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.
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。.
Keywords: Acupoint catgut embedding; Intestinal mucosal epithelial barrier; Protein kinase C; Tight junction protein; Ulcerative colitis.