J.Acupunct.Tuina.Sci.2011,9(1):21—25
D0I:10.1007/sl1726.0l1.0462一z
s~ ial Top.c s dy
M oxibustion at Front..M n Point of Abdomen for Intestinal
Dysbacteri0sis in Rats
艾灸腹部募穴调整大鼠肠道菌群失调的实验研究
Wang Shudong(王树 东) ,Cheng Zedong(成泽东) ,Jin Di(金迪) ,Chen Yiguo(陈以国)
1 School ofAcupuncture and Tuina,Liaoning University ofTraditional Chinese Medicine,Shenyang 110847,
PR.China
2 Hospital Affiliated to Liaoning University of Traditional Chinese Medicine,Shenyang 1 10847,E R.China
[Abstract]Objective:To study the moxibustion at Guanyuan(cv 4)and Tianshu(ST 25)on intestinal
dysbacteriosis in rats.M ethods:Fifty W istar rats,clearing grade,were ran domly divided into 5 groups,a normal
group,a model group,a medical group,and a Guanyuan(cv 4)group and a Tianshu(ST 25)group,with 10 rats
in each group.The rats were treated with Lincomycin Hydrochloride via intragastric administration for
developing models.Th e model rats were treated with medicine and moxibustion respectively.O.1 g fresh rat feces
in each group were cultured on the selective culture medium of bifidobacterium species(BS),lactobacillus(LBS),
enterobacteriaceae(EB)and enterococcus(EC).Th e growth and quantity of the bacterial colony were tested by
biochemical identification tubes and turbidimetry.Results:Moxibustion at Guanyuan (CV 4)increased BS and
LBS while moxibustion at Tianshu(ST 25)improved EB and EC.Conclusion:Moxibustion at Front-Mu points
of different body parts selectively regulated advantaged probiotics for treating intestinal dysbacteriosis.
【Key Words】 Moxibustion Therapy;Moxa Stick Moxibustion;Points.Front—Mu;Bifidobacterium;
Lactobacillus;Enterobacteriaceae;Enterococcus
【摘要】目的:观察艾灸关元、天枢对实验性大鼠肠道菌群失调的影响。
:将 50只清洁级wistar大
鼠随机分为正常组(Norn1al group)、模型8~l(Model group)、药物~/(Medical group)、关元组(CV 4 group)、
天枢组(sT 25 group),每组 10只。用大量盐酸林可霉素灌胃造模,造模成功后,分别进行药物治疗和艾灸治
疗。1个疗程后,采取各组大鼠新鲜粪便 0.1 g,应用双歧杆菌(Bifidobacterium Species。BS)、乳酸杆菌
(Lactobacillus,LBS)、肠杆菌(Enterobacteriaceae,EB)、肠球菌(Enterococcus,Ec)选择性培养基进行细菌
培养,生化鉴定管和比浊法检测不同菌落生长情况和各组菌落数量。结果:艾灸关元穴使Bs、LBS数量有
所增加;艾灸天枢穴使EB、EC数量有所增加。结论:艾灸不同部位的募穴可以选择性调整肠道优势益生
菌群,从而治疗肠道菌群失调症。
【关键词】灸法;艾条灸;募穴;双歧杆菌;乳酸杆菌;肠杆菌:肠球菌
【中图分类号1 R2—03 【文献标志码】A
Microorgan isms that inhabit in the gastrointestinal
tract of healthy human are called intestinal flora.
Intestinal flora is an integral part of the intemal
environment to benefit the organism without harm,
and form a large an d complex mi croecosystem in the
gastrOintestinal tract,bringing about balance in
quantity and quality.The homeostasis of auality and
quantity for intestinal flora would be broken if there
were chan ges in the internal an d external environment,
resulting in clinical symptoms.which is known as
intestinal dysbacteriosis.Antibiotics abuse was one of
the key causes for the intestinal dysbacteriosis.In this
study, the alteration resulted from the abuse of
antibiotics was treated with moxibustion at the
Front—M u point of the abdomen.
1 Materials
Author:Wang Shudong,master of medicine,teaching assistant,
wsdeasts@sina.com . . . .
