the organism from environmentalantigens Respiratory epithelial cells are bound together bydifferent cellular junctions as described in Heijink et al [10]Gap junctions in particular are membrane channels formedby the union of two hemichannels belonging to adjacentcells these hemichannels are also known as connexons eachof them is composed of six connexins that are four-passtransmembrane proteins [11] Gap junctions have multiplefunctions by allowing the direct passage of ions secondmessengers and metabolites among cells Therefore gapjunctions allow an essential communication that maintainsnormal functions of tissues and organs [12]These communi-cations are important in all physiological cellular processes

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 631758 12 pageshttpdxdoiorg1011552015631758

2 Oxidative Medicine and Cellular Longevity

such as cell growth cell development cell differentiation andcell death and its absence leads to changes in the cellularhomeostasis [13] In addition connexins are known to affectcell migration and adhesion [14ndash16]

In the last few decades many studies have shown thatpolyphenols and oxidant scavengers can contrast CS inducedoxidative stress [17ndash20] Corilagin is a polyphenol memberof the tannin family extracted from different plants likeDimocarpus longan [21] and Geranium thunbergii [22] Thisnatural compound seems to have beneficial effects in sev-eral cardiovascular disorders hypertension thrombosis oratherosclerosis [23] but it is also known for its antiprolifera-tive and antitumor effects [24] Zhao et al have demonstratedthe anti-inflammatory properties of corilagin Their studyshows that corilagin is able to block NF120581B activation andits nuclear translocation demonstrating anti-inflammatorycharacteristic In addition it has been shown that corilagindecreases the production of proinflammatory cytokines likeTNF-120572 IL-1120573 IL-6 iNOS and COX-2 [25] Furthermoresome studies have evidenced the ability of corilagin to inhibitIL-8 production by blocking TNF-120572 induced NF120581B nucleartranslocation [26] It has been also demonstrated that cori-lagin is hepatoprotective as it acts like radical scavenger forsuperoxide anion and peroxyl radicals In addition corilagininhibits ROS production from leukocytes as well as freeradicals formation and lipid peroxidation in mitochondria[27]

In our study we evaluated the damaging effects of CSon lung epithelial cells in particular we investigated CS-modulation of gap junctions and connexin expression andwealso determined whether corilagin could counteract the nox-ious effect of CS For our purpose we have used Calu-3 cellline grown in air-liquid interface which has been reportedto be a good model to study biological agents on humanairway epithelial cells functions and structures Indeed thismodel has been extensively used for lung pathophysiologyand therapeutics studies and it exhibits many characteristicsof the primary airway cell cultures thanks to its ability to formstable cell-to-cell junctions [28]

2 Materials and Methods

21 Cell Culture and Treatment Calu-3 cells cultured in75 cm2 plastic flasks (BD Falcon USA) were grown inDulbeccorsquos modified Eaglersquos medium (Lonza Milan Italy)supplemented with 10 fetal bovine serum (EuroCloneMilan Italy) 2mM l-glutamine (Lonza Milan Italy) andantibiotics (100UImL penicillin and 100 120583gmL strepto-mycin) (Lonza Milan Italy) They were maintained at 37∘Cin a humidified 5 CO

2atmosphere Upon reaching 90

confluence cells were harvested using 002 trypsinEDTA(Lonza Milan Italy) After trypsinisation Calu-3 cells wereseeded at a density of 1 times 106 cellsmL onto 04120583m pore size415 cm2 surface area polyethylene terephthalate Transwellcell culture inserts (BD Falcon USA) placed in 6-well cellculture plates (BD Falcon USA)When the cells reached 90of confluence and were pretreated with corilagin 10 120583M for24 hr then the culture media were changed and the cells wereexposed to CS

Corilagin was a gift by the China National Institute forthe Control of Pharmaceutical and Biological Products Thepowder was dissolved in ethanol and the 10 120583M solution wasadded to the cells (final ethanol concentration 01)

22 CS Exposure Cells were exposed to CS for 50min infresh serum-free media (as previously described [29]) whilecontrol cells were exposed to filtered air (50min) CS wasgenerated by burning one research cigarette (12mg tar 11mgnicotine) using a vacuum pump to draw air through theburning cigarette and leading the smoke stream over the cellculture as described previously [29] After the exposure (airor CS) fresh media supplemented with 10 FBS were addedto the cells

23 Cytotoxicity Determination After CS exposure culturemedia were collected at different time points (50min 6 hr12 hr and 24 hr) Cytotoxicitywas determined by LDHreleasein the media measured by enzymatic assay in the first stepNAD+ is reduced to NADHH+ by the LDH-catalyzedconversion of lactate to pyruvate in the second step thecatalyst (diaphorase) transfers HH+ from NADHH+ totetrazolium salt which is reduced to formazan The amountof LDH in the supernatant was determined and calculatedaccording to kit instructions (EuroClone Milan Italy) Alltests were performed in triplicate and assays were repeatedthree times independently

24 Protein Extraction At each time point cells werewashed with ice-cold PBS and lysed in ice-cold lysis buffer(20mM Tris pH 8 150mM NaCl 1 Triton X-100 1mMsodium orthovanadate 1 120583gmL leupeptin 1 120583gmL apro-tinin 1 120583gmL pepstatin 10 120583gmL PMSF and 5mM 120573-glycerophosphate) (Sigma Milan Italy) After centrifugation(13500 rpm 15min at 4∘C) the supernatants were collectedProteins concentration was determined by the method ofBradford (BioRad Milan Italy)

25Western Blot Analysis Sixty 120583g boiled proteinwas loadedonto 10 sodium dodecyl sulphate-polyacrylamide elec-trophoresis gels and separated by molecular size Gels wereelectroblotted onto nitrocellulose membranes and then wereblocked for 1 hr in Tris-buffered saline pH 75 containing05 Tween 20 and 3 milk Membranes were incubatedovernight at 4∘C with the appropriate primary antibodiesCx40 (Santa Cruz Biotechnology Inc USA) Cx43 (SantaCruz Biotechnology Inc USA) 4HNE (Chemicon USA)and 120573-actin (Cell Signalling Celbio Milan Italy) The mem-branes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr and the boundantibodies were detected by chemiluminescence (BioRadMilan Italy) 120573-actin was used as loading control Images ofthe bands were digitized and the densitometry analysis wasperformed using ImageJ software

26 Electrophoretic Mobility Shift Assay (EMSA) Elec-trophoretic Mobility Shift Assay (EMSA) was performedas previously described [30 31] Briefly double-strandedsynthetic oligodeoxynucleotides mimicking two functional

Oxidative Medicine and Cellular Longevity 3

NF120581B binding sites displaying different sequence (A sense51015840-GTT CTG GGA TTT CCC CCG AT-31015840 B sense 51015840-CAG CAG GAA CGT CCC AGA GAA-31015840) [32] have beenemployed Oligodeoxynucleotides were labeled with 12057432-P-ATP using 10 units of T4-polynucleotide-kinase (Kinase-MAX Ambion) in 500mMTris-HCl pH 76 100mMMgCl

