雷公藤多苷对溃疡性结肠炎大鼠TLR4/MyD88非依赖信号通路的作用研究

Regulatory effect of Tripterygium wilfordii polycoride( TWP) towards TLR4/MyD88 independent pathway in TNBS / ethanol ulcerative colitis( UC) rat model

为了研究雷公藤多苷(TWP)对三硝基苯磺酸(TNBS)/乙醇溃疡性结肠炎(UC)大鼠TLR4/My D88非依赖信号通路的调控作用,采用了TNBS/乙醇联合灌肠的方法建立TNBS/乙醇UC大鼠模型。模型建立成功后,将90只雄性Wistar大鼠随机分为正常对照组,模型对照组,TWP低、中、高剂量组(3,6,12 mg·kg^(-1)),硫唑嘌呤(AZA)组(6 g·kg^(-1)),每组15只。各组分别给予相应药物连续灌胃14 d。每隔3 d评估UC大鼠疾病活动指数(DAI)。14 d后解剖所有大鼠,留取相应结肠组织观察各组大鼠结肠组织大体及镜下病理表现,并对其进行评分。采用Western blot法和RT-PCR法检测UC大鼠结肠组织中TLR4/My D88非依赖信号通路相关分子(TLR4,TRAM,TRIF,NF-κB,IFN-γ)在mRNA及蛋白水平的表达情况。结果提示,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,TWP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。RT-PCR及Western blot实验均提示,与正常对照组相比,模型对照组中TLR4/My D88非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)。与模型对照组相比,TWP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TWP高剂量组中各节点分子mRNA及蛋白表达水平显著低于模型对照组(P<0.05)。与AZA组相比,TWP高剂量组对该信号通路上游因子(TLR4,TRAM,TRIF,NF-κB)mRNA及蛋白表达水平的抑制作用略好于AZA组,而对该信号通路的末端炎症因子IFN-γmRNA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。因此,在TNBS/乙醇UC大鼠模型中,My D88非依赖信号通路参与了炎症活动的调控,TWP可以通过抑制TLR4/My D88非依赖信号通路抑制IFN-γ的释放,发挥抗炎作用,其作用强度与剂量呈正相关。

In order to study the regulatory effect of Tripterygium wilfordii polycoride（ TWP） towards TLR4 /My D88 independent pathway in TNBS / ethanol ulcerative colitis（ UC） rat model,TNBS / ethanol enema was adopted to build TNBS / ethanol UC rat model.After the successful modeling procedure,90 male Wistar rats are were divided into 6 groups,including namely normal group,model group,TWP low,middle,high dose groups（ 3,6,12 mg·kg^（-1）） and azathioprine（ AZA） group（ 6 g·kg^（-1））,with 15 rats in each group. All rats in each group were administrated with corresponding medicines for 14 days. After 14 days of administration,corresponding colon tissues were taken for general and microscopic evaluation. Western blotting analysis and RT-PCR were adopted to test the mRNA and protein expressions of TLR4 / My D88 independent pathway-related molecules,namely TLR4,TRAM,TRIF,NF-κB and IFN-γ. The results showed that DAI,general and microscopic evaluations all indicated that TNBS / ethanol UC rat model was successful. TWP can improve UC-related clinical manifestation and heal colonic mucosa,which was equal to AZA. RT-PCR and WB results showed that the expression of TLR4 / My D88 independent pathway-related molecules in model group were significantly superior to that in normal group at either mRNA or protein level（ P 0. 01）. Compared with model group,TWP can inhibit the expression of each node in TLR4 / My D88 independent pathway in a dose-dependent manner. The inhibitory effect of TWP with high dose towards the above molecules was inferior to that in model group at either mRNA or protein level（ P 0. 05）. The inhibitory effect of TWP with high dose towards upstream molecules of TLR4 / My D88 independent pathway（ TLR4,TRAM,TRIF,NF-κB） was slightly superior to AZA group at either mRNA or protein level. However,such inhibitory effect towards terminal inflammatory cytokines（ IFN-γ） was inferior to AZA group at either mRNA or protein level. All the above differences had no statistical significance. Therefore,in TNBS / ethanol UC rat model,TLR4 / My D88 independent pathway took part in regulating inflammation. TWP exerted its anti-inflammation effect by inhibiting the expression of TLR4 / My D88 independent pathway in a dose-dependent manner.