Effect of Zusanli (ST 36) moxibustion on rat mesenteric microvascu-lar system
Hongqing Xia, Ling Guan, Yiling Yangaa
Hongqing Xia, Ling Guan, Yiling Yang, Department ofAcupuncture and Moxibustion, the General Hospital of Chi-nese People's Liberation Army, Beijing 100853, ChinaSupported by Natural Science Foundation-funded Project:the Discussion of Laws And Mechanisms of Moxibustion Zu-sanli (ST 36) To Stimulate Research Targeted Surface Ther-mal Effects (No. 30973795)Correspondence to: Associate prof. Ling Guan, Depart-ment of Acupuncture and Moxibustion, the General Hospi-tal of Chinese People's Liberation Army, Beijing 100853, Chi-na. [email protected]: +86-13520575830Accepted: April 13, 2014
AbstractOBJECTIVE: To investigate the effect of moxibus-tion on Zusanli (ST 36) on visceral-mesenteric ves-sels by observing circulation.
METHODS: Forty-five SD rats were randomly as-signed to a moxibustion, electroacupuncture (EA),and blank group. In the moxibustion group, heatstimulation of moxibustion to the Zusanli (ST 36) ar-ea of normal rats was performed for 15 min. In theEA group, needles were inserted into the Zusanli(ST 36) and lateral point [0.5 cm lateral from Zusanli(ST 36)] for 15 min. The blank group was not givenany treatment. We continuously monitored mesen-teric microvascular changes with in vivo microscop-ic video.
RESULTS: Moxibustion and EA to Zusanli (ST 36) in-crease the diameter of mesenteric arterioles and ve-nules (P<0.05). There were no obvious changes inthe blank group. Fine arterial diameter peaked at12 min in the moxibustion group, while it peaked
at 15 min in the EA group.
CONCLUSION: The stimulation of moxibustion andacupuncture to Zusanli (ST 36) has immediate ef-fects on expanding the microvasculature. This dila-tion may be the mechanism of the gastrointestinaleffect of Zusanli (ST 36).
INTRODUCTIONAcupuncture and moxibustion affects the overall regu-lation of the nerve, vascular, endocrine, and immunenetwork to achieve therapeutic effects, but its mecha-nism is complicated.1 However, vascular mechanismslikely play an important part. In our previous study,we found that moxibustion on Zusanli (ST 36) cancause temperature changes of the inner canthus, peri-oral area, nasal, forehead, supraclavicular fossa, mam-mary gland, and groin, where the blood is abundant.However, there was no obvious conduction of heat onthe surface of body, suggesting that the numerous func-tions of Zusanli (ST 36) are related to improved circu-lation.2
Zusanli (ST 36) is one of the most important and wide-ly studied acupoints on the human body, and is usedin a wide range of disease treatment. Current researchon Zusanli (ST 36) involves mostly acupuncture, andinvestigates central and peripheral effects. For example,research has shown that acupuncture on Zusanli (ST36) can cause widespread activation of brain regions.3
The outer peripheral effects include inhibiting acutegastric mucosal injury,4 reducing inflammatory re-
sponse, preventing infection,5 reducing asthma at-tacks,6 suppressing lung ischemia-reperfusion injury,changing the levels of certain cytokines in rats,7 pro-moting colonic transmission function,8 and loweringblood pressure.9
There are also many studies investigating the effect ofacupuncture on the stomach and intestine. For exam-ple, acupuncture can increase gastric blood flow perfu-sion,10 promote the recovery of bowel function aftergastrointestinal surgery,11 and prevent and relieve nau-sea and hiccups in cancer patients after chemothera-py.12 Moreover, catgut implantation at acupoints has areliable curative effect for constipation.13 This study in-vestigates the effects of moxibustion at Zusanli (ST 36)on the gastrointestinal system. The effect of moxibus-tion at Zusanli (ST 36) on visceral-mesenteric vesselswere immediately and visually observed. We aimed tofurther confirm moxibustion's role in improving circu-lation of the internal organs, and possible neural regula-tion mechanisms.