2

50mM DTT 1mM spermidine and 1mM EDTA in thepresence of 50 mCi 12057432-P-ATP in a volume of 20120583L for60 minutes at 37∘C Reaction was brought to 150mM NaCland 150 ng complementary oligodeoxynucleotide was addedReaction temperature was increased to 100∘C for 5 minutesand left diminishing to room temperature overnight Bindingreactions were set up as described elsewhere [30] in a totalvolume of 20 120583L containing buffer TF plus 5 glycerol1mM dithiothreitol 5 ng of human NF120581B p50 protein anddifferent concentrations of corilagin After incubation of20min at room temperature 025 ng of 32P-labeled oligonu-cleotides was added to the samples for further 20min atroom temperature and then they were electrophoresed atconstant voltage (200V) under low ionic strength conditions(025x TBE buffer 22mM Tris borate 04mM EDTA) on6 polyacrylamide gels Gels were dried and subjected tostandard autoradiographic procedures [30]

27 Protein Carbonyls The levels of proteins carbonyl groupswere determined by OxyBlot (Chemicon USA) Briefly afterderivatization of carbonyl groups to dinitrophenylhydrazone(DNP-hydrazone) by reaction with dinitrophenylhydrazine(DNPH) the DNP-derivatized protein samples were sep-arated by polyacrylamide gel electrophoresis followed byWestern blotting as previously described [33]

28 Immunoprecipitation Cell lysates containing 300 120583g ofprotein were mixed with Dynabeads protein G (InvitrogenUSA) and 2 120583g of polyclonal antibody against Cx40 Fol-lowing immunoprecipitation of Cx40 the proteins were sep-arated by SDS-PAGE electrotransferred in a nitrocellulosemembranes and immunoblotted with 4HNE antibody

29 Preparation of Cytoplasmic and Nuclear Extracts forWestern Blotting For cytoplasmic and nuclear extracts cellswere resuspended in 100 120583L of hypotonic buffer containing10mmolL HEPES (pH 79) 10mmolL KCl 15mmolLMgCl

2 03 Nonidet P-40 05mmolL dithiothreitol

05mmolL phenylmethylsulphonylfluoride proteaseinhibitor cocktail 1mmolL orthovanadate and 5mmolL120573-glycerophosphate The lysates were incubated for 15minon ice and then centrifuged at 1500timesg for 5min at 4∘C forcollection of the supernatant containing cytosolic proteinsPellets containing nuclei were resuspended in 50120583L ofextraction buffer containing 20mmolL HEPES (pH 79)15mmolL MgCl

2 06molL KCl 02mmolL EDTA 20

glycerol 05mmolL phenylmethylsulfonyl fluoride proteaseinhibitor cocktail 1mmolL orthovanadate and 5mmolL120573-glycerophosphate and then were incubated for 30minon ice Samples were then centrifuged at 13000 rpm for15min to obtain supernatants containing nuclear fractionsProtein samples were separated by polyacrylamide gel

electrophoresis followed by Western blotting NF120581B (SantaCruz Biotechnology Inc USA) was used as primaryantibody

210 RT-qPCR (Reverse Transcription Quantitative Real-TimePCR) Total RNA from each sample was extracted with theAURUM Total RNA Mini Kit with DNAse digestion (Bio-Rad Laboratories Inc USA) according to themanufacturerrsquosrecommended procedure After solubilization in RNAase-free water total RNA was quantified by Bio-Rad SmartSpecPlus spectrophotometer (Bio-Rad Laboratories Inc USA)First-strand cDNA was generated from 1120583g of total RNAusing iScript cDNA Synthesis Kit (Bio-Rad Laboratories IncUSA) Primer pairs were obtained fromPrimerBank from theReal-Time PCR Primer and Probe Database RTPrimerDBto hybridise to unique regions of the appropriate genesequence The reverse transcriptase (RT) PCR reactions werecarried out using 1 120583L of cDNA in a 15 120583L total volumeof PCR buffer (Invitrogen Milan Italy) containing 3mMMgCl

2 300 120583M dNTPs and 300 nM of appropriate primers

Taq polymerase (035U) was also added The amplificationreactions were carried out in a thermal gradient cycler(Bio-Rad Laboratories Inc USA) for 40 cycles Each cycleconsists of denaturation for 30 s at 94∘C annealing for 30 sat 60∘C and extension for 30 s at 72∘C A final extensionstep at 72∘C for 5min terminates the amplification Foreach amplification two controls were performed (i) RT-PCRmixture with no reverse transcriptase to control for genomicDNA contamination and (ii) PCR mixture with no cDNAtemplate to check for possible external contamination A5 120583L sample of the PCR reaction was electrophoresed on anethidium bromide containing 2 agarose gel by the use ofthe Bio-Rad Subcell GT system Quantitative Real-Time PCR(qPCR) was performed using SYBR Green on iQ5MulticolorReal-Time PCR Detection System (Bio-Rad LaboratoriesInc USA) The final reaction mixture contained 1120583L ofcDNA 300 nM of each primer 75120583L of iQ SYBR GreenSupermix (Bio-Rad Laboratories Inc USA) and RNAse-freewater to complete the reaction mixture volume to 15 120583L Allreactions were run as triplicates The QPCR was performedwith a hot-start denaturation step at 95∘C for 3min andthen was carried out for 40 cycles at 95∘C for 10 s and at60∘C for 20 s The fluorescence was read during the reactionby the Opticon Monitor 3 software (Bio-Rad LaboratoriesInc USA) allowing a continuous monitoring of the amountof PCR products Primers are initially used to generate astandard curve over a large dynamic range of starting cDNAquantity which allows calculating the amplification efficiency(a critical value for the correct quantification of expressiondata) for each of the primer pairs The melting curve analysiswas performed at the end of each experiment to verifythat a single product for primer pair is amplified (data notshown) As to control experiments gel electrophoresis wasalso performed to verify the sizes of the amplified QPCRproducts Ribosomal proteins L13a (RPL13a) L11a (RPL11a)and GAPDH were used in our experiments as internalstandards Samples were compared using the relative cyclethreshold (CT) method The fold increase or decrease wasdetermined relative to a control after normalising to RPL13a


Table 1 Cellular viability as measured by trypan blue assay

Samples Cell viability ()Control 89 plusmn 15CS 50min 78 plusmn 12CS 6 hr 69 plusmn 21CS 24 hr 62 plusmn 18

(internal standard) The formula 2 minus ΔΔCT was used whereΔCT is gene of interest CT (RPL13A CT) and ΔΔCT is ΔCTexperimental (ΔCT control)

211 Ultrastructural Analysis Cells were scraped and col-lected in 01M cacodylate buffer (pH 74) and then spun in15mL tubes at 2000timesg for 5min Pellets were fixed with 25glutaraldehyde in 01M sodium cacodylate buffer for 4 hr at4∘CThey were then washed with 01M cacodylate buffer (pH74) three times and postfixed in 1 osmium tetroxide and01M cacodylate buffer at pH 74 for 1 hr at room temperatureThe specimens were dehydrated in graded concentrations ofethanol and embedded in epoxide resin (Agar Scientific 66ACambridge Road Stansted Essex CM24 8DA UK)

Cells were then transferred to latex modules filled withresin and subsequently thermally cured at 60∘C for 48 hr

Semithin sections (05ndash1120583m thickness) were cut usingan ultramicrotome (Reichard Ultracut S Austria) stainedwith toluidine blue and blocks were selected for thinningUltrathin sections of about 40ndash60 nm were cut and mountedonto formvar-coated copper grids These were then double-stained with 1 uranyl acetate and 01 lead citrate for30min each and examined under a transmission electronmicroscope Hitachi H-800 (Tokyo Japan) at an acceleratingvoltage of 100KV