C0rresp0nding AuthOr: Chen Yiguo, pmfessoL doctoral 1.1 A IlIInaIS
suDervi 0r 。 。 ’ Fifty Wistar rats (25 males and 25 females)
weighed(2001-_20)g,clearing grade,were supplied by
@Shanghai Research Institute ofAcupuncture and Meridian and Springer-Verlag Berlin Heidelberg 2011 l·2l·
J.Acupunct.Tuina.Sci.201l,9(1):21-25
the Experimental Animal Center of Liaoning
University of Traditional Chinese M edicine and raised
in the Lab of the Acupuncture and Tuina College in
the university.The rats were free to eat and drink.The
lab was good in lighting and not draughty,clean and
quiet.with the temperature ofaround 22℃
1-2 Reagents and medicine
Lincomycin Hydrochloride of Lingyan Brand
(H32021365.batch number 070308)was purchased
from Suzhou No.6 Pharmaceutical Factory,Jiangsu
W uzhong M edicine Group. Live Bifidobacterium
Preparation(capsule)was from Lizhu Pharmaceutical
Factory,Lizhu Group.stored at 4℃ for use.
1-3Agar
TPY agar (batch number 2007 1023)was used for
selective culture of bifidobacterium species (BS).
MRS agar(batch number 2008032 1 1 was for selective
culture of lactobacillus(LBS .Sorbitol—MacConkey
agar (batch number 20o80324一l 1 was for
enterobacteriaceae(EB)while Pfizer enterococcus
selective agar (batch number 20080428)was for
enterococcus (EC). A11 the agars above were
purchased from Hangzhou Baisi Biotechnology
Compan y for modulation.
1.4 Equipm ents
The Biochernical Technology Experiment Center
for Basic Immunology in the Liaoning University of
Traditional Chinese M edicine supplied the
equipments as follows: Anthos 2010 ELIASA
(Austria), Jinghong electrothermal constant
temperature blast drying oven (DHG一9070A.
Shanghai), Hengyu electrothermal constant
temperature incubator(HH.B 11.500。Shanghai Yuejin
Medical Apparatus and Instruments Factory),the
imaging system of BX5 1 system microscope(Japan),
DP70 digital microscope camera(Japan),and Sanyo
autoclave rSANYoM[LS一3780,Japan).
Anaerobic bag,oxygen indicator and 2.5 L sealed
culture tank made by Mitsubishi Gas Chemistry Co.,
Ltd.were purchased from Beijing Bingyang Science
an d Technology Co.,Ltd.
2 M ethods
2.1 Groups
W istar rats were normally raised for a week,freely
able to eat and d打nk.The rats were randomly divided
into 5 groups,norm al group,model group,med ical
group,Guangyuan (CV 4)group and Tianshu(ST 25)
group,with 10 ratsin eachgroup.
2.2 Model
The rats in each group,except the normal group,
were treated with intragastric admi nistration of
Lincomycin Hydrochloride at 5 000 mg/kg in the
consecutive 5 d.Th e rats suffered diarrhea,significant
increase in frequency of defecation,and watery feces。
indicating the degree of dVsbacteriosis caused by
an tibiotics decontamination.
2.3 Animal therapy
Th e rats in the medical group were washed with
physiological saline solution of live bifidobacterium
preparation on the quantity of 0.5 rnL/d one day per
week,starting from the 6 d afterthe model reduced .
The rats in the Guanyuan (CV 4)group and
Tianshu(ST 25)group were treated with smokeless
moxa stick for warrrl moxibustion by copper
moxibustion gun.The treatment was carried out once
a day and 20 min per time per week for a treatment
course.The acupoints of Guanyuan(cv 4 point and
Tianshu (ST 25)point were located according to
experimental acupuncture sciencet¨
.
2.4 M ed ia preparations
M RS agar was weighed for 66.24 g and dissolved
in 1 000 mL distilled water witll 1.32 mL glacial
acetic acid.Th e an tigrading mixture was heated and
agitated then cooled to 50℃ to po ur into a sterile
culture dish for use.TPY agar was weighed for 46.4 g.
dissolved in l 000 mL distilled water,subpackaged
into a triangular flask.autoclaved at 121℃ for 15 min
an d poured into sterile culture dish for use.Tl1e
procedure for Pfizer agar was 58 g dissolving in
1 000 mL distilled water,subpackage,autoclaving,
and pouring into culture dish.Sorbito1.MacConkey
agar was weighed for 55 g and dissolved in l 00O mL
distilled water,subpackaged into triangular nask.
autoclaved at 121℃ for 15 min and cooled to 50℃.
added 1 mL of 0.05% sterile potassium nitrite solution
per 20 mL agar solution,and po ured into sterile
culturedishforuse.
