燮理汤对溃疡性结肠炎小鼠炎症因子及肠黏膜屏障损伤的影响

Effects of Xie Li(燮理)Decoction on Inflammatory Factors and Intestinal Mucosal Barrier Damage in Mice with Ulcerative Colitis

目的:探讨燮理汤对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(Ulcerative colitis, UC)小鼠的保护作用及其作用机制。方法:60只C57BL/6小鼠随机分为正常对照组、模型对照组、柳氮磺吡啶肠溶片0.6 g/kg组和燮理汤1.67、3.34、6.68 g/kg组,以DSS溶液自由饮周期循环的方式复制慢性UC模型小鼠。正常对照组小鼠在整个试验期间自由饮用蒸馏水,其余各组在第1 d~7 d、22 d~28 d和43 d~49 d自由饮用1.5%的DSS溶液,其余时间自由饮用蒸馏水。在试验的第22 d起给予相应药物治疗,连续42 d。试验期间每日记录小鼠的一般状况和体质量,观察疾病活动指数(DAI)评分变化;ELISA法检测血清白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和髓过氧化物酶(MPO)的含量;HE染色观察结肠组织病理形态学以及病理损伤评分变化;免疫荧光法检测结肠组织中闭锁蛋白(Occludin)和闭锁小带蛋白-1(ZO-1)的蛋白表达;蛋白免疫印迹(Western blot)法检测结肠组织中粘蛋白2(MUC2)、肠三叶因子3(TFF3)和缺氧诱导因子-1α(HIF-1α)的蛋白表达。结果:与正常对照组比较,模型对照组小鼠一般状况下降,体质量减轻,结肠长度显著缩短(P<0.01),DAI评分、IL-6、TNF-α和MPO含量均显著升高(P<0.01),结肠黏膜结构破坏,炎性细胞严重浸润,病理评分显著升高(P<0.01),结肠组织中ZO-1、Occludin、MUC2和TFF3蛋白表达显著下调,HIF-1α蛋白表达明显上调(P<0.01);与模型对照组比较,燮理汤各剂量组小鼠状况明显好转,体质量增加,结肠长度显著增加(P<0.01),DAI评分以及IL-6、TNF-α和MPO含量均显著降低(P<0.01),结肠黏膜病理损伤明显减轻,病理评分显著降低(P<0.01),结肠组织中ZO-1和Occludin蛋白表达均显著上调(P<0.01),并可不同程度地调控MUC2、TFF3和HIF-1α蛋白表达。结论:燮理汤对UC小鼠有较好的治疗作用,其作用机制可能与减轻炎症反应,保护肠黏膜屏障有关。

Objective:To explore the protective effect and potential mechanism of Xie Li(燮理)decoction on dextran sodium sulfate(DSS)-induced ulcerative colitis(UC)in mice.Methods:Sixty C57BL/6 mice were randomly divided into a normal group,a model group,a entericcoated sulfasalazine tablets(0.6 g/kg)group,and Xie Li decoction(1.67 g/kg,3.34 g/kg and 6.68 g/kg)groups.The mouse model of chronic UC was established by repeated oral administration of DSS-supplemented water.The mice in normal group had free access to distilled water throughout the experiment,while the mice in rest groups were exposed to 1.5%DSS from the 1st to 7th day,22th to 28th day and 43th to 49th day,and to distilled water in the rest days.The corresponding medications were given from the 22th day of the experiment for 42 successive days.The general condition,body mass and disease activity index(DAI)scores of mice were recorded daily during the experiment.Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and myeloperoxidase(MPO)in serum.Hematoxylin-eosin(HE)staining was used to detect the changes in pathological morphology and histopathological score(HS)of colon.Immunofluorescence assay was used to detect the protein expressions of occludin and zonula occludens-1(ZO-1)in colon.Western blot was used to determine the protein expressions of hypoxia inducible factor-1α(HIF-1α),mucin 2(MUC2)and intestinal trefoil factor 3(TFF3)in colon.Results:Compared with the normal group,general status,body mass and the colon length were significant decreased in the mice of the model group of(P<0.01),while the DAI score and the contents of IL-6,TNF-αand MPO were significantly increased(P<0.01).The structure of colon mucosa was damaged,with obvious inflammatory cell infiltration,while the HS score was significantly increased(P<0.01).In colon tissue,the protein expressions of ZO-1,occludin,MUC2 and TFF3 were significantly down-regulated(P<0.01),while the protein expression of HIF-1αwas significantly up-regulated(P<0.01).Compared with model group,the general status was improved,the body mass,and the colon length were increased significantly in mice of all Xie Li decoction groups(P<0.01).The DAI score and the contents of IL-6,TNF-αand MPO were significantly decreased(P<0.01).The pathological injury of colon was obviously alleviated and HS was significantly decreased(P<0.01).In colon tissue,the protein expressions of ZO-1 and occludin were siginificantly up-regulated(P<0.01),and the protein expressions of MUC2,TFF3 and HIF-1αwere also enhanced to vary degree.Conclusion:Xie Li decoction has the therapeutic effect on UC mice,and the mechanism may be related to inhibiting inflammatory response and protecting intestinal mucosal barrier.