复方蜥蜴散不同微粒组合剂干预大鼠溃疡性结肠炎模型TLRs/MyD88信号通路及下游炎症因子的实验研究

Experimental study on the effect of combined reagent of different particles of Compound Lizards Powder on TLRs/MyD88 signaling pathways and downstream inflammatory factors in rat model of ulcerative colitis

目的探讨复方蜥蜴散不同微粒组合剂干预大鼠溃疡性结肠炎(UC)模型TLRs/My D88信号通路中肠组织髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子6(TRAF6)蛋白表达、下游炎症因子肿瘤坏死因子-α(TNF-α)含量及其对UC的作用机制。方法雄性SD大鼠84只,随机分为6组,即正常对照组、模型组、柳氮磺吡啶片治疗组、蜥蜴散80目、100目、80+100目等量混合治疗组,分别编号A^F组。A组予0.9%Na Cl溶液灌肠处理,B^F组均采用三硝基苯磺酸(TNBS)灌肠诱导制备UC模型。造模成功后,治疗组(C^F组)分别给予对应药物灌胃治疗,A组及B组给予0.9%Na Cl溶液灌胃治疗,连续14 d,频次相同。14 d后麻醉全部大鼠,行腹主动脉取血,离心低温保存,取血后取结肠组织病变处包埋。采用HE染色观察大鼠结肠黏膜病理改变,采用免疫组化法检测My D88、TRAF6蛋白的原位表达,采用酶联免疫吸附试验(ELISA试验)检测血清TNF-α水平。结果与正常对照组比较,其他组My D88、TRAF6蛋白表达量及TNF-α水平升高,差异均有统计学意义(P<0.05);各治疗组与模型组比较,各项指标水平均下降,差异有统计学意义(P<0.05);复方蜥蜴散各治疗组与柳氮磺吡啶片治疗组比较,复方蜥蜴散80+100目组治疗效果最佳,差异有统计学意义(P<0.05),蜥蜴散80目、100目组与柳氮磺吡啶片治疗组比较,差异无统计学意义(P>0.05);复方蜥蜴散各治疗组间比较,80+100目等量混合组各项指标水平明显降低,差异有统计学意义(P<0.05)。结论复方蜥蜴散不同微粒组合剂能抑制TLRs/My D88信号通路下游信号传递分子My D88、TRAF6蛋白表达及降低下游炎症因子TNF-α水平,提示其治疗溃疡性结肠炎的主要作用机制在于阻断溃疡性结肠炎的炎症反应,调节免疫,恢复肠道屏障,维持肠道稳态,促进肠黏膜修复。

Objective To investigate the effect of combined reagent of different particles of Compound Lizards Powder on the protein expression of myeloid differentiation factor88（MyD88） and tumor necrosis factor receptor-associated factor-6（TRAF6） that in TLRs/MyD88 signaling pathway and the content of downstream inflammatory factors tumor necrosis factor-α（TNF-α） in rat model of ulcerative colitis（UC）,and discuss its mechanisms on UC.Methods 84 male SD rats were randomly divided into six groups as the normal control group,model group,sulfasalazine tablets（SASP） treatment group,treatment groups of Compound Lizards Powder filtrated through 80 mesh,100 mesh,80＋ 100 mesh equivalent mix respectively,14 rats in each group.The control group was received the enema treatment with 0.9% NaCl solution,and the other groups were received the enema treatment with three nitrobenzene sulfonic acid（TNBS） to establish the UC model.After successful modeling,the control and model group were administered intragastrically with 0.9% NaCl solution and the other groups were administered intragastrically with the corresponding drugs,with a course of 14 days.After treatment for 14 days,all rats were anesthetized and the blood were collected from abdominal aorta and preserved at low temperature after centrifugation.The lesion of colon tissue were obtained and embedded.The pathological changes of colonic mucosa were observed by HE staining.The situ protein expressions of MyD88 and TRAF6 in colon tissues were determined by immunohistochemistry.The level of serum TNF-α was detected by enzyme linked immunosorbent assay（ELISA）.Results Compared with the normal control group,the protein expression of MyD88 and TRAF6 and the level of TNF-α in the other groups were increased,with statistically significant differences（P〈0.05）.Compared with the model group,the levels of all indicators in each treatment group were decreased,with statistically significant differences（P〈0.05）.Comparison of SASP treatment group and treatment groups of Compound Lizards Powder,the treatment effects of the Compound Lizards Powder filtrated through 80＋100 mesh equivalent mix were best,with statistically significant differences（P〈0.05）.There were no statistically significant differences on the effects between the SASP treatment group and treatment group of Compound Lizards Powder filtrated through 80 mesh or 100 mesh（P〉0.05）.Comparison of treatment groups of Compound Lizards Powder,the levels of all indicators were obviously decreased in the treatment group of Compound Lizards Powder filtrated through 80＋100 mesh equivalent mix,with statistically significant differences（P〈0.05）.Conclusion The combined reagent of different particles of Compound Lizards Powder could inhibit the protein expression of MyD88 and TRAF6 that in TLRs/MyD88 signaling pathway and decrease the level of downstream inflammatory TNF-α,which suggested that its main mechanism in the treatment of ulcerative colitis were blocking the inflammatory reaction,regulating immunity,restoring intestinal barrier,maintaining intestinal homeostasis and promoting intestinal mucosal repair.