葛根芩连汤调控MMP-9/p38 MARK途径修复溃疡性结肠炎小鼠肠黏膜上皮屏障功能

Gegen Qinliantang Regulates MMP-9/p38 MARK Pathway to Repair Intestinal Mucosal Barrier Function in Mice with Ulcerative Colitis

目的:探讨葛根芩连汤对溃疡性结肠炎(UC)小鼠肠黏膜上皮屏障功能的保护作用,并通过基质金属蛋白酶-9(MMP-9)/p38丝裂原活化蛋白激酶(p38 MAPK)信号通路探究其治疗UC的作用机制。方法:48只雌性C57BL/6小鼠随机分为正常组、模型组、柳氮磺吡啶组(0.3 g·kg^(-1))及葛根芩连汤高、中、低剂量组(2.84,1.42,0.71 g·kg^(-1))。采用3%葡聚糖硫酸钠(DSS)溶液构建UC小鼠模型,葛根芩连汤和柳氮磺吡啶于造模后第8天灌胃给药,连续7 d,正常组给予等量生理盐水处理。末次给药后取结肠组织,苏木素-伊红(HE)染色观察结肠组织病理变化,免疫组化(IHC)观察结肠组织紧密连接(TJ)蛋白,如闭合蛋白(Occludin),闭锁连接蛋白-1(ZO-1)的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织中肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β),MMP-9 mRNA表达,蛋白免疫印迹法(Western blot)检测结肠组织中磷酸化(p)-p38 MAPK,p38 MAPK和MMP-9蛋白的表达。结果:与正常组比较,模型组小鼠体质量显著下降(P<0.01),疾病活动指数(DAI)评分显著升高(P<0.01),结肠黏膜上皮破损并可见黏膜和黏膜下层炎细胞浸润明显,Occludin,ZO-1蛋白表达显著降低(P<0.01),TNF-α,IL-1β,MMP-9 mRNA相对表达量显著升高(P<0.01),p-p38 MAPK和MMP-9蛋白表达显著升高(P<0.01);与模型组比较,柳氮磺吡啶组、葛根芩连汤各剂量组小鼠体质量和DAI评分均有明显改善(P<0.05,P<0.01),结肠组织损坏明显改善,Occludin,ZO-1蛋白明显增多(P<0.05,P<0.01),TNF-α,IL-1β,MMP-9 mRNA相对表达量显著下降(P<0.01),p-p38MAPK和MMP-9蛋白表达显著下降(P<0.01),其中葛根芩连汤各组中以中剂量组的变化最为明显。结论:葛根芩连汤能够通过抑制MMP-9和炎性细胞因子TNF-α,IL-1β的表达,阻断p38 MAPK信号通路的激活,增加TJ蛋白的表达,从而修复肠道黏膜屏障功能。

Objective:To explore the protective effect of Gegen Qinliantang on the intestinal mucosal epithelial barrier function of ulcerative colitis(UC)mice,and to explore its mechanism of action in the treatment of ulcerative colitis via matrix metallopeptidase-9(MMP-9)/p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway. Method: The 48 female C57 BL/6 mice were randomly divided into normal group,model group,sulfasalazine group(0.3 g·kg^(-1))and Gegen Qinliantang high,medium and low dose groups(2.84,1.42,0.71 g·kg^(-1)). The UC murine model was established by 3% dextran sulfate sodium(DSS).Gegen Qinliantang and sulfasalazine were intragastrically administered on the 8 thday after the model was established for 7 days,and the normal group was treated with the same amount of normal saline. Colon tissues were collected after the last administration,and the pathological changes of colon tissues were detected by hematoxylin-eosin(HE)staining. The expression of tight junction(TJ)proteins such as Occludin and zonula occludens-1(ZO-1)in colon tissues was detected by immunohistochemistry(IHC),and the expression levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and MMP-9 mRNA in colon tissues were detected by Real-time polymerase chain reaction(Real-time PCR). The expression of phosphorylated p38 MAPK(p-p38 MAPK),p38 MAPK and MMP-9 protein in colon tissues was detected by Western blot. Result:Compared with normal group,the body weight of mice decreased(P<0.01)and disease activity index(DAI)score increased significantly(P<0.01)in model group,the colon tissues of the model group were damaged more obviously,the expression of occludin and ZO-1 proteins in model group was significantly reduced(P<0.01),and the relative expression levels of TNF-α,IL-1β,and MMP-9 mRNA in model group were significantly increased(P<0.01),the expression of p-p38 MAPK and MMP-9 in model group was significantly increased(P<0.01). Compared with model group,the body mass and DAI score of the sulfasalazine group and Gegen Qinliantang group were significantly improved(P<0.05,P<0.01),the colonic tissues damage were significantly improved,and the expression of Occludin and ZO-1 protein was significantly increased(P<0.05,P<0.01),the relative expression levels of TNF-α,IL-1β,and MMP-9 mRNA were significantly decreased(P<0.01),and the expression of p-p38 MAPK and MMP-9 was significantly decreased(P<0.01). The changes in the middle dose group were the most obvious among the various dose groups of Gegen Qinliantang. Conclusion: Gegen Qinliantang repairs the intestinal mucosal barrier function by inhibiting the expressions of MMP-9 and inflammatory cytokines such as TNF-α and IL-1β,blocking the activation of the p38 MAPK signaling pathway,and increasing the expressions of tight junction protein.