艾灸腹部募穴调整大鼠肠道菌群失调的实验研究

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． 【1】Li ZR．Experimental acupuncture science．Beijing：China Press of Traditional Chinese M edicine，2007：i． r21 Dong XZ，Cai M Y．Manual of common determinative bacteriology．Beijing：Science Press，2001：2． [3】Guan YZ，Wang AL，Li J．Experimental techniques in microbiology．Beijing：Chemical Industry Press，2006：1． 【4】Jia wX．Medical microbiology．Beijing：People’s Medical Publishing House．2o05：7． 【5]Zhuang GL，Mou ZQ，Liu XM．Observation of clinical effects of acupuncture on postoperative alteration of intestinal flora．Shan Xi Lin Chuang Yi Yao，20ol，10(8)： 623。624． 【6】Tang XS．Thinking about needling Zusanli(ST 36)and moxibustion at Guanyuan(cv 4、．Shan Dong Zhong Yi Za Zhi，20o2，2 1(4)：245—246． f71 Ju XD，Sui H．1'reatment of 1o0 cases of functional diarrhea with acupuncture．Nei Meng Gu Yi Yao，2010，29(2)：32—33． f81 Li F，Cui XM．Treatment of chronic diarrhea with acupuncture．Xian Dai Zhong Xi Yi Jie He Za Zhi，2009． 1 8(30)：3685—3686． 【9】Xu YQ，Dong Y Clinical function and application of bifidobacterium．Zhong Guo Xian Dai Yao Wu Ying Y_ong， 2007．1(7)：61—62． 【10】Wang XM，Shi Z，Ma XP,Zhou CL，Qin XD，Liu HR，Wu HG Thinking and methods of moxibustion regulation of pattern recognition receptors and signal transduction in inflammatory bowel disease．Shang Hai Zhen Jiu Za Zhi． 2010．29(3)：136—139． 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 academic publication on Chinese traditional acupuncture—moxibustion science，initiated in 1982，sponsored by Shanghai Academy of TCM an d Shan ghai Society of Acupuncture and Moxibustion，an d undertaken by Shan ghai Research Institute of Acupuncture and M eridian ．It won excellent scientific an d technologic publication prizes awarded bv the Ministry of Science an d Technology of the People’s Republic of China， Science and Technology Commission of Shanghai M unicipality,an d Shanghai Association of Science an d Technology respectively,and was selected as double—hundred periodical of Chinese Journal Phalan x in 20o1．All its articles are recorded in the Chinese Joumal Database．the Chinese Biomedical Journal an d Literature Database． Address：No．650，South Wanping Road，Shanghai 200030，P R．China Telephone(Fax)：0086-021—643821 81 E-mail：zhenjiush@126．tom；shzj@chinajourna1．net．ca @Shanghai Research Institute of Acupuncture and Meridian and Springer-Verlag Berlin Heidelberg 20 1 1 l·25·