212 Statistical Analysis For each of the variables tested two-way analysis of variance (ANOVA) was used A significantresult was indicated by a 119875 value lt 005 Data are expressedas mean plusmn SD of triplicate determinations obtained in 5independent experiments

3 Results

31 Cytotoxic Effect of CS on Human Lung Epithelial Calu-3 Cells The first step of the study was to evaluate Calu-3cytotoxicity induced by CS After CS exposure media werecollected at different time points (from 50min to 24 hr) andthe lactate dehydrogenase (LDH) release from the cells wasmeasured As shown in Figure 1 CS induced a significantrelease of LDH already after 50min of CS exposure (2-fold)and this effect was even more pronounced after 24 hr leadingto 7-fold increase of LDH release with respect to the airexposed cells (the same results were obtained by trypan blueexclusion assay Table 1)

32 CS Exposure Induced Cells Detachment Ultrastructuralstudy was performed to better evaluate the status of the cellsexposed to CS and by transmission electronic microscopy

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Figure 1 CS induces toxicity in Calu-3 cells Cells were exposedto CS for 50 minutes and media were collected at different timepoints (50minndash24 hr) The graph shows evidence of toxicity in cellsexposed toCS asmeasured by LDHrelease in themediaData shownare representative of five independent experiments 119875 lt 005 lowastversus C

(TEM) As shown in Figure 2 control cells (C) appeared witha normal cellular structure and a clear cell-to-cell contactAfter CS exposure the cells started to form internal vesicles(50min 2 hr) and to lose intercellular contacts in a time-dependent manner (6 and 24 hr) as evidenced by the redarrows in the representative image

33 Evidence of Oxidized Proteins in Calu-3 Cells Exposedto CS Calu-3 cells exposed to CS showed increase levels ofoxidative stress as determined by the protein oxidation andlipid peroxidation products formation Carbonyl proteins(Figure 3(a)) levels were significantly higher immediatelyafter CS exposure reaching the highest levels 50minutes afterCS exposure In addition CS induced the formation of 4-hydroxynonenal (4HNE) protein adducts (Figure 3(b)) afterCS exposure in a time-dependent manner (2-fold at 50minand 6-fold at 24 hr)

34 CS Induced the Activation of NF120581B As NF120581B activationis determined by the translocation of p65p50 subunits tothe nucleus Western blotting for p65 subunit was performedon nuclear protein extracts from Calu-3 cells exposed to CSAs shown in Figure 4 CS determined a 2-fold increase inp65 nuclear protein levels at the earlier time points after CSexposure On the contrary no evidence of NF120581B activationwas found at the later time points (ie 12 and 24 hr exposure)suggesting an early NF120581B activation by CS exposure

35 Corilagin Reduces the Cytotoxicity Induced by CS Expo-sure The next step of our study was to investigate whethercorilagin could have a protective effect against CS inducedtoxicity in Calu-3 cells The dose of corilagin was chosenbased on the literature and on preliminary studies whereits toxicity was evaluated in the 0ndash100 120583M concentrationsrange (data not shown) As shown in Figure 5 corilagin


C 62 24CS (hr)

50998400

Figure 2 Cells lose intercellular contact after CS exposure Control cells are well assembled and show the presence of densifications in thecontact regions between them (see black arrows) At later time points (12 and 24 hr) the loss of cell-to-cell contact is clear as shown by redarrows Pictures are taken at different magnifications

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Figure 3 CS promotes proteins oxidation and lipid peroxidation WBs in the top panel are representative of five independent experimentsQuantification of carbonyl proteins is shown in the bottom left panel (a) and quantification of 4HNE is shown in the bottom right panel (b)Data are expressed as arbitrary units (average of five independent experiments 119875 lt 005 lowast versus C)

pretreatment reduced about 30ndash50 the cytotoxicity inducedby CS exposure as measured by LDH release compared tocontrol cells (Figure 5)

36 Pretreatment with Corilagin Prevents CS Induced Loss ofCell-to-Cell Contacts As shown in Figure 6 pretreatmentwith 10 120583M of corilagin prevented CS induced loss of cell-to-cell contacts This effect was evident at all the time points asshown by the black arrows in Figure 6

37 Pretreatment with Corilagin Prevents CS Induced ProteinsOxidation Calu-3 cells pretreated with corilagin showed sig-nificantly decreased levels of carbonyl proteins induced byCSof about 50 during the analyzed time points (Figure 7(a))The same effect was observed for the formation of 4HNE-protein adducts as shown in Figure 7(b) with a level of 4HNEalmost back to the control levels in the cells pretreated withcorilaginThese data confirm that corilagin operates on earlyCS-mediated changes (see in particular Figure 7(a))


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Figure 4 CS exposure activates NF120581B WB in the top panelis representative of five independent experiments In the bottompanel is shown the quantification of nuclear NF120581B protein levelsData are expressed as arbitrary units (average of five independentexperiments 119875 lt 005 lowast versus C) Lamin B1 was used as loadingcontrol

38 Corilagin Prevents CS Induced Connexin 40 ExpressionAs connexin 40 (Cx40) is a protein present in cellular gapjunctions we evaluated whether its expression is affected byCS exposure in Calu-3 cells As shown in Figure 8(a) theprotein levels of Cx40 decreased upon CS exposure startingat 12 hr while corilagin pretreatment partially prevented thisdecrease In parallel as it is depicted in Figure 8(b) Cx40gene expression started to increase significantly in a time-dependent manner after 6 hr from CS exposure (7-fold) andit returned to the control levels at the later time points (12ndash24 hr) in cells pretreated with corilagin

This effect was not noticed for connexin 43 (Cx43)another protein present in the gap junctions as it is shownin Figure 9

39 Corilagin Prevents CS Induced Cx40-4HNE Adduct For-mation Since 4HNE can bind to the proteins ndashSH groupsand Cx40 contains several cysteine amino acids (ndashSH group)the formation of Cx40-4HNE adducts was evaluated byimmunoprecipitation upon CS exposure withwithout thepresence of corilagin As shown in Figure 10 there was a clearand significant formation of 4HNE-Cx40 adducts (25-fold)after CS exposure and this effect was clearly prevented bycorilagin pretreatment

310 Corilagin Inhibits CS Induced NF120581B Activation Thereason for analyzing the effects of corilagin on NF120581B was

C 6 12 24CS (hr)

509984000123456789

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Figure 5 Corilagin pretreatment modulates the toxic effect ofCS The graph shows LDH release after CS exposure from cellspretreated or not with corilagin Lower LDH levels are evident atall time points in cells pretreated Data is representative of fiveindependent experiments 119875 lt 005 lowast versus 0 hr wo corilagin10 120583M (24 hr) versus wo corilagin 10120583M (24 hr)

based on (a) the evidence that CS induces activation ofNF120581B (see Figure 4) and (b) the reported finding thatthe promoter of the connexin 40 gene (GJA5) has NF120581Bconsensus binding sequences suggesting a NF120581B dependenttranscriptional regulation [33] In addition corilagin wasreported to prevent NF120581B activation and regulate NF120581Bdependent gene expression in a different experimental modelsystem [26] we have analyzed whether also in this cellularand experimental model corilagin could prevent CS inducedNF120581B activation and function Indeed as it is shown inFigure 11 cells pretreated with corilagin showed a clear andsignificant inhibition of NF120581B activation induced by CS (4-fold at 50min and 2-fold at 6ndash24 hr) as measured by p65nuclear translocation Furthermore corilagin was able toinhibit although with a slightly different efficiency the NF120581Binteraction to oligonucleotides mimicking the two majorNF120581B consensus sequences (Figures 11(b) and 11(c))