MATERIALS AND METHODS
Animals and groupingForty-five healthy male three-month-old SD rats,weighing 240-270 g, were provided by the People'sLiberation Army General Hospital Medical LaboratoryAnimal Center (SCXK [Beijing]-2012-0001). Feedingwas adapted to standard rodent animal feeding cage, atroom temperature 20℃± 2℃ , with light for 12 h. Aspreviously described,14,15 we randomized mice using Ex-cel and the random function "Rand." The rats were di-vided into moxibustion, electroacupuncture (EA), andcontrol groups, with 15 rats in each. Differences in thenumber and weight of rats in each group were not sta-tistically significant (P>0.05). During the experiment,the disposal for animals conformed to the "GuidingOpinions on kind of experimental animals" publishedby the Republic of China Ministry of Science in 2006.
Main equipmentBiological microscope (Olympus BH-2, Olympus Cor-poration, Tokyo, Japan) and Image-Pro image analysissoftware (Media Cybernetics, Silver Spring, MD,USA) were provided by Pathophysiology Departmentof the PLA General Hospital. Moxa stick (the 4thsmokeless moxa) was produced by Ai Shang MoxaCo., Ltd., (Guangzhou, China), Huatuo electroacu-puncture device (SDZ-V-type) by Suzhou MedicalSupplies Company Limited (Suzhou，China), and ster-ile acupuncture needles (0.25 mm × 25 mm) by Gui-zhou Ande Medication Appliance., Ltd., (Guizhou,China).Surgical methods before the experiment, rats were fast-ed for 12 h, with normal water. Animal experimentswere conducted according to the mesenteric microcir-culation in vivo observation method.16 After using 20%
urethane (0.7 mL/100 g) intramuscular anesthesia, ratswere fixed on rat-fixing plates, and incised 1-2 cmalong the ventral midline. With a small tweezers, someof the small intestine was gently raised, and the mesen-teric vascular distribution areas where the blood vesselswere tiny and less fatty were located. Then, the ratswere flipped into a lateral position, fixed them on themesenteric microcirculation observation box, and even-ly separated the mesentery, avoiding overlap andstretch. The mesentery was placed on the microscopestage with a 37℃ saline drip to maintain the tempera-ture and humidity of the mesentery.
Treatment methods of each groupCommon laboratory animal acupoints were located inreference to Experimental Acupuncture Science.17 Zusan-li (ST 36) is posterolateral to the knees, about 5 mmbelow the fibular head. In the moxibustion group, hairat the point was removed to allow easy penetration ofmoxibustion heat. Suspended moxibustion was usedwith a No. 4 (4 mm) moxa stick 1.5 cm away from thepoint. In the EA group, needles were inserted 0.7 cmdeep into the Zusanli (ST 36) and lateral point (0.5 cmlateral from ST 36) EA was connected with a frequen-cy of 4-16 Hz and intensity to slight trembling. Theblank group not given any treatment. Moxibustion andEA groups were treated for 15 min, and their videoswere recorded 5 min before the treatment, and 15 minduring and after the treatment. The blank group wasrecorded for 35 min.
Outcome measuresCapillary diameter: using Image-Pro image analysissoftware (Media Cybernatics, Silver Spring, MD,USA), the inside diameters of arterioles and venuleswere measured every 3 min. The measured capillarieswere 30-120 μm in diameter, accompanied by fine ar-terio-venous tissue, and without cluttered surroundingblood vessels.
Statistical analysisSPSS 13.0 statistical software (SPSS Inc., Chicago, IL,USA) was used for data analysis. The paired t-test wasused to compare the changes of each group before, dur-ing and after the treatments. Analysis of variance for re-peated measures data was used for comparison amonggroups. If it satisfied the spherical requirements, name-ly P>0.05, the direct analysis of variance was used, ifnot, ε correction was required. P<0.05 was consideredstatistically significant.
