解毒活血方灌肠对溃疡性结肠炎小鼠TLR4相关通路蛋白及炎症因子表达的影响

Effects of Jiedu Huoxue Recipe Enema on Expressions of TLR4 Related Signal Pathway Proteins and Inflammatory Factors in Ulcerative Colitis Mice

目的探讨解毒活血方灌肠治疗对溃疡性结肠炎(UC)小鼠Toll样受体4(TLR4)、β干扰素TIR结构域衔接蛋白(TRIF)、干扰素调节因子3(IRF3)通路蛋白及相关炎症因子表达的影响。方法雄性C57BL/6小鼠80只,根据随机数字表法分为中药组、西药组、模型组和空白组,每组20只。中药组、西药组、模型组小鼠饮用3%葡聚糖硫酸钠(DSS)溶液7天,空白组小鼠正常饮水。造模成功后中药组以12.24 g/kg中药蒸馏水混悬液灌肠,西药组给予1.33 g/kg美沙拉秦灌肠剂灌肠,模型组、空白组给予等量生理盐水灌肠,每天1次,连续给药7天。记录小鼠体重、粪便性状及便血情况,计算疾病活动指数(DAI),检测血清白细胞介素18(IL-18)、白细胞介素10(IL-10)含量,采用Western Blot检测结肠组织TLR4、TRIF、IRF3通路蛋白表达及实时荧光定量PCR法检测mRNA水平。结果与空白组比较,模型组DAI升高(P<0.01),血清IL-10降低(P<0.01),IL-18升高(P<0.01),结肠组织TLR4、TRIF、IRF3蛋白及mRNA表达升高(P<0.01);与模型组比较,西药组、中药组结肠病理改善,DAI降低(P<0.01),血清IL-10升高(P<0.01),中药组IL-18降低(P<0.01),西药组TLR4蛋白、中药组TLR4、TRIF、IRF3蛋白表达降低(P<0.01),西药组、中药组TLR4、TRIF、IRF3mRNA表达降低(P<0.01);与西药组比较,中药组结肠病理改善,DAI降低(P<0.01),血清IL-10升高(P<0.01),IL-18降低(P<0.05),中药组TLR4、IRF3蛋白表达降低(P<0.05),TLR4、IRF3mRNA表达降低(P<0.05)。结论解毒活血灌肠方可下调TLR4、TRIF、IRF3信号通路,抑制促炎因子释放,增加抗炎因子水平,从而减轻肠道炎症反应,促进结肠黏膜修复。

Objective To observe the effect of Jiedu Huoxue Recipe(JDHXR) enema on expressions of Toll-like receptor 4(TLR4)/TIR-domain-containing adaptor inducing interferon-β (TRIF)/interferon regulatory factor 3(IRF3) signal pathway proteins and related inflammatory factors in mice with ulcerative colitis(UC). Methods Eighty male C57 BL/6 mice were divided into Chinese medicine group, Western medicine group, model group, and blank group by random digit table, 20 mice in each group. Mice in Chinese medicine group, Western medicine group, and blank group were administered with 3% sodium dextran sulfate(DSS) solution for 7 days, while mice in blank group drank water normally. After successful modeling, mice in Chinese medicine group were given 12.24 g/kg JDHXR enema. Those in Western medicine group were given 1.33 g/kg Mesalachin enema. And mice in the model group and the blank group were given the same amount of normal saline enema. All medication was performed once per day for 7 consecutive days. Body weight, fecal traits, and blood in stool of mice were recorded to calculate daily activity index(DAI). The levels of serum IL-18 and IL-10 and mRNA and protein expression levels of TLR4, TRIF, and IRF3 pathways in colon tissues were detected by Western Blot and real time fluorescence quantitative PCR. Results Compared with the blank group, DAI of model group increased(P<0.01), serum IL-10 decreased(P<0.01), IL-18 increased(P<0.01), protein and mRNA expressions of TLR4, TRIF, and IRF3 in colon tissue increased(P<0.01). Compared with the model group, the pathological colon conditions were improved, DAI decreased(P<0.01), and serum levels of IL-10 increased(P<0.01) in Western medicine and Chinese medicine group. IL-18 decreased(P<0.01) in Chinese medicine group. TLR4 protein decreased(P<0.01) in Western medicine group. The expressions of TLR4, TRIF, and IRF3 protein decreased(P<0.01) in Chinese medicine group. mRNA expressions of TLR4, TRIF, IRF3 decreased in both Western medicine and Chinese medicine group(P<0.01). Compared with Western medicine group, colon pathologies were improved, DAI decreased(P<0.01), serum IL-10 increased(P<0.01), IL-18 decreased(P<0.05), protein expressions of TLR4 and IRF3 decreased(P<0.05), and mRNA expressions of TLR4 and IRF3 decreased(P<0.05) in Chinese medicine group. Conclusion JDHXD enema down-regulated TLR4, TRIF, and IRF3 signaling pathways, inhibited the release of pro-inflammatory factors, and increased the levels of antiinflammatory factors, so as to reduce intestinal inflammatory response and promote colon mucosal repair.