4 Discussion

The results presented in this study underlined the effectsthat CS has on lung epithelial cells with focus on cellularjunctions and how a polyphenolic compound corilagin canattenuate these effects

The respiratory tract is a complex system important forthe process of respiration and it allows the direct contactbetween human body and external oxidative environmentthat can be noxious to lung tissues Indeed lung functionalityis maintained thanks to the RTLF (respiratory tract liningfluid) which is rich in antioxidants compounds and to thecompact epithelium composed of cells containing multiplejunctions [34]

Therefore lung epithelium when intact is able to protectthe respiratory tract from outdoor stressors [35] Nowadaysmany studies focus on the composition of the environmentalair as source of damage for human health Pollutants such as


C 2 6 24

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Figure 6 Corilagin prevents the loss of cell-to-cell contacts induced by CS CS exposure leads to the formation of vesicles inside the cellspiled structures keeping cells together are still visible after 2 6 and 24 hr from exposure (see black arrows) Pictures are taken at differentmagnifications

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Figure 7 CS induced oxidative markers are modulated by corilagin The WBs shown on the top are representative of five differentexperiments Quantification of carbonyl proteins is shown in the bottom left panel (a) and quantification of 4HNE is shown in the bottomright panel (b) Data are expressed as arbitrary units (average of five independent experiments 119875 lt 005) lowast versus 0 hr wo corilagin 10 120583M(24 hr) versus wo corilagin 10 120583M (24 hr)

ozone [36] ambient particles [37] and cigarette smoke (CS)[38] are among the most toxic to which living organisms arecontinuously exposed

It is well documented that CS is toxic on airway cellsespecially due to oxidative damage [39] In our study weused LDH as a marker of cytotoxicity in Calu-3 cells andwe observed increased levels of LDH in cells exposed to CSconfirming the noxious effect that CS has on the airwaysepithelium LDH is released from cells mostly after damageto the cellular membrane and often is correlated to necrosiscellular process In our experimental procedure as previouslypublished [29] the cell number did not vary in a significantmanner as demonstrated by the trypan blue assay thereforewe have analyzed the cells by TEM in order to evaluate thecellular morphology changes induced by CS Of interest wasthe fact that the cells exposed to CS clearly showed a loss in

cell-to-cell contactThis result is in linewith previouswork byUpham et al showing that CS inhibited the formation of gapjunctions via the activation of extracellular receptor kinasein liver epithelial cells of WB-F344 rat [40] In additiona previous work has shown that oxidative stress inducedby cigarette smoke extract (CSE) or H

2O2(which is also

present in CS) is able to induce the opening of gap junctionhemichannels this will cause membrane depolarization andthe opening of the hemichannels will facilitate the entry oftoxic molecules that in turn can injure the cells [41] In ourstudy we were not able to detect a significant cell death butthis could be just a matter of timing and CS doses In factin the study of Ramachandran the authors have used a highdose of H

2O2(1mM) and CSE concentrated that has a quite

different composition from freshly smoked CS used in ourexperiments In addition in our previous work we detected a


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Figure 8 Corilagin reestablishes connexin 40 protein and gene levels after CS exposure WBs in the top left panels are representative of fiveindependent experiments Bands quantification is shown in the bottom left panel (a) 120573-actin was used as loading control Gene expressionis shown in the right panel (b) Data are expressed as arbitrary units (average of five independent experiments 119875 lt 005) lowast versus 0 hr wocorilagin 10120583M (24 hr) versus wo corilagin 10120583M (24 hr)

concentration of H2O2in CS ranging from 30 to 100120583M [37]

which ismuch lower than the one used by Ramachandra et al[41]

Gap junctions and connexins in particular are importantnot only in cellular communication but also in cell-to-celladhesion Among the most representative connexins in thelung there are connexin 40 (Cx40) and connexin 43 (Cx43)[42 43] We have shown that CS was able to decrease thelevels of Cx40 in a time-dependent manner and this is inparallel with the induction of new Cx40 mRNA which couldbe interpreted as a protective cellular response of the cellsto the loss of the protein In addition this confirms the factthat the cells were alive and able to start the new transcriptsWhether the CS-mediated effects on Cx40 and Cx43 are NF-120581B dependent remains to be determined

Our work is in agreement with many other studiesshowing that oxidative stress in specific CS is able toinduce the activation of NF120581B [44] Its activation can bemeasured indirectly by analyzing the nuclear translocationof its cytoplasmic subunits among which there is p65 Wehave shown that the nuclear level of p65 in Calu-3 cells afterCS exposure was significantly increased demonstrating theactivation of NF120581B by CS in airway epithelial cells On theother hand we have demonstrated that corilagin inhibitsNF120581B activation and has important effects on the interactionsbetween NF120581B and DNA As far as NF120581B activation it ispossible that this is the consequence of CS induced oxidative

stress as demonstrated by the increased levels of proteincarbonyls formation and also by the increased levels of4HNE-protein adductsWe have observed that when the cellswere exposed to CS for 50min the levels of carbonyl proteinsincreased quickly indicating that CS led to the oxidation ofproteins Similar response was observed in the case of 4HNEadducts as CS induced lipid peroxidation and formation of120572120573-unsaturated aldehydes

Our study suggests another possiblemechanismbywhichCS can affect gap junction besides the one elegantly proposedby Ramachandra et al that is the loss of gap junction is aconsequence of the formation of 4HNE-connexin adducts Infact since CS induced the formation of 4HNE this aldehydeis able to bind to Cys His or Lys residues via Michaeladdition that are present in large amount in Cx40 Our datademonstrated that Cx40was able to form adducts with 4HNEand this could be the reason of the protein loss that oncemodified by 4HNE can be degraded by the proteasome[37] This effect seems to be related only to Cx40 in factneither Cx43 protein levels nor its mRNA levels were affectedby CS exposure This is corroborated by a study showingthat connexin expression is frequently disrupted in responseto lung pathology for instance during the acute phase oflung injury Cx43 expression does not decrease while Cx40expression decreases [45]

The connexins when present in the same cells can formheterodimers in the gap junctions channels as demonstrated


0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400

Figure 9 Effect of CS and corilagin inCx43 protein levels Top panelshows representative WBs of five independent experiments Bandsquantification is shown in the bottom panel 120573-actin was used asloading control

for Cx40Cx43 Cx37Cx43 and Cx40Cx37 [46] thereforeit is possible that the loss of just one kind of connexin inour case Cx40 can affect the functionality of several gapjunctions

A recent study confirms anyways the importance of Cx40in lung structure in fact using animal KO for Cx40 theauthors have shown that the animals developed severe lungabnormalities such as fibrosis and altered alveolar remodeling[47]

In the second part of our study the possible protectiveeffect of the polyphenol corilagin on CS induced cell damageand loss of gap junctions has been evaluated Corilagin isa polyphenolic compound known for its strong ability ofinhibiting NF120581B activation [26] The results of the studyconfirm that corilagin inhibits CS induced NF120581B activationThe protective effect of corilagin against lung injury ina bleomycin model has been recently demonstrated [48]together with its ability to quench free radicals and inhibitNF120581B [49 50] In addition pretreatment with corilagin wasable to counteract CS induced protein oxidation and lipidperoxidation as determined by carbonyl proteins formationand 4HNE proteins adducts Furthermore corilagin was alsoable to prevent the loss of Cx40 most likely thanks to its

