王欢;朱莹;娄余;
WANG Huan;ZHU Ying;LOU Yu;Yueyang hospital affiliated to Hunan University of Chinese Medicine;The First Affiliated Hospital of Hunan University of Chinese Medicine;Graduate School of Hunan University of Chinese Medicine;

• 1:湖南中医药大学附属岳阳医院
• 2:湖南中医药大学第一附属医院
• 3:湖南中医药大学研究生院

摘要(Abstract)：

目的:观察穴位埋线对"虚、瘀"状态下溃疡性结肠炎(UC)模型大鼠肠黏膜上皮屏障的影响,探讨其改善UC的作用机制。方法:雄性SD大鼠随机分为正常组12只与造模组48只。造模组采用腺嘌呤、番泻叶分阶段灌胃制造大鼠"虚、瘀"状态,接着用5%2,4,6-三硝基苯磺酸(TNBS,100 mg/kg)加0.25 mL 50%乙醇灌肠建立UC大鼠模型。将造模成功的大鼠随机分为模型组、柳氮磺吡啶组和穴位埋线组,每组10只。穴位埋线组予以双侧"天枢""足三里""膈俞""脾俞""肾俞""大肠俞"埋线治疗,14 d埋线1次,共3次。柳氮磺吡啶组予以柳氮磺吡啶灌胃,每日1次,共42 d。观察大鼠一般状态及体质量变化,采用肉眼及HE染色观察大鼠结肠组织外观及病理变化,ELISA法检测各组大鼠血清中D乳酸(D-LA)、二胺氧化酶(DAO)和蛋白激酶C(PKC)含量,荧光定量PCR法检测各组大鼠结肠组织中肠黏膜紧密连接蛋白occludin、 claudin8、cingulin、zonulin的mRNA水平,Western blot法检测各组大鼠结肠组织中occludin、claudin8、cingulin、 zonulin和PKC的蛋白表达水平。结果:与正常组比较,模型组大鼠体质量显著降低(P<0.01),结肠组织明显肿胀,肠壁充血,结肠黏膜大量炎性细胞浸润,结肠黏膜损伤指数评分显著升高(P<0.01),血清中D-LA、DAO和PKC含量显著升高(P<0.01),结肠组织内occludin、claudin8、cingulin mRNA和蛋白表达水平均显著降低(P<0.01),zonulin mRNA和蛋白表达水平显著升高(P<0.01),PKC蛋白表达水平显著升高(P<0.01)。与模型组比较,柳氮磺吡啶组和穴位埋线组大鼠体质量显著升高(P<0.01),结肠病变明显减轻,黏膜损伤指数评分显著降低(P<0.05),血清中D-LA、DAO和PKC含量显著降低(P<0.01),结肠组织内occludin、claudin8、cingulin mRNA和蛋白表达水平显著增高(P<0.05,P<0.01),zonulin mRNA和蛋白表达水平显著降低(P<0.01),PKC蛋白表达水平显著降低(P<0.01)。穴位埋线组和柳氮磺吡啶组各指标比较差异无统计学意义(P>0.05)。结论:穴位埋线可调节上皮紧密连接,修复肠黏膜上皮屏障,促进肠黏膜愈合,其作用机制可能与抑制PKC的激活有关。
Objective To observe the effect of acupoint catgut embedding on intestinal mucosal epithelial injury, and expression of epithelial tight junction proteins and mRNAs and protein kinase C(PKC) protein(intestinal mucosal epithelial barrier-related molecules) in rats with experimental ulcerative colitis(UC) of "deficiency-stasis" type, so as to explore its mechanisms underlying improvement of UC. Methods Male SD rats were randomly divided into normal control, UC model, medication(sulfasalazine, SASP) and catgut-embedding groups. The "deficiency-stasis" type UC model was established by gavage of adenine(10 mL/kg) for 2 weeks, cold Folium Sennae(10 mL/kg) for 2 weeks, and then enema of mixture solution of 5% trinitro-benzene-sulfonic acid(100 mg/kg) + 50% ethyl alcohol. Rats of the medication group received gavage of SASP. The catgut-embedment was applied to bilateral "Tianshu"(ST23),"Zusanli"(ST36), "Geshu"(BL17), "Pishu"(BL20), "Shenshu"(BL23) and "Dachangshu"(BL25), once every two weeks, 3 times altogether. The body mass was recorded, and histopathological changes of the colon tissues were observed after H.E. staining. The contents of serum D-lactic acid(D-LA), diamine oxidase(DAO) and protein kinase C(PKC) were detected by ELISA. The expression levels of occludin, claudin-8, cingulin and zonulin mRNAs and proteins and PKC protein of the colon tissues were detected by using quantitative real-time PCR and Western blot, separately. Results Compared with the control group, the body mass and expression levels of colonic occludin, claudin-8 and cingulin mRNAs and proteins were significantly lower(P<0.01), and the colonic mucosa damage index(CMDI) score, contents of serum D-LA, DAO and PKC, as well as the expression levels of zonulin mRNA and protein and PKC protein were significantly higher in the model group(P<0.01). In comparison with the model group, the body mass, and the expression levels of occludin, claudin-8 and cingulin mRNAs and proteins were significantly higher(P<0.05, P<0.01), whereas the CMDI score, expression levels of zonulin mRNA and protein and PKC protein were remarkably lower in both the medication group and acupoint embedding groups(P<0.05,P<0.01). No significant differences were found between the acupoint embedding and medication groups in all the indexes mentioned above(P>0.05). H.E. staining showed markedly swollen, disordered arrangement of intestinal mucosal cells, and hemorrhage with infiltration of inflammatory cells in the colonic tissues after modeling, which was relatively milder in both medication and acupoint embedding groups. Conclusion Acupoint catgut embedding can reduce colonic tissue injury in UC rats, which may be related to its functions in regulating the expression of intestinal epithelial tight junction proteins and mRNAs and in inhibiting the activation of PKC protein.

