目的 研究雷公藤多苷片(TWPT)对2,4,6-三硝基苯磺酸(TNBS)/乙醇溃疡性结肠炎(UC)大鼠微小RNA(miR-146a、miR-146b)及TLR4/MyD88依赖信号通路的调控作用。方法 采用TNBS/乙醇灌肠法制备UC大鼠模型。将90只雄性Wistar大鼠随机分为对照组,模型组,TWPT低、中、高剂量(30、60、120 mg/kg)组,硫唑嘌呤(AZA,60 mg/kg)组,每组15只。各组分别ig给予相应药物连续14 d。进行各组大鼠结肠组织大体及镜下病理评分。采用qRT-PCR法检测miR-146a和miR-146b的表达情况;Western blotting法和RT-PCR法检测大鼠结肠组织中TLR4/MyD88依赖信号通路相关分子(TLR4、MyD88、TRAF-6、NF-κB、TNF-α、IL-1β)在mRNA及蛋白水平的表达情况。结果 DAI评分,大体及镜下表现和评分均提示TNBS/乙醇UC大鼠模型造模成功,TWPT对UC大鼠临床症状的改善及黏膜愈合具有一定作用,该作用与AZA相比相当或强于AZA。qRT-PCR结果提示:与对照组相比,模型组中miR-146a和miR-146b表达明显增加(P<0.01);与模型组相比,TWPT及AZA均可显著抑制miR-146a和miR-146b的表达(P<0.01),其中TWPT中剂量组对2者的抑制作用最强。RT-PCR及Western blotting实验均提示:与对照组相比,模型组中TLR4/MyD88依赖信号通路相关的分子无论在mRNA还是蛋白水平表达均显著升高(P<0.01);与模型组相比,TWPT呈剂量依赖性地抑制该信号通路上各节点分子mRNA及蛋白水平的表达,其中TWPT高剂量组中各节点分子mRNA及蛋白表达水平低于模型组(P<0.05)。与AZA组比较,TWPT高剂量组对该信号通路上游因子(TLR4、MyD88、TRAF-6、NF-κB)mRNA及蛋白表达水平的抑制作用略好于AZA组,而对末端炎症因子TNF-α及IL-1β mRNA及蛋白水平的抑制作用却略逊于AZA组,但上述2种差异均无统计学意义(P>0.05)。结论 在TNBS/乙醇UC大鼠模型中,TWPT能通过抑制miR-146a和miR-146b的表达,并能抑制TLR4/MyD88依赖信号通路及炎症因子IL-1β及TNF-α释放,对信号通路及炎症因子的作用强度与剂量呈正相关。

Objective To study the regulatory effect of Tripterygium wilfordii Polycoride Tadlet (TWPT) towards miR-146a, miR-146b, and TLR4/MyD88 dependent signaling pathway in TNBS/ethanol ulcerative colitis (UC) rat model. Methods TNBS enema was adopted to build TNBS/ethanol UC rat model. After the modeling procedure, 90 male Wistar rats were divided into six groups, including normal, model, low-, mid-, high-dose TWPT, and azathioprine (AZA) groups, and each for 15 rats. All rats in each group were administered with corresponding medicines for 14 d. After 14 d administration, corresponding colon tissues were taken to undergo general and microscopic evaluation. qPCR was adopted to test the expression of miR-146a and miR-146b. Western blotting analysis and RT-PCR were adopted to test the mRNA and protein expression levels of TLR4/MyD88 dependent signaling pathway related molecular, including TLR4, MyD88, TRAF-6, NF-κB, TNF-α, and IL-1β. Results DAI, general and microscopic evaluation all showed that TNBS/ethanol UC rat model was successfully established. TWPT could improve UC-related clinical manifestation and promote the colonic mucosa healing procedure and such effect was equal to AZA. qRT-PCR showed that the expression of miR-146a and miR-146b in model group was significantly superior to that in normal group (P<0.01). Compared with the model group, TWPT and AZA could significantly inhibit the expression of miR-146a and miR-146b (P<0.01). The mid-dose TWPT showed the strongest inhibitory effect. RT-PCR and Western blotting results showed that the expression of TLR4/MyD88 dependent signaling pathway related molecular in model group was significantly superior to that in normal group either in mRNA or protein levels (P<0.01). Compared with model group, TWPT could inhibit the expression of each spot in TLR4/MyD88 dependent signaling pathway in a dose-dependent manner. The inhibitory effect of high-dose TWPTtowards the above molecular was superior to that in model group either in mRNA or protein levels (P<0.05). The inhibitory effect of high-dose TWPT towards upstream molecular of TLR4/MyD88 dependent signaling pathway (TLR4/MyD88/TRAF-6/NF-κB) was slightly superior to that in AZA group either in mRNA or protein levels. However, such inhibitory effect towards terminal inflammatory cytokines (TNF-α and IL-1β) was slightly inferior to that in AZA group either in mRNA or protein levels. All the above differences had no statistical significance (P>0.05). Conclusion In TNBS/ethanol UC rat model, TWPT could inhibit the expression of miR-146a, miR-146b, and TLR4/MyD88 dependent signaling pathway. The inhibitory effect of TWPTtowards pathway and inflammatory cytokines shows a dose-dependent manner.

国家自然科学基金资助项目(81273903)