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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。
To investigate the regulatory effect of TWP on TLR4 / My D88 independent signaling pathway in trinitrobenzene sulfonic acid (TNBS) / ethanol ulcerative colitis (UC) rats, TNBS / ethanol combination enema Methods The TNBS / ethanol UC model was established. After successful establishment of the model, 90 male Wistar rats were randomly divided into normal control group, model control group, TWP low, medium and high dose groups (3, 6, 12 mg · kg -1), azathioprine (AZA) group (6 g · kg -1), 15 rats in each group. Each group were given the corresponding drug for 14 days. UC rat disease activity index (DAI) was assessed every 3 days. After 14 days, all the rats were dissected, and the corresponding colon tissues were taken for observing the gross and microscopic pathological findings of the colon tissues in each group and scoring them. Western blot and RT-PCR were used to detect the expression of TLR4 / My D88 independent signaling pathway related molecules (TLR4, TRAM, TRIF, NF-κB and IFN-γ) in UC rats. The results suggest that the DAI score, gross and microscopic findings and scores suggest successful TNBS / ethanol UC rat model, TWP may play a role in the improvement of clinical symptoms and mucosal healing in UC rats, which is comparable to that of AZA or Stronger than AZA. Both RT-PCR and Western blot suggested that TLR4 / My D88 independent signaling pathway-related molecules in model control group were significantly increased at both mRNA and protein levels (P <0.01) compared with the normal control group. Compared with the model control group, TWP inhibited the mRNA and protein expression of each node in a dose-dependent manner, and the mRNA and protein expression of each node in TWP high-dose group was significantly lower than that of the model control group (P <0.05). Compared with the AZA group, the TWP high dose group had slightly better inhibitory effect on the mRNA and protein expression of TLR4, TRAM, TRIF and NF-κB in AZA group, The inhibitory effect of IFN-γmRNA and protein level was slightly lower than that of AZA group, but the above two differences were not statistically significant. Therefore, My D88-independent signaling pathway is involved in the regulation of inflammatory activity in TNBS / ethanol UC rat model. TWP can exert anti-inflammatory effect by inhibiting the release of IFN-γ by inhibiting TLR4 / My D88-independent signaling pathway Intensity and dose was positively correlated.