针刺疗法调节IBS-D大鼠肠道菌群结构和促进肠紧密连接的作用研究
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摘要
【目的】探讨针刺改变肠道菌群结构及促进肠紧密连接对腹泻型肠易激综合征(IBS-D)大鼠的作用机制。【方法】将48只Wistar大鼠随机分为4组,即正常组、模型组、西药组和针刺组,每组各12只。通过强行应激法(CAS)刺激构建IBS-D大鼠模型,西药组予匹维溴铵混悬液灌胃,每日15 mg/kg;针刺组双侧取穴:ST25(天枢),SP15(大横),ST36(足三里),ST37(上巨虚),LR3(太冲)以及GV20(百会),针刺深度3 mm,留针20 min,每隔5 min行针1次;正常组、模型组、针刺组每日灌服生理盐水;正常组、模型组、西药组每日予同针刺组相同固定束缚时间。以上处理方法,1次/d,连续干预14 d。观察各组大鼠排便和腹泻的情况,并行粪便16 s测序分析大鼠肠道菌群变化,结肠组织行Real-time PCR法和免疫组化法检测紧密连接蛋白ZO-1、Occludin基因表达量。【结果】与模型组比较,针刺组腹泻症状改善,菌群多样性升高(P<0.05),乳酸杆菌科(Lactobacillaceae)和双歧杆菌科(Bifidobacteriaceae)丰度上升(P<0.01),结肠紧密连接基因ZO-1和Occludin表达量显著升高(P<0.01),差异菌群与紧密连接蛋白表达呈正相关。【结论】针刺可调节IBS-D肠道菌群结构,增加菌群多样性及上调肠屏障相关益生菌丰度,并介导ZO-1和Occludin的表达。
Objective To explore the therapeutic mechanism for acupuncture in improving the structure of gut microbiota and promoting intestinal tight junction in diarrhea-predominant irritable bowel syndrome(IBS-D)rats.Methods Forty-eight rats were randomly divided into 4 groups,namely normal group,model group,western medicine group and acupuncture group,12 rats in each group. The IBS-D model was established by chronic and acute stress(CAS). After modeling, the western medicine group was given intragastric administration of Pinaverium Bromide suspension at dosage of 15 mg·kg~(-1)·d~(-1). The acupuncture group was given acupuncture at bilateral acupoints of Tianshu(ST25),Daheng(SP15),Zusanli(ST36),Shangjuxu(ST37),Taichong(LR3)and Baihui(GV20),acupuncture depth being 3 mm,retaining of the needle for 20 min,manipulating the needle once per 5 min. The normal group,model group,and acupuncture group were given intragastric administration of normal saline,and the normal group,model group,and western medicine group were given the same constrain time as the acupuncture group. Once per day,the intervention covered 14 days. The symptoms of defecation and diarrhea in various groups were observed,the changes of the gut microbiota were detected by feces 16 s sequence analysis, and the mRNA and protein expression levels of tight junction protein ZO-1 and Occludin in colonic tissues were determined by real-time PCR and immunohistochemistry,respectively. Results Compared with the model group,the symptom of diarrhea was improved in acupuncture group,the microbial diversity was increased(P < 0.05), the abundance of Lactobacillaceae and Bifidobacteriaceae was increased(P < 0.01), and the expression levels of colonic tight junction genes of ZO-1 and Occludin were increased(P < 0.01). There was positive correlation between differential flora and expression of colonic tight junction proteins. Conclusion Acupuncture has effects on regulating the structure of gut microbiota,increasing the microbial diversity and upregulating the abundance of probiotics related to intestinal barrier as well as mediating the expression of ZO-1 and Occludin in IBS-D rats.
Objective To explore the therapeutic mechanism for acupuncture in improving the structure of gut microbiota and promoting intestinal tight junction in diarrhea-predominant irritable bowel syndrome(IBS-D)rats.Methods Forty-eight rats were randomly divided into 4 groups,namely normal group,model group,western medicine group and acupuncture group,12 rats in each group. The IBS-D model was established by chronic and acute stress(CAS). After modeling, the western medicine group was given intragastric administration of Pinaverium Bromide suspension at dosage of 15 mg·kg~(-1)·d~(-1). The acupuncture group was given acupuncture at bilateral acupoints of Tianshu(ST25),Daheng(SP15),Zusanli(ST36),Shangjuxu(ST37),Taichong(LR3)and Baihui(GV20),acupuncture depth being 3 mm,retaining of the needle for 20 min,manipulating the needle once per 5 min. The normal group,model group,and acupuncture group were given intragastric administration of normal saline,and the normal group,model group,and western medicine group were given the same constrain time as the acupuncture group. Once per day,the intervention covered 14 days. The symptoms of defecation and diarrhea in various groups were observed,the changes of the gut microbiota were detected by feces 16 s sequence analysis, and the mRNA and protein expression levels of tight junction protein ZO-1 and Occludin in colonic tissues were determined by real-time PCR and immunohistochemistry,respectively. Results Compared with the model group,the symptom of diarrhea was improved in acupuncture group,the microbial diversity was increased(P < 0.05), the abundance of Lactobacillaceae and Bifidobacteriaceae was increased(P < 0.01), and the expression levels of colonic tight junction genes of ZO-1 and Occludin were increased(P < 0.01). There was positive correlation between differential flora and expression of colonic tight junction proteins. Conclusion Acupuncture has effects on regulating the structure of gut microbiota,increasing the microbial diversity and upregulating the abundance of probiotics related to intestinal barrier as well as mediating the expression of ZO-1 and Occludin in IBS-D rats.