RESULTS
Changes in fine arterial diameter during and afterthe treatment in each groupIn the moxibustion and EA groups, there were signifi-cant differences in fine artery diameter during and af-
ter treatment compared with that before treatment (allP<0.05). Comparing different groups in the same peri-od, it was found that the thin artery diameter value ofthe EA group was significantly higher than that of con-trol group (P<0.05). Moreover, thin artery diametervalues of the moxibustion group 9, 12, and 15 min in-to treatment, and 3 and 12 min after treatment weresignificantly higher than those of the control group (allP<0.05). There were no significant differences (all P>0.05) between the two groups during other periods.Meanwhile, although changes of fine artery diameterin the EA group were greater than that of the moxibus-tion group, there were no significant differences (all P>0.05) between the two groups (Table 1).
Time to peak of fine arterial diameter changesFine arterial diameter peaked at 12 min in the moxi-bustion group, but 15 min in the EA group (Figure 1).
Changes in fine vein diameter during and aftertreatment in each groupIn the moxibustion and EA groups, there were signifi-cant differences in fine vein diameter during and aftertreatment compared with that before treatment (P<0.05). Only the fine vein diameter of the moxibustiongroup was significantly higher than that of the controlgroup (P<0.05), and in other periods during and aftertreatment, diameter changes of the moxibustion andEA groups were significantly higher than that of the
Group
Blank
Moxibustion
EA
F value
P value
n
15
15
15
-
-
3 min aftertreatment
44±14
55±9ab
58±16ab
4.828
0.013
6 min aftertreatment
44±14
54±9a
60±17ab
4.892
0.012
9 min aftertreatment
44±15
54±9a
58±16ab
4.062
0.024
12 min aftertreatment
44±14
54±10ab
59±15ab
5.256
0.009
15min aftertreatment
44±15
53±10a
59±15ab
4.531
0.017
Group
Blank
Moxibustion
EA
F value
P value
n
15
15
15
-
-
Beforetreatment
44±14
48±9a
55±15ab
2.528
0.092
3 min duringtreatment
44±14
52±10a
57±16ab
3.711
0.033
6 min duringtreatment
44±14
53±9a
58±14ab
4.329
0.02
9 min duringtreatment
44±15
54±9ab
58±15ab
4.639
0.015
12 min duringtreatment
44±14
56±9ab
60±13ab
6.512
0.003
15 min duringtreatment
44±15
55±9ab
60±16ab
5.362
0.008
Table 1 Changes in fine arterial diameter during and after treatment in each group (μm, x̄ ±s)
Notes: moxibustion group: suspended moxibustion was used with a No. 4 (4 mm) moxa stick 1.5 cm away from the point; EA (electroacu-puncture) group: needles were inserted 0.7 cm deep into the Zusanli (ST 36) and lateral point, with a frequency of 4-16 Hz and intensityof slight trembling; blank group did not receive intervention. EA: electroacupuncture. aP<0.05, compared with prior treatment of the samegroup; result of Mauchly test of sphericity is 0.345, P>0.05, which meets the spherical assumption; bP<0.05, compared with the controlgroup during the same period.
Figure 1 Fine arterial diameter changes in each group during and after treatment (μm)Moxibustion group: suspended moxibustion was used with a No. 4 (4 mm) moxa stick 1.5 cm away from the point; EA (electroacu-puncture) group: needles were inserted 0.7 cm deep into the Zusanli (ST 36) and lateral point, with a frequency of 4-16 Hz and in-tensity of slight trembling; blank group did not receive intervention. arepresents the time of therapy discontinuation.
control group (all P<0.05). Although fine artery diame-ter changes of the moxibustion group were greaterthan that of the EA group, there were no significantdifferences (all P>0.05) between the two groups (Table2).
Time to peak of fine vein diameter changesFine vein diameter peaked at 12 min in the moxibus-tion group, but 15 min in the EA group (Figure 2).
Image analysisThe images of all groups before, during, and after treat-ment were captured by Image-Pro image analysis soft-ware. In the moxibustion(Figure 3) and EA group (Fig-
ure 4) , the diameters of the fine vein and fine artery di-ameters increased during the treatment. Then diame-ters of the vessels decreased after treatment. However,in the blank group (Figure 5), there no obvious change.