0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast

W corilagin 10120583M (24hr)Wo corilagin 10120583M (24hr)

Figure 10 Corilagin reduces the formation of 4HNE-Cx40 adductsinduced by exposure to CS WB in the top panel is representative offive independent experiments Bands quantification is shown in thebottom panel Data are expressed as arbitrary units average of fiveindependent experiments 119875 lt 005 lowast versus C versus CS

free radical quenching properties therefore preventing theformation of Cx40-4HNE-protein adducts

In parallel Cx40 gene expression appeared to be lessenhanced by CS when Calu-3 cells were pretreated with thepolyphenol as a consequence of the effect of corilagin onpreventing Cx40 protein loss Interestingly the promoter ofconnexin 40 (GJA5) hasNF-120581B consensus binding sequencessuggesting aNF120581B dependent transcriptional regulation [33]However further work is necessary to verify whether thereduced transcription of Cx40 in the presence of corilaginis dependent on the inhibitory effects of corilagin on NF-120581B In any case and whatever the molecular basis of thecorilagin-mediated effects is TEM analysis showed thattreatment with corilagin prevented the loss of cell-to-celljunctions enforcing the theory of Cx40ndashCx43 heterodimersgap junctions presence in airway epithelium since Cx43protein expression was not affected by CS exposure Ofnote is that in the present study corilagin was used at theconcentration of 10120583Mwhich although used in other studiespresent in the literature [24 26] could be a dose difficult toreach in the lung tissue due to its bioavailabilityTherefore wecould suggest the use of corilagin by nebulization to avoid itsdegradation in the GI tract and to reach higher doses in therespiratory tract

Altogether the results presented in our study showedthat CS is able to damage airway epithelium integrity bycausing cellular junctions loss through oxidative damage


CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1

Corilagin 10120583M (24hr)

C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400

800 (nM)(minus) (minus)

lowast

(c)

Figure 11 Effects of corilagin on NF120581B 24 hr of pretreatment with corilagin decreases the activation of NF120581B induced by CS WB shown inthe top panel (a) is representative of five independent experiments In the bottom of panel (a) is shown the quantification of nuclear NF120581Bprotein levels Data are expressed as arbitrary units (average of five independent experiments 119875 lt 005 lowast versus C) Lamin B1 was usedas loading control Corilagin inhibits the interaction between NF120581B and oligonucleotides mimicking NF120581B binding sites (panel (b) NF120581Bconsensus sequence A panel (c) NF120581B consensus sequence B) EMSA analysis was performed on binding reactions conducted in the presenceof the indicated nM concentrations of corilagin

and NF120581B activation and that corilagin thanks to itsanti-inflammatory and free radical scavenging characteris-tics prevents CS induced cell damage

Conflict of Interests

The authors declare that there is no conflict of interests


Acknowledgment

This work was supported by a contribution from ChiesiFoundation 2011 to Monica Borgatti

References

[1] B J Ingebrethsen ldquoAerosol studies of cigarette smokerdquo RecentAdvances in Tobacco Science vol 12 pp 54ndash142 1986

[2] W A Pryor D G Prier and D F Church ldquoElectron-spinresonance study ofmainstream and sidestream cigarette smokenature of the free radicals in gas-phase smoke and in cigarettetarrdquo Environmental Health Perspectives vol 47 pp 345ndash3551983

[3] B Messner and D Bernhard ldquoSmoking and cardiovascular dis-ease mechanisms of endothelial dysfunction and early athero-genesisrdquo Arteriosclerosis Thrombosis and Vascular Biology vol34 no 3 pp 509ndash515 2014

[4] C Sticozzi A Pecorelli G Belmonte and G ValacchildquoCigarette smoke affects ABCAl expression via liver X receptornuclear translocation in human keratinocytesrdquo InternationalJournal of Molecular Sciences vol 11 no 9 pp 3375ndash3386 2010

[5] K Kunchithapautham C Atkinson and B Rohrer ldquoSmokeexposure causes endoplasmic reticulum stress and lipid accu-mulation in retinal pigment epithelium through oxidative stressand complement activationrdquo Journal of Biological Chemistryvol 289 no 21 pp 14534ndash14546 2014

[6] W H Evans and P E M Martin ldquoGap junctions structure andfunctionrdquo Molecular Membrane Biology vol 19 no 2 pp 121ndash136 2002

[7] R P Rainey I G Gillman X Shi et al ldquoFluorescent detectionof lipid peroxidation derived protein adducts upon in-vitrocigarette smoke exposurerdquo Toxicology Mechanisms and Meth-ods vol 19 no 6-7 pp 401ndash409 2009

[8] K Avezov A Z Reznick and D Aizenbud ldquoOxidative damagein keratinocytes exposed to cigarette smoke and aldehydesrdquoToxicology in Vitro vol 28 no 4 pp 485ndash491 2014

[9] H Xue K Sun W Xie et al ldquoEtanercept attenuates short-termcigarette-smoke-exposure-induced pulmonary arterial remod-elling in rats by suppressing the activation of TNF-120572NF-120581Bsignal and the activities of MMP-2 and MMP-9rdquo PulmonaryPharmacology andTherapeutics vol 25 no 3 pp 208ndash215 2012

[10] I HHeijink SM Brandenburg J ANoordhoekD S PostmaD-J Slebos and A J M van Oosterhout ldquoCharacterisation ofcell adhesion in airway epithelial cell types using electric cell-substrate impedance sensingrdquo European Respiratory Journalvol 35 no 4 pp 894ndash903 2010

[11] K Husgafvel-Pursiainen ldquoGenotoxicity of environmentaltobacco smoke a reviewrdquoMutation ResearchReviews in Muta-tion Research vol 567 no 2-3 pp 427ndash445 2004

[12] D B Alexander and G S Goldberg ldquoTransfer of biologicallyimportant molecules between cells through gap junction chan-nelsrdquo Current Medicinal Chemistry vol 10 no 19 pp 2045ndash2058 2003

[13] D A Goodenough J A Goliger and D L Paul ldquoConnexinsconnexons and intercellular communicationrdquo Annual Reviewof Biochemistry vol 65 pp 475ndash502 1996

[14] H A Dbouk R MMroue M E El-Sabban and R S TalhoukldquoConnexins a myriad of functions extending beyond assemblyof gap junction channelsrdquo Cell Communication and Signalingvol 7 article 4 2009

[15] M Vinken E Decrock L Leybaert et al ldquoNon-channel fun-ctions of connexins in cell growth and cell deathrdquo Biochimica etBiophysica Acta vol 1818 no 8 pp 2002ndash2008 2012

[16] T Kojima M Murata M Go D C Spray and N SawadaldquoConnexins induce and maintain tight junctions in epithelialcellsrdquo Journal of Membrane Biology vol 217 no 1ndash3 pp 13ndash192007

[17] C Luchese E C Stangherlin B M Gay and C W NogueiraldquoAntioxidant effect of diphenyl diselenide on oxidative damageinduced by smoke in rats involvement of glutathionerdquo Ecotox-icology and Environmental Safety vol 72 no 1 pp 248ndash2542009