引文
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[10] Zeisel M B,Dhawan P,Baumert T F. Tight junction proteins in gastrointestinal and liver disease[J]. Gut,2018,31(6):906.
[11] Martinez C, Vicario M, Ramos L, et al. The jejunum of diarrhea-predominant irritable bowel syndrome shows molecular alterations in the tight junction signaling pathway that are associated with mucosal pathobiology and clinical manifestations[J]. Ame J Gastroenterol,2012,107(5):736.
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[13] Ling X, Ling L P, Wei X D, et al. Protective Effects of bifidobacterium on intestinal barrier function in LPS-induced enterocyte barrier injury of Caco-2 monolayers and in a rat NEC model[J]. PloS one,2016,11(8):e161635.
[14]曹健,吴耀南.吴耀南教授治疗腹泻型肠易激综合征经验浅述[J].中医药通报,2014,13(2):30.
[15]沈淑华,黄宣,吕宾,等.腹泻型肠易激综合征证型及证候要素的文献研究[J].中华中医药杂志,2013,28(5):1538.
[16]张声生,汪红兵,李振华,等. 360例腹泻型肠易激综合征的聚类分析及证候特征研究[J].中华中医药杂志,2010,25(8):1183.
[17]吴晓亮,王烨林,孙建华,等.针刺治疗腹泻型肠易激综合征肝郁脾虚证临床观察及其对Th1/Th2的影响[J].中国针灸,2013,33(12):1057.
[18]李静.调神健脾针法治疗腹泻型肠易激综合征的临床疗效评价及脑fMRI改变的研究[D].南京:南京中医药大学,2017.
[2] Pei L, Geng H, Chen H, et al. Acupuncture for irritable bowel syndrome:study protocol for a multicenter randomized controlled trial[J]. Trials,2018,19(1):529.
[3]孙路强,张微,魏韬,等.针灸对肠道菌群影响的研究进展[J].湖南中医杂志,2018,34(1):190.
[4] Zou N, Lv H, Li J, et al. Changes in brain G proteins and colonic sympathetic neural signaling in chronic-acute combined stress rat model of irritable bowel syndrome(IBS)[J]. Translational Research,2008,152(6):283.
[5]张铮铮,张进领,陈惠军,等.加味柴胡疏肝散对腹泻型肠易激综合征模型大鼠结肠黏膜胆囊收缩素的影响[J].中药新药与临床药理,2013,24(5):473.
[6] Simren M,Tack J. New treatments and therapeutic targets for IBS and other functional bowel disorders[J]. Nat Rev Gastroenterol Hepatol,2018,15(10):589.
[7] Collins S M. A role for the gut microbiota in IBS[J]. Nat Rev Gastroenterol Hepatol,2014,11(8):497.
[8] Pei L, Chen H, Guo J, et al. Effect of acupuncture and its influence on visceral hypersensitivity in IBS-D patients:Study protocol for a randomized controlled trial[J]. Medicine,2018,97(21):e10877.
[9] Wang X,Qi Q,Wu H,et al. Moxibustion modulates the gut microbiota in rats with irritable bowel syndrome[J]. Gastroenterology,2017,152(5):S632.
[10] Zeisel M B,Dhawan P,Baumert T F. Tight junction proteins in gastrointestinal and liver disease[J]. Gut,2018,31(6):906.
[11] Martinez C, Vicario M, Ramos L, et al. The jejunum of diarrhea-predominant irritable bowel syndrome shows molecular alterations in the tight junction signaling pathway that are associated with mucosal pathobiology and clinical manifestations[J]. Ame J Gastroenterol,2012,107(5):736.
[12] Eun C S,Kim Y S,Han D S,et al. Lactobacillus casei prevents impaired barrier function in intestinal epithelial cells[J].APMIS:acta pathologica, microbiologica, et immunologica Scandinavica,2011,119(1):49.
[13] Ling X, Ling L P, Wei X D, et al. Protective Effects of bifidobacterium on intestinal barrier function in LPS-induced enterocyte barrier injury of Caco-2 monolayers and in a rat NEC model[J]. PloS one,2016,11(8):e161635.
[14]曹健,吴耀南.吴耀南教授治疗腹泻型肠易激综合征经验浅述[J].中医药通报,2014,13(2):30.
[15]沈淑华,黄宣,吕宾,等.腹泻型肠易激综合征证型及证候要素的文献研究[J].中华中医药杂志,2013,28(5):1538.
[16]张声生,汪红兵,李振华,等. 360例腹泻型肠易激综合征的聚类分析及证候特征研究[J].中华中医药杂志,2010,25(8):1183.
[17]吴晓亮,王烨林,孙建华,等.针刺治疗腹泻型肠易激综合征肝郁脾虚证临床观察及其对Th1/Th2的影响[J].中国针灸,2013,33(12):1057.
[18]李静.调神健脾针法治疗腹泻型肠易激综合征的临床疗效评价及脑fMRI改变的研究[D].南京:南京中医药大学,2017.