关键词(KeyWords)： 穴位埋线;溃疡性结肠炎;肠黏膜上皮屏障;紧密连接蛋白;蛋白激酶C
Acupoint catgut embedding;Ulcerative colitis;Intestinal mucosal epithelial barrier;Tight junction protein;Protein kinase C

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(No.81874466);; 湖南省中医药管理局重点课题(No.201903)

作者(Author): 王欢;朱莹;娄余;
WANG Huan;ZHU Ying;LOU Yu;Yueyang hospital affiliated to Hunan University of Chinese Medicine;The First Affiliated Hospital of Hunan University of Chinese Medicine;Graduate School of Hunan University of Chinese Medicine;

Email:

DOI: 10.13702/j.1000-0607.200388

参考文献(References)：

• [1] MAGRO F,GIONCHETTI P,ELIAKIM R,et al.Third European evidence-based consensus on diagnosis and management of ulcerative colitis.part 1:definitions,diagnosis,extra-intestinal manifestations,pregnancy,cancer surveillance,surgery,and ileo-anal pouch disorders[J].J Crohns Colitis,2017,11(6):649-670.
• [2] KAPLAN G G,NG S C.Understanding and preventing the global increase of inflammatory bowel disease[J].Gastroenterology,2017,152(2):313-321.
• [3] KAMM M A.Rapid changes in epidemiology of inflammatory bowel disease[J].Lancet,2018,390(10114):2741-2742.
• [4] BEVIVINO G,MONTELEONE G.Advances in understan-ding the role of cytokines in inflammatory bowel disease[J].Expert Rev Gastroenterol Hepatol,2018,12(9):907-915.
• [5] DE SOUZA H S,FIOCCHI C.Immunopathogenesis of IBD:current state of the art[J].Nat Rev Gastroenterol Hepatol,2016,13(1):13-27.
• [6] PARK J H,PEYRIN-BIROULET L,EISENHUT M,et al.IBD immunopathogenesis:a comprehensive review of inflammatory molecules[J].Autoimmun Rev,2017,16(4):416-426.
• [7] CAMMAROTA G,IANIRO G,CIANCI R,et al.The involvement of gut microbiota in inflammatory bowel disease pathogenesis:potential for therapy[J].Pharmacol Ther,2015,149:191-212.
• [8] CAPALDO C T,POWELL D N,KALMAN D.Layered defense:how mucus and tight junctions seal the intestinal barrier[J].J Mol Med (Berl),2017,95(9):927-934.
• [9] 陈盼碧，陈静，崔瑾，等.穴位埋线法对原发性痛经大鼠神经-内分泌-免疫网络的调控与影响[J].针刺研究，2018,43(1):29-33.
• [10] 朱莹，袁伟建，姚红艳.穴位埋线对溃疡性结肠炎患者血清IL-2受体及T淋巴细胞亚群的影响[J].中国中西医结合杂志，2003,23(1):58-59.
• [11] 朱莹，袁伟建，白晓明.穴位埋线对溃疡性结肠炎淋巴细胞凋亡调控蛋白的影响[J].中医杂志，2007,48(6):526-528.
• [12] 于千惠，李丽红，卢雨微，等.穴位埋线联合艾灸对溃疡性结肠炎大鼠结肠黏膜的抗炎修复作用[J].针刺研究，2020,45(4):305-309.
• [13] 郑洁，朱莹，高昂，等.脾肾阳虚型溃疡性结肠炎大鼠模型的研制[J].中国中西医结合消化杂志，2016,24(3):182-187.
• [14] MORRIS G P,BECK P L,HERRIDGE M S,et al.Hapten-induced model of chronic inflammation and ulceration in the rat colon[J].Gastroenterology,1989,96(2):795-803.
• [15] 吴阳阳，刘明江，殷韶杰，等.针刺对溃疡性结肠炎大鼠氧化应激和内质网应激的影响[J].针刺研究，2020,45(1):8-14.
• [16] 霍金，赵冏琪，袁永，等.穴位埋线疗法作用机制的研究现状[J].中国针灸，2017,37(11):1251-1254.
• [17] 温淑婷，PHAM Ba Tuyen,刘凤斌，等.穴位埋线治疗溃疡性结肠炎的选穴规律[J].中医药导报，2019,25(15):38-42.
