四君子汤合痛泻要方对感染后肠易激综合征肝郁脾虚证小鼠肠道免疫及黏膜屏障功能的影响

Effects of Sijunzi Decoction and Tongxie Yaofang on intestinal immunity and mucosal barrier function of PI-IBS mice with liver depression and spleen deficiency syndrome

[目的]探讨四君子汤合痛泻要方对感染后肠易激综合征(PI-IBS)肝郁脾虚证小鼠肠道免疫及黏膜屏障功能的影响。[方法]将50只雄性NIH小鼠随机分为空白组、感染2周组、感染8周组、2周治疗组、8周治疗组,每组10只。除空白组外,其余各组小鼠采用慢性束缚应激+过度疲劳+饮食失节+旋毛虫感染建立PI-IBS肝郁脾虚证模型,2周治疗组、8周治疗组分别在感染旋毛虫2周及8周后给予四君子汤合痛泻要方灌胃治疗6周。观察小鼠一般状况并对小鼠肝郁脾虚症状进行评价;通过腹壁回撤反应(AWR)评分评估小鼠内脏敏感性变化;比较各组小鼠粪便含水百分数变化;取末端回肠及近端结肠组织,苏木精-伊红(HE)染色观察小鼠肠道炎症情况;酶联免疫吸附实验(ELISA)测定小鼠血清中白细胞介素-10(IL-10)、干扰素(IFN-γ)、白介素-17(IL-17)细胞因子水平;逆转录-聚合酶链反应法(RT-PCR)检测结肠组织中IL-10、IFN-γ、IL-17的mRNA表达;蛋白免疫印迹法(Western blot)检测结肠组织中闭合蛋白(occludin)、紧密粘连蛋白-1(ZO-1)的蛋白表达。[结果]感染2周组、感染8周组小鼠出现肝郁脾虚症状,AWR评分及粪便含水百分数较空白组均显著增高(P<0.05)。感染2周组回肠及结肠组织可见明显炎性损伤,感染8周组肠道组织炎症基本恢复至正常。两组小鼠血清及结肠组织中IFN-γ、IL-17的含量及mRNA表达明显升高,IL-10的含量及mRNA表达显著降低(P<0.05),occludin、ZO-1的蛋白表达明显减少(P<0.05)。2周治疗组、8周治疗组小鼠AWR评分及粪便含水百分数较治疗前显著改善(P<0.05),肝郁脾虚症状明显减轻。2周治疗组小鼠结肠组织炎症损伤较治疗前明显改善。两组小鼠血清及结肠组织中IFN-γ、IL-17的含量及mRNA表达较治疗前明显降低,IL-10的含量及mRNA表达明显升高(P<0.05),同时明显上调了occludin、ZO-1的蛋白表达(P<0.05)。[结论]四君子汤合痛泻要方对肝郁脾虚型PI-IBS小鼠急性及慢性感染期均具有明显的治疗作用,其机制可能是通过调节肠道细胞因子免疫失衡,恢复肠黏膜紧密连接蛋白表达水平,保护肠黏膜屏障功能,降低肠黏膜通透性。

[Objective]To investigate the effects of Sijunzi Decoction combined with Tongxie Yaofang on intestinal immunity and mucosal barrier function in mice with irritable bowel syndrome(PI-IBS)of liver depression and spleen deficiency type.[Methods]Fifty male NIH mice were randomly divided into blank group,2-week infection group,8-week infection group,2-week treatment group and 8-week treatment group,with 10 mice in each group.In addition to the blank group,the other groups were used to establish PI-IBS model of liver depression and spleen deficiency type by chronic restraint stress,excessive fatigue,diet disorder and trichinella infection.The treatment groups were given Sijunzi Decoction and Tongxieyaofang for 6 weeks after 2 and 8 weeks of infection with trichinella spiralis respectively.The general condition of mice was observed and the symptoms of liver depression and spleen deficiency were evaluated;the visceral sensitivity was evaluated by abdominal wall withdrawal response(AWR)score;the intestinal motility was evaluated by calculating the percentage of water in feces;the intestinal inflammation was observed by HE staining in terminal ileum and proximal colon tissue;IL-10,IFN-γand IL-17 cell factors in serum were determined by ELISA.RT-PCR was used to detect the mRNA expression of IL-10,IFN-γand IL-17,and Western blot was used to detect the protein expression of occludin and ZO-1.[Results]The symptoms of liver depression and spleen deficiency were found in the 2-week and 8-week-old infection groups,and the AWR score and fecal water content percentage were significantly higher than those in the blank group(P<0.05).In the 2-week group,the ileum and colon tissue showed obvious inflammatory injury,while in the 8-week group,the inflammation of intestinal tissue returned to normal.The content and mRNA expression of IFN-γand IL-17 in serum and colon tissue of the two groups were significantly increased,the content and mRNA expression of IL-10 were significantly decreased(P<0.05),and the protein expressions of occludin and ZO-1 were significantly decreased(P<0.05).AWR score and fecal water content percentage of mice in 2-week and 8-week treatment groups were significantly improved(P<0.05),and symptoms of liver depression and spleen deficiency were significantly reduced.The inflammatory injury of colon tissue in 2-week treatment group was significantly improved compared with that before treatment.The content and mRNA expression of IFN-γand IL-17 in serum and colon tissue of the two groups were significantly lower than those before treatment,while the content and mRNA expression of IL-10 were significantly increased(P<0.05),and the protein expressions of occludin and ZO-1 were significantly up-regulated(P<0.05).[Conclusion]Sijunzi Decoction combined with Tongxie Yaofang has obvious therapeutic effect on acute and chronic infection of PI-IBS mice with liver depression and spleen deficiency.The mechanism may be through regulating cellular immune imbalance,restoring the expression of tight junction protein in intestinal mucosa,protecting intestinal mucosal barrier function and reducing intestinal mucosal permeability.