[18] N Holzer K F Braun S Ehnert et al ldquoGreen tea protectshuman osteoblasts from cigarette smoke-induced injury pos-sible clinical implicationrdquo Langenbeckrsquos Archives of Surgery vol397 no 3 pp 467ndash474 2012

[19] J A D S Jaques P H Doleski L G Castilhos et al ldquoFree andnanoencapsulated curcumin prevents cigarette smoke-inducedcognitive impairment and redox imbalancerdquo Neurobiology ofLearning and Memory vol 100 pp 98ndash107 2013

[20] C Sticozzi G Belmonte F Cervellati et al ldquoResveratrol pro-tects SR-B1 levels in keratinocytes exposed to cigarette smokerdquoFree Radical Biology and Medicine vol 69 pp 50ndash57 2014

[21] Y Sudjaroen W E Hull G Erben et al ldquoIsolation and char-acterization of ellagitannins as the major polyphenolic com-ponents of Longan (Dimocarpus longan Lour) seedsrdquo Phyto-chemistry vol 77 pp 226ndash237 2012

[22] S Okabe M Suganuma Y Imayoshi S Taniguchi T Yoshidaand H Fujiki ldquoNew TNF-120572 releasing inhibitors geraniin andcorilagin in leaves of acer nikoense megusurino-kirdquo Biologicaland Pharmaceutical Bulletin vol 24 no 10 pp 1145ndash1148 2001

[23] W G Duan Z Q Shen M Yan Y Yun and L Y ZhangldquoCorilagin a promising natural product to treat cardiovasculardiseasesrdquo in Phytopharmacology andTherapeutic Values II J NGovil V K Singh and S K Mishra Eds pp 163ndash172 2008

[24] D K-P Hau G-Y Zhu A K-M Leung et al ldquoIn vivoanti-tumour activity of corilagin on Hep3B hepatocellularcarcinomardquo Phytomedicine vol 18 no 1 pp 11ndash15 2010

[25] L Zhao S-L Zhang J-Y Tao et al ldquoPreliminary explo-ration on anti-inflammatory mechanism of Corilagin (beta-1-O-galloyl-36-(R)-hexahydroxydiphenoyl-d-glucose) in vitrordquoInternational Immunopharmacology vol 8 no 7 pp 1059ndash10642008

[26] R Gambari M Borgatti I Lampronti et al ldquoCorilagin isa potent inhibitor of NF-kappaB activity and downregulatesTNF-alpha induced expression of IL-8 gene in cystic fibrosisIB3-1 cellsrdquo International Immunopharmacology vol 13 no 3pp 308ndash315 2012

[27] S Kinoshita Y Inoue S Nakama T Ichiba and Y AniyaldquoAntioxidant and hepatoprotective actions of medicinal herbTerminalia catappa L from Okinawa Island and its tannincorilaginrdquo Phytomedicine vol 14 no 11 pp 755ndash762 2007

[28] Y Zhu A Chidekel and T H Shaffer ldquoCultured human airwayepithelial cells (Calu-3) amodel of human respiratory functionstructure and inflammatory responsesrdquo Critical Care Researchand Practice vol 2010 Article ID 394578 8 pages 2010

[29] G Valacchi P A Davis E M Khan et al ldquoCigarette smokeexposure causes changes in Scavenger Receptor B1 level and dis-tribution in lung cellsrdquoThe International Journal of Biochemistryamp Cell Biology vol 43 no 7 pp 1065ndash1070 2011

[30] M Borgatti L Breda R Cortesi et al ldquoCationic liposomesas delivery systems for double-stranded PNA-DNA chimeras


exhibiting decoy activity against NF-120581B transcription factorsrdquoBiochemical Pharmacology vol 64 no 4 pp 609ndash616 2002

[31] M Borgatti I Lampronti A Romanelli et al ldquoTranscriptionfactor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sitesrdquo Journal of Biologi-cal Chemistry vol 278 no 9 pp 7500ndash7509 2003

[32] S Galardi N Mercatelli M G Farace and S A Ciafre ldquoNF-120581kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cellsrdquoNucleic Acids Research vol 39 no 9 pp 3892ndash3902 2011

[33] G Gao and S C Dudley Jr ldquoRedox regulation NF-120581B andatrial fibrillationrdquo Antioxidants and Redox Signaling vol 11 no9 pp 2265ndash2277 2009

[34] A van der Vliet and C E Cross ldquoInnate antioxidant defensesystems in the respiratory tractrdquo BioFactors vol 15 no 2ndash4 pp83ndash86 2001

[35] S Prasad C M Hogaboam and G Jarai ldquoDeficient repairresponse of IPF fibroblasts in a co-culture model of epithelialinjury and repairrdquo Fibrogenesis and Tissue Repair vol 7 no 1article 7 2014

[36] R Castagna P A Davis V T Vasu et al ldquoNitroxide radicalTEMPO reduces ozone-induced chemokine IL-8 production inlung epithelial cellsrdquo Toxicology in Vitro vol 23 no 3 pp 365ndash370 2009

[37] N D Magnani T Marchini V Vanasco D R Tasat S Alvarezand P Evelson ldquoReactive oxygen species produced by NADPHoxidase and mitochondrial dysfunction in lung after an acuteexposure to Residual Oil Fly Ashesrdquo Toxicology and AppliedPharmacology vol 270 no 1 pp 31ndash38 2013

[38] C Sticozzi G Belmonte A Pecorelli et al ldquoCigarette smokeaffects keratinocytes SRB1 expression and localization via H

2O2

production and HNE protein adducts formationrdquo PLoS ONEvol 7 no 3 Article ID e33592 2012

[39] KAoshibaMKoinumaN Yokohori andANagai ldquoImmuno-histochemical evaluation of oxidative stress in murine lungsafter cigarette smoke exposurerdquo Inhalation Toxicology vol 15no 10 pp 1029ndash1038 2003

[40] B L Upham L Blaha P Babica et al ldquoTumor promotingproperties of a cigarette smoke prevalent polycyclic aromatichydrocarbon as indicated by the inhibition of gap junctionalintercellular communication via phosphatidylcholine-specificphospholipase Crdquo Cancer Science vol 99 no 4 pp 696ndash7052008

[41] S Ramachandra L-H Xie S A John S Subramaniam andR Lal ldquoA novel role for connexin hemichannel in oxidativestress and smoking-induced cell injuryrdquo PLoS ONE vol 2 no8 article e712 2007

[42] H Hennemann T Suchyna H Lichtenberg-Frate et alldquoMolecular cloning and functional expression of mouse con-nexin40 a second gap junction gene preferentially expressed inlungrdquo The Journal of Cell Biology vol 117 no 6 pp 1299ndash13101992

[43] F J Martin and A S Prince ldquoTLR2 regulates gap junctionintercellular communication in airway cellsrdquo The Journal ofImmunology vol 180 no 7 pp 4986ndash4993 2008

[44] R van den Berg G R M M Haenen H van den Berg and ABast ldquoTranscription factor NF-120581B as a potential biomarker foroxidative stressrdquo British Journal of Nutrition vol 86 no 1 ppS121ndashS127 2001

[45] S Rignault J-A Haefliger B Waeber L Liaudet and F FeihlldquoAcute inflammation decreases the expression of connexin 40in mouse lungrdquo Shock vol 28 no 1 pp 78ndash85 2007

[46] E Oviedo-Orta B R Kwak and W Howard Evans ConnexinCell Communication Channels Roles in the Immune System andImmunopathology CRC Press 2013