·22·l@Shanghai Research Institute ofAcupuncture and Meridian and Springer-Verlag Berlin Heidelberg 201 1
J.Acuounct.Tuina.Sci.201 1,9(1):21—25
2.5 Animal sampling,bacterial culture and
measurement
2.5.1 Animal sampling
The flesh feces of each rat in every group were
weighed at 0.1 g and diluted with O.9 mL
physiological saline(10% WⅣ )in the sterile bottles
with beadings at the end of a treatment course.Th e
bottles were oscillated at 200 times per minute for
15min.
2.5.2 Bacterial culture
The 40 lxL of each oscillated samples were dropped
to the MRS agar medium for LBS,TPY agar medium,
Pfizer enterococcus selective agar medium for EC as
well as Sorbitol—M acConkey agar medium for EB and
pushed by L—shaped rod. The inoculations were
carried out from high dilution to low dilution(10
times to 10-。times).Two drops of the diluents at l0
t0 10。o dilution were inoculated to the corresponding
media with the fluid volume of 1/47 mL.Th e
inoculated media were standing for 20 min an d the
EB and EC were incubated in 37℃ incubator for 24h
while BS and LBS were blocked in an anaerobic jar
with an an aerobic bag an d an oxygen indicator an d
anaerobically incubated in 37℃ incubator for 48—
72 h.
2.5.3 M easure ment
Th e probiotics in each media were dissolved in
4 mL distilled water to examine the OD value of the
bacterial colony in each group by ELIASA in
turbidimetry for comparison of the intergroup
difference.Mean while,the bacteria in each group
were identified in gram s stain and biochemical
method【 ’ .Th e typical bacterial colonies were picked
by aseptic oese and inoculated into corresponding
assessor to incubate at 37℃ in baking oven for 48.
72 h.刀,en the colors of different id~ntificationo tubes
were observed.Th e anaerobic bacteria in the assessor
were sealed by glycerin.
11.5.Analysis of variance and q test were used for
group mean —comparison.Significan ce was defined by
P<0.O5.
3 Results
3.1 M oxibustion influencing typical probiotics in
intestinal tract of experimental rats
3.1.1 Moxibustion at Guanyuan(CV 41 point
influencing BS and LBS in intestinal tract
Th e comparisons ofthe quan tities ofBS an d LBS
in intestinal tract of the rats in the Guanyuan (CV 4、
group and medical group had statistical significan ce
fP<0.05). indicating that the moxibustion at
Guanyuan fCV 4)point increased the quantities of BS
and LBS(table l1.
Table1.ComparisonofBSandLBSineachgroup( )
Note:Compared with the model group,1)P<0.05;compared with
the medical group,2)P<0.05
3.1-2 Moxibustion at Tianshu(ST 25)point
influencing EB and EC in intestinal tract
lble 2 showed the comparisons of the quan tities
of BS an d LBS in intestinal tract of the rats in
Guanyuan (CV 4)group and medical group with
statistical significance( <0.05),indicating that the
moxibustion at Tianshu(ST 25)point increased the
quan tities ofEB an d EC.
Table2.ComparisonofEBandECineachgroup( 蝴)
Note:Compared with the model group,1)P<0.05;compared with
themedicalgroup,2)P<0.05
3.2 Stain of typical bacterial colony from intestinal
tract of experim ental rats
2.6 Statistics
Data were expressed as me mean ± standard Fig.1 showed the stain of typical bacterial colony
deviation( ±s).The data were analyzed with SPSS from intestinal tract of experimental rats.
@Shanghai Research Institute ofAcupuncture and Meridian and Springer—Verlag Berlin Heidelberg 201 1 I·23·
J.Acuounct.Tuina.Sci.201l,9(11:21.25
BS LBS EB EC
Fig.1.Typical bacterial colony stain(Grams stain,xl 000)
3.3 Colonial morphology and biochemical
identification
The features of colonial morphology on MRS agar
media were medium size,smooth surface,whitish,
round and irregular margins. The colonial
morphology on TPY agar media was smal1.
transparent,regular margins,mill( white,soft an d
delicate.For Pfizer enterococcus selective agar media,
the colonial morphology was gray,opaque,smooth
and smal1.The colonial morphology on Sorbito1.