• [18] 乔翠霞，赵刚，张立泽，等.基于Toll样受体4/髓样分化因子88/核因子-κB通路探讨电针对溃疡性结肠炎大鼠的干预机制[J].针刺研究，2020,45(3):180-187.
• [19] UNGARO R,MEHANDRU S,ALLEN P B,et al.Ulcerative colitis[J].Lancet,2017,389(10080):1756-1770.
• [20] K?NIG J,WELLS J,CANI P D,et al.Human intestinal barrier function in health and disease[J].Clin Transl Gas-troenterol,2016,7(10):e196.
• [21] PARIKH K,ANTANAVICIUTE A,FAWKNER-CORBETT D,et al.Colonic epithelial cell diversity in health and inflammatory bowel disease[J].Nature,2019,567(7746):49-55.
• [22] 叶毅君，陆虞荪，周雪，等.针刺对5-氟尿嘧啶所致肠黏膜屏障破坏大鼠的保护作用[J].针刺研究，2016,41(2):95-99.
• [23] 臧凯宏，吴建军，秦红岩，等.黄芪多糖对溃疡性结肠炎大鼠肠道黏膜屏障的影响[J].中药材，2017,40(1):208-211.
• [24] FURUSE M,HIRASE T,ITOH M,et al.Occludin:a novel integral membrane protein localizing at tight junctions[J].J Cell Biol,1993,123(6 Pt 2):1777-1788.
• [25] AL-SADI R,KHATIB K,GUO S H,et al.Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier[J].Am J Physiol Gastrointest Liver Physiol,2011,300(6):G1054-G1064.
• [26] MA T M,XU N,MA X D,et al.Moxibustion regulates inflammatory mediators and colonic mucosal barrier in ulcerative colitis rats[J].World J Gastroenterol,2016,22(8):2566-2575.
• [27] SUZUKI T.Regulation of intestinal epithelial permeability by tight junctions[J].Cell Mol Life Sci,2013,70(4):631-659.
• [28] TAMURA A,HAYASHI H,IMASATO M,et al.Loss of claudin-15,but not claudin-2,causes Na+deficiency and glucose malabsorption in mouse small intestine[J].Gastroen-terology,2011,140(3):913-923.
• [29] FUJITA H,CHIBA H,YOKOZAKI H,et al.Differential expression and subcellular localization of claudin-7,-8,-12,-13,and -15 along the mouse intestine[J].J Histochem Cytochem,2006,54(8):933-944.
• [30] YANO T,MATSUI T,TAMURA A,et al.The association of microtubules with tight junctions is promoted by cingulin phosphorylation by AMPK[J].J Cell Biol,2013,203(4):605-614.
• [31] FASANO A,NOT T,WANG W,et al.Zonulin,a newly discovered modulator of intestinal permeability,and its expression in coeliac disease[J].Lancet,2000,355(9214):1518-1519.
• [32] FASANO A.Zonulin and its regulation of intestinal barrier function:the biological door to inflammation,autoimmunity,and cancer[J].Physiol Rev,2011,91(1):151-175.
• [33] FASANO A.Intestinal permeability and its regulation by zonulin:diagnostic and therapeutic implications[J].Clin Gastroenterol Hepatol,2012,10(10):1096-1100.
• [34] DODANE V,KACHAR B.Identification of isoforms of G proteins and PKC that colocalize with tight junctions[J].J Membr Biol,1996,149(3):199-209.
• [35] NISHIZUKA Y.Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C[J].Science,1992,258(5082):607-614.
• [36] CLARKE H,MARANO C W,PERALTA SOLER A,et al.Modification of tight junction function by protein kinase C isoforms[J].Adv Drug Deliv Rev,2000,41(3):283-301.