[47] M Koval M Billaud A C Straub et al ldquoSpontaneous lungdysfunction and fibrosis inmice lacking connexin 40 and endo-thelial cell connexin 43rdquoTheAmerican Journal of Pathology vol178 no 6 pp 2536ndash2546 2011

[48] Z Wang Q-Y Guo X-J Zhang et al ldquoCorilagin attenuatesaerosol bleomycin-induced experimental lung injuryrdquo Interna-tional Journal ofMolecular Sciences vol 15 no 6 pp 9762ndash97792014

[49] X-R Dong M Luo L Fan et al ldquoCorilagin inhibits the doublestrand break-triggered NF-120581B pathway in irradiated microglialcellsrdquo International Journal of MolecularMedicine vol 25 no 4pp 531ndash536 2010

[50] Y-J Guo L Zhao X-F Li et al ldquoEffect of Corilagin onanti-inflammation in HSV-1 encephalitis and HSV-1 infectedmicrogliasrdquoEuropean Journal of Pharmacology vol 635 no 1ndash3pp 79ndash86 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


such as cell growth cell development cell differentiation andcell death and its absence leads to changes in the cellularhomeostasis [13] In addition connexins are known to affectcell migration and adhesion [14ndash16]

In the last few decades many studies have shown thatpolyphenols and oxidant scavengers can contrast CS inducedoxidative stress [17ndash20] Corilagin is a polyphenol memberof the tannin family extracted from different plants likeDimocarpus longan [21] and Geranium thunbergii [22] Thisnatural compound seems to have beneficial effects in sev-eral cardiovascular disorders hypertension thrombosis oratherosclerosis [23] but it is also known for its antiprolifera-tive and antitumor effects [24] Zhao et al have demonstratedthe anti-inflammatory properties of corilagin Their studyshows that corilagin is able to block NF120581B activation andits nuclear translocation demonstrating anti-inflammatorycharacteristic In addition it has been shown that corilagindecreases the production of proinflammatory cytokines likeTNF-120572 IL-1120573 IL-6 iNOS and COX-2 [25] Furthermoresome studies have evidenced the ability of corilagin to inhibitIL-8 production by blocking TNF-120572 induced NF120581B nucleartranslocation [26] It has been also demonstrated that cori-lagin is hepatoprotective as it acts like radical scavenger forsuperoxide anion and peroxyl radicals In addition corilagininhibits ROS production from leukocytes as well as freeradicals formation and lipid peroxidation in mitochondria[27]

In our study we evaluated the damaging effects of CSon lung epithelial cells in particular we investigated CS-modulation of gap junctions and connexin expression andwealso determined whether corilagin could counteract the nox-ious effect of CS For our purpose we have used Calu-3 cellline grown in air-liquid interface which has been reportedto be a good model to study biological agents on humanairway epithelial cells functions and structures Indeed thismodel has been extensively used for lung pathophysiologyand therapeutics studies and it exhibits many characteristicsof the primary airway cell cultures thanks to its ability to formstable cell-to-cell junctions [28]

2 Materials and Methods

21 Cell Culture and Treatment Calu-3 cells cultured in75 cm2 plastic flasks (BD Falcon USA) were grown inDulbeccorsquos modified Eaglersquos medium (Lonza Milan Italy)supplemented with 10 fetal bovine serum (EuroCloneMilan Italy) 2mM l-glutamine (Lonza Milan Italy) andantibiotics (100UImL penicillin and 100 120583gmL strepto-mycin) (Lonza Milan Italy) They were maintained at 37∘Cin a humidified 5 CO

2atmosphere Upon reaching 90

confluence cells were harvested using 002 trypsinEDTA(Lonza Milan Italy) After trypsinisation Calu-3 cells wereseeded at a density of 1 times 106 cellsmL onto 04120583m pore size415 cm2 surface area polyethylene terephthalate Transwellcell culture inserts (BD Falcon USA) placed in 6-well cellculture plates (BD Falcon USA)When the cells reached 90of confluence and were pretreated with corilagin 10 120583M for24 hr then the culture media were changed and the cells wereexposed to CS

Corilagin was a gift by the China National Institute forthe Control of Pharmaceutical and Biological Products Thepowder was dissolved in ethanol and the 10 120583M solution wasadded to the cells (final ethanol concentration 01)

22 CS Exposure Cells were exposed to CS for 50min infresh serum-free media (as previously described [29]) whilecontrol cells were exposed to filtered air (50min) CS wasgenerated by burning one research cigarette (12mg tar 11mgnicotine) using a vacuum pump to draw air through theburning cigarette and leading the smoke stream over the cellculture as described previously [29] After the exposure (airor CS) fresh media supplemented with 10 FBS were addedto the cells

23 Cytotoxicity Determination After CS exposure culturemedia were collected at different time points (50min 6 hr12 hr and 24 hr) Cytotoxicitywas determined by LDHreleasein the media measured by enzymatic assay in the first stepNAD+ is reduced to NADHH+ by the LDH-catalyzedconversion of lactate to pyruvate in the second step thecatalyst (diaphorase) transfers HH+ from NADHH+ totetrazolium salt which is reduced to formazan The amountof LDH in the supernatant was determined and calculatedaccording to kit instructions (EuroClone Milan Italy) Alltests were performed in triplicate and assays were repeatedthree times independently

24 Protein Extraction At each time point cells werewashed with ice-cold PBS and lysed in ice-cold lysis buffer(20mM Tris pH 8 150mM NaCl 1 Triton X-100 1mMsodium orthovanadate 1 120583gmL leupeptin 1 120583gmL apro-tinin 1 120583gmL pepstatin 10 120583gmL PMSF and 5mM 120573-glycerophosphate) (Sigma Milan Italy) After centrifugation(13500 rpm 15min at 4∘C) the supernatants were collectedProteins concentration was determined by the method ofBradford (BioRad Milan Italy)

25Western Blot Analysis Sixty 120583g boiled proteinwas loadedonto 10 sodium dodecyl sulphate-polyacrylamide elec-trophoresis gels and separated by molecular size Gels wereelectroblotted onto nitrocellulose membranes and then wereblocked for 1 hr in Tris-buffered saline pH 75 containing05 Tween 20 and 3 milk Membranes were incubatedovernight at 4∘C with the appropriate primary antibodiesCx40 (Santa Cruz Biotechnology Inc USA) Cx43 (SantaCruz Biotechnology Inc USA) 4HNE (Chemicon USA)and 120573-actin (Cell Signalling Celbio Milan Italy) The mem-branes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr and the boundantibodies were detected by chemiluminescence (BioRadMilan Italy) 120573-actin was used as loading control Images ofthe bands were digitized and the densitometry analysis wasperformed using ImageJ software

26 Electrophoretic Mobility Shift Assay (EMSA) Elec-trophoretic Mobility Shift Assay (EMSA) was performedas previously described [30 31] Briefly double-strandedsynthetic oligodeoxynucleotides mimicking two functional



2





















3 Results



0

1

2

3

4

5

6

7

8

9

C 6 12 24

LDH

rele

ase (

au)

CS (hr)50998400

lowastlowastlowast

lowast







C 62 24CS (hr)

50998400


0

100

200

300

400

500

600

700

800

C 24

CS (hr)

lowast

lowast

50998400

Carb

onyl

pro

tein

leve

ls (a

u

of c

ontro

l)

C 2450998400CS (hr)