M acConkey agar media was in different sizes,untidy
margins.asperity,white and opaque.111e colors of
vaccinated biochemical identification tube and non
vaccinated biochemical identification tubes were
compared after 24—48 h.The changed color indicated
positive while unchan ged color was for negative,
concluding to the results as identification tubes
instruction。 The biochemical identification was
performed according to the Manual of Common
Determinative Bacteriology an d the Experimental
Techniques in MicrobiologyIjJ.
4 Discussion
Norm ally, the relative equilibrium between gut
flora an d human internal/external environment
secures human health.Many physical chemi cal and
biological factors, such as an tiauxin, radioactive
substances, non—green rood, surgery, exarmne,
hormone,lmmunosuppresslve agents and cytotoxlc
drug . can result in the mi croflora imbalance.
Stressful work and a fast paced life enhan ced the
possibility of intestinal dysbacteriosis.The intestinal
dysbacteriosis may result in plenty of diseases such as
diarrhea, constipation, liver disease, decreased
immunity,progeria,and tumor,and then induce
diseases of other tissues an d organ ics,being one of
the principal diseases for health threats.In the study,
the relation of intestinal dysbactriosis and chronic
diarrhea was discussed depending on animal
experiment.Th e intestinal dysbacteriosis in rats was
induced by feeding them large doses of antibiotics for
a long time.Th e results showed that the change in
quantity of enterobacteria and imbalance of ratio
diminished the immuneit~rand clearance function of
normal flora, leading to a large population of
pathogenic bacteria to stimulate the intestinal mucosa
an d diarrhea occured as a result.Chronic diarrhea
may induce and improve the intestinal dysbacteriosis.
Chronicdiarrheaand alterationofintestinal florawere
reciprocal causationl5】.
Tianshu(ST 25).the Front-Mu point of the large
intestine,and Guanyuan (CV 4),the Front—Mu point
of the small intestine.were investigated in the study.
”Shu” mean s the keY for ascending lucidity an d
descending turbidity,playing a role in invigorating
spleen to resolve dampness.”Guan ”means hiding and
”Yuan ”means the qi of nephroyin an d nephroyang.
Guanyuan(cv 4、point relates with the uterus and
scrotum, which holds the qi of nephroyin an d
nephroyang with the function of adjusting the
Th oroughware and Conception Vessels,warming
kidney,fastening essence,an d regulating qi an d blood.
Moxibustion at the two Front-Mu points regulated the
functional disorders of the large intestine and small
intestine induced by intestinal floraI J.
The warm moxibustion at Guanyuan (CV 4)and
Tianshu(ST 25)improved the diarrhea and regulated
the intestinal flora.Guanyuan fCV 4)effectively
increased the quantity of anaerobic bacteria flora(the
BS and the LBS)while Tianshu(ST 25)significantly
improved the quantity of aerobic bacteria flora(the
BS and the LBS).balancing the intestinal flora to the
normal range.The Front—Mu points.with a close
relationship to intestinal flora,were in every organ.
· 24 ·I@Shanghai Research Institute ofAcupuncture and Meridian and Springer-Verlag Berlin Heidelberg 2011
J.Acupunct.Tuina.Sci.201 1,9(1):21—25
Intestinal flora formed large and complex
microecosystem. Different Front—M u points in
different organs selectively regulated probiotics
groups and inhibited pathogenic bacteria with
targeting features that different Front—Mu points
specifically regulated some kinds of bacteria flora
from different intestinal segments when they were
heated.The warrrl moxibustion at the Front—M u points
【Guanyuan(CV 4)point and Tianshu(ST 25)point】
selectively adjusted probiotics group for treating
intestinal dysbacteriosis.The warlTl moxibustion at
Front—Mu points of the abdomen had a positive effect
on intestinal mucosa protection,mucus secretion and
gastrointestinal motility to recover the homeostasis of
the quantity an d quality of various flora and improve
the growth of probiotics in the intestines[9,10】
.
M oxibustion is a safe and effective treatment for
intestinal dysbacteriosis.
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Translator:Wang Zhen(王臻)
Received Date:December 6,2010
Introduction to Shanghai Journal ofAcupuncture and Moxibustion
Shanghai Journal of Acupuncture and Moxibustion (Monthly,
CN 31一l317/R,ISSN l0o5—0957,64 pages)is a modern professional and
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science,initiated in 1982,sponsored by Shanghai Academy of TCM an d
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