DISCUSSIONMoxibustion is one of the primary means of treatingthe disease in Traditional Chinese Medicine. Ling Shustates that "if the needle is not suitable, moxibustion isappropriate,"18 implying that moxibustion and acu-puncture have similar effects for treatment and preven-tion. Zusanli (ST 36) has efficacy for gastrointestinal
Group
Blank
Moxibustion
EA
F value
P value
n
15
15
15
-
-
Beforetreatment
67±20
87±18b
80±18
4.345
0.059
3 min duringtreatment
67±19
92±19ab
86±19ab
6.839
0.003
6 min duringtreatment
67±20
93±19ab
86±18ab
7.529
0.002
9 min duringtreatment
67±20
94±20ab
86±18ab
7.658
0.001
12 min duringtreatment
67±19
93±19ab
86±20ab
7.012
0.002
15 min duringtreatment
67±20
93±19ab
88±18ab
7.974
0.001
Group
Blank
Moxibustion
EA
F value
P value
n
15
15
15
-
-
3 min aftertreatment
67±20
95±18ab
87±19ab
8.44
0.001
6 min aftertreatment
67±20
95±19ab
87±19ab
8.622
0.001
9 min aftertreatment
67±20
95±19ab
85±19ab
7.959
0.001
12 min aftertreatment
67±19
95±19ab
86±18ab
8.867
0.001
15 min aftertreatment
67±20
93±18ab
86±18ab
7.601
0.002
Table 2 Changes in fine vein diameter during and after treatment in each group (μm, x̄ ±s)
Notes: moxibustion group: suspended moxibustion was used with a No. 4 (4 mm) moxa stick 1.5 cm away from the point; EA (electroacu-puncture) group: needles were inserted 0.7 cm deep into the Zusanli (ST 36) and lateral point, with a frequency of 4-16 Hz and intensityof slight trembling; blank group did not receive intervention. EA: electroacupuncture. aP<0.05, compared with prior treatment of the samegroup; result of Mauchly test of sphericity is 0.312, P>0.05, which meets the spherical assumption; bP<0.05, compared with the controlgroup during the same period.
Figure 2 Fine venous diameter changes of each group during and after treatment (μm)Moxibustion group: suspended moxibustion was used with a No. 4 (4 mm) moxa stick 1.5 cm away from the point; EA (electroacu-puncture) group: needles were inserted 0.7 cm deep into the Zusanli (ST 36) and lateral point, with a frequency of 4-16 Hz and in-tensity of slight trembling; blank group did not receive intervention. EA: electroacupuncture. arepresents the time of therapy dis-continuation.
C
a
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diseases in clinic. In previous studies, we used moxibus-tion on body points and observed blood flow on thebody surface with an infrared camera. We found thatmoxibustion on Zusanli (ST 36) could stimulate ther-mal image changes on specific parts of the body sur-face, particularly the inner canthus, perioral area, nasal,forehead, supraclavicular fossa, mammary gland, andgroin, where the blood is abundant. Research has alsoshown that moxibustion stimulating Zusanli (ST 36)and Hegu (LI 4) both could cause a T-shaped hot tem-perature zone (blood distribution range) on the face.2
We aimed to investigate whether the moxibustion ef-fects are only because of increased local blood flow.The results of this study showed the Zusanli (ST 36)moxibustion group had a larger mesenteric microvascu-lar diameter during and 15 min after moxibustion thanthat before moxibustion. Compared with the EAgroup, the moxibustion group's small artery changewas less than that in the EA group, while the venulechange was greater. We also found that moxibustionand EA at Zusanli (ST 36) caused the vascular diame-ter to change within 0-3 min. This effect is a reflectionof the hypothesis that the body points "feel stimulat-
ed-the central signal integration-react,". When moxi-bustion and electrical acupuncture stimulate the rat,the peripheral receptors feel warm and electrical stimu-lation. The afferent nerves pass signals to the centralnervous system. Through signal integration of the cen-tral somatosensory, applied to the respective blood ves-sels which perform as diameter increases.Its mecha-nism needs further study.Studies have found that stimulating Zusanli (ST 36)can cause gastrointestinal two-way adjustment, whichis related to neural and humoral regulation. The mainadjusting nervous systems are the central nervous sys-tem, autonomic nervous system, and the enteric ner-vous system. Li et al 19 studied the characteristics of pe-ripheral afferent nerve discharges evoked by manualacupuncture and EA of Zusanli (ST 36) in rats. Theyfound that acupuncture stimulation Zusanli (ST 36)can induce peripheral nerve domination in the regionof discharge. Li et al 20 investigated the central nervoussystem, autonomic nervous system, and enteric ner-vous system neural mechanisms affecting gastrointesti-nal function. They found that gastrointestinal functionwas regulated by coordination of the internal and exter-
A B C
FAFV FAFV FAFV
Figure 5 Changes in vessel diameter in the blank groupA: before treatment; B: during treatment; C: after treatment. FA: fine artery; FV: fine vein.