(a)

C 2450998400CS (hr)

0

100

200

300

400

500

600

700

800

900

lowast

lowast

C 24CS (hr)50998400

4H

NE

leve

ls (a

u

of c

ontro

l)

(b)






0

50

100

150

200

250

C 6 12 24

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

CS (hr)50998400

C 6 12 24

CS (hr)

50998400

67kDa

65kDa

Lamin B1

lowast

lowast

NF120581B p65






C 6 12 24CS (hr)

509984000123456789

LDH

rele

ase (

au)

lowast lowastlowast

lowast




4 Discussion





C 2 6 24

CS (hr)

50998400

Corilagin 10120583M


0

100

200

300

400

500

600

700

800

Carb

onyl

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6CS (hr)

50998400

lowast

lowast


(a)

4HN

E le

vels

(au

o

f con

trol)

0100200300400500600700800900

C 24CS (hr)

50998400

lowast

lowast


(b)





2O2(which is also





Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2





















3 Results



0

1

2

3

4

5

6

7

8

9

C 6 12 24

LDH

rele

ase (

au)

CS (hr)50998400

lowastlowastlowast

lowast







C 62 24CS (hr)

50998400


0

100

200

300

400

500

600

700

800

C 24

CS (hr)

lowast

lowast

50998400

Carb

onyl

pro

tein

leve

ls (a

u

of c

ontro

l)

C 2450998400CS (hr)

(a)

C 2450998400CS (hr)

0

100

200

300

400

500

600

700

800

900

lowast

lowast

C 24CS (hr)50998400

4H

NE

leve

ls (a

u

of c

ontro

l)

(b)






0

50

100

150

200

250

C 6 12 24

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

CS (hr)50998400

C 6 12 24

CS (hr)

50998400

67kDa

65kDa

Lamin B1

lowast

lowast

NF120581B p65






C 6 12 24CS (hr)

509984000123456789

LDH

rele

ase (

au)

lowast lowastlowast

lowast




4 Discussion





C 2 6 24

CS (hr)

50998400

Corilagin 10120583M


0

100

200

300

400

500

600

700

800

Carb

onyl

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6CS (hr)

50998400

lowast

lowast


(a)

4HN

E le

vels

(au

o

f con

trol)

0100200300400500600700800900

C 24CS (hr)

50998400

lowast

lowast


(b)





2O2(which is also





Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results



0

1

2

3

4

5

6

7

8

9

C 6 12 24

LDH

rele

ase (

au)

CS (hr)50998400

lowastlowastlowast

lowast







C 62 24CS (hr)

50998400


0

100

200

300

400

500

600

700

800

C 24

CS (hr)

lowast

lowast

50998400

Carb

onyl

pro

tein

leve

ls (a

u

of c

ontro

l)

C 2450998400CS (hr)

(a)

C 2450998400CS (hr)

0

100

200

300

400

500

600

700

800

900

lowast

lowast

C 24CS (hr)50998400

4H

NE

leve

ls (a

u

of c

ontro

l)

(b)






0

50

100

150

200

250

C 6 12 24

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

CS (hr)50998400

C 6 12 24

CS (hr)

50998400

67kDa

65kDa

Lamin B1

lowast

lowast

NF120581B p65






C 6 12 24CS (hr)

509984000123456789

LDH

rele

ase (

au)

lowast lowastlowast

lowast




4 Discussion





C 2 6 24

CS (hr)

50998400

Corilagin 10120583M


0

100

200

300

400

500

600

700

800

Carb

onyl

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6CS (hr)

50998400

lowast

lowast


(a)

4HN

E le

vels

(au

o

f con

trol)

0100200300400500600700800900

C 24CS (hr)

50998400

lowast

lowast


(b)





2O2(which is also





Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C 62 24CS (hr)

50998400


0

100

200

300

400

500

600

700

800

C 24

CS (hr)

lowast

lowast

50998400

Carb

onyl

pro

tein

leve

ls (a

u

of c

ontro

l)

C 2450998400CS (hr)

(a)

C 2450998400CS (hr)

0

100

200

300

400

500

600

700

800

900

lowast

lowast

C 24CS (hr)50998400

4H

NE

leve

ls (a

u

of c

ontro

l)

(b)






0

50

100

150

200

250

C 6 12 24

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

CS (hr)50998400

C 6 12 24

CS (hr)

50998400

67kDa

65kDa

Lamin B1

lowast

lowast

NF120581B p65






C 6 12 24CS (hr)

509984000123456789

LDH

rele

ase (

au)

lowast lowastlowast

lowast




4 Discussion





C 2 6 24

CS (hr)

50998400

Corilagin 10120583M


0

100

200

300

400

500

600

700

800

Carb

onyl

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6CS (hr)

50998400

lowast

lowast


(a)

4HN

E le

vels

(au

o

f con

trol)

0100200300400500600700800900

C 24CS (hr)

50998400

lowast

lowast


(b)





2O2(which is also





Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

C 6 12 24

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

CS (hr)50998400

C 6 12 24

CS (hr)

50998400

67kDa

65kDa

Lamin B1

lowast

lowast

NF120581B p65






C 6 12 24CS (hr)

509984000123456789

LDH

rele

ase (

au)

lowast lowastlowast

lowast




4 Discussion





C 2 6 24

CS (hr)

50998400

Corilagin 10120583M


0

100

200

300

400

500

600

700

800

Carb

onyl

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6CS (hr)

50998400

lowast

lowast


(a)

4HN

E le

vels

(au

o

f con

trol)

0100200300400500600700800900

C 24CS (hr)

50998400

lowast

lowast


(b)





2O2(which is also





Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C 2 6 24

CS (hr)

50998400

Corilagin 10120583M


0

100

200

300

400

500

600

700

800

Carb

onyl

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6CS (hr)

50998400

lowast

lowast


(a)

4HN

E le

vels

(au

o

f con

trol)

0100200300400500600700800900

C 24CS (hr)

50998400

lowast

lowast


(b)





2O2(which is also





Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Wocorilagin

Wcorilagin

CS (hr)

0

20

40

60

80

100

120

C 6 12 24CS (hr)

50998400

Cx40

pro

tein

leve

ls(a

u

of c

ontro

l)

C 6 12 2450998400

lowast lowast


Cx40(40kDa)

120573-actin(45kDa)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

6 12 24C 50998400

CS (hr)

Cx40

gene

expr

essio

n (a

u

of c

ontro

l)

lowast

lowast

lowast

lowast


(b)










0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

C 6 12 2450998400

Cx43(43kDa)

(45kDa)

Wocorilagin

Wcorilagin

CS (hr)


Cx43

pro

tein

leve

ls (a

u

of c

ontro

l)

120573-actin

CS (hr)C 6 12 2450998400





0

50

100

150

200

250

300

C CSCorilagin

C CS

+CS

6hr

Cx4

0-4H

NE

addu

cts (

au

o

f con

trol)

lowast

lowast







CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CS (hr)

0

20

40

60

80

100

120

C 6 12 24

CS (hr)

Lamin B1


C 2412650998400


65kDa

67kDa

lowast

lowast

lowastlowast

50998400

Nuc

lear

NF120581

B le

vels

(au

o

f con

trol)

NF120581B p65

(a)

625

125

25 50 100

200

400

800

(nM)(minus) (minus)

lowast

(b)

625

125

25 50 100

200

400


lowast

(c)






Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgment


References









































2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2O2



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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