CBA
FAFVFAFV
FAFV
Figure 4 Changes in vessel diameter in the EA (electroacupuncture) groupA: before treatment; B: during treatment; C: after treatment. Needles were inserted 0.7 cm deep into the Zusanli (ST 36) and later-al point, with a frequency of 4-16 Hz and intensity of slight trembling. FA: fine artery; FV: fine vein.
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FA FV FA FVFA FV
A B C
Figure 3 Changes in vessel diameter in the moxibustion groupA: before treatment; B: during treatment; C: after treatment. Suspended moxibustion was used with a No. 4 (4 mm) moxa stick 1.5 cmaway from the point. FA: fine artery; FV: fine vein.
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Xia HQ et al. / Experimental Study
nal nervous system and humoral factors. Hu et al 21
found that EA at Zusanli (ST 36) alleviates intestinalischemia-induced free radical injury in rats that hadbeen scalded, and the mechanism might be related tothe cholinergic anti-inflammatory pathway. Studies al-so found that acupuncture at Zusanli (ST 36) can im-prove neural regulation, promote gastrointestinal motil-ity, and improve gastrointestinal function.22
Stimulating Zusanli (ST 36) can improve gastrointesti-nal function possibly via regulating the central nervoussystem.23-27 In fact, the stimulation of other remotestomach channel points is associated with the centralnervous system and can influence intestinal func-tion.28-30 The central nervous system adjusts mesentericmicrocirculation via the sympathetic and parasympa-thetic nervous systems. For example, Song et al 31 stud-ied the effects of capsaicin on induced action potentialof inferior mesenteric nerves in rats. They found thatcapsaicin's influence on the mesentery is affected bythe sympathetic and parasympathetic nervous systems.Kong et al 32 showed that activation of the sympatheticnervous system plays an important role in regulating in-testinal immunity. It is also thought that the stomachis mainly affected by the vagus nerve, and the small in-testine is affected by sympathetic nerve activity.33
Therefore, the mesenteric microcirculation is chieflyregulated by the sympathetic and parasympathetic ner-vous systems after the signal is sent by the central ner-vous system and passes along the efferent nerve fiber toregular bowel function.34,35
In summary, after moxibustion on Zusanli (ST 36) innormal rats for 15 min, the fine arteriovenous diame-ter was larger compared with that before treatment.Both moxibustion and EA could increase fine mesen-teric artery and vein diameter, but changes in arterialdiameter were slightly smaller, but small changes in ve-nous diameter greater in the moxibustion group thanthat in the EA group. Moxibustion can produce effectson both the body surface and deep organs. Our ob-served changes in the fine mesenteric vascular diameterof microcirculation might be related to regulation ofthe central nervous system. Stimulating points on therat might cause peripheral nerve structures around thepoint to generate afferent impulses and release acetyl-choline, causing excitement of the nerve endings. Thisexcitement spreads along the spinal cord to the centralnervous system, where the information is integrated,and then the information spreads downward throughthe efferent system to sympathetic and parasympathet-ic mesentery. This signal could cause the release ofadrenaline and noradrenaline to adjust intestinal vascu-lar systolic and diastolic function, thus affecting the in-testine microcirculation. This may be an importantmechanism of moxibustion on Zusanli (ST 36) thatimpacts the mesenteric microcirculation.
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