粪菌移植对小鼠实验性结肠炎TLR4信号通路及肠黏膜屏障的影响

目的探讨粪菌移植(FMT)对溃疡性结肠炎(UC)小鼠肠黏膜屏障的影响及可能机制。方法小鼠饮用2.0%葡聚糖硫酸钠(DSS)溶液构建小鼠UC模型;50只成年雄性C57BL/6J小鼠,随机留取10只取粪便(这10只不参与后续的实验),其余40只称重、编号,随机分为空白对照组(Con组)、DSS模型对照组(Model组)、美沙拉嗪组(Model+5-ASA组)和粪菌液组(Model+FMT组),每组10只,Con组和Model组均给予0.9%NaCl溶液灌肠,给药组分别给予美沙拉嗪、粪便滤液灌肠;评估疾病活动指数(DAI)、各组结肠组织病理情况,用透射电镜检测各组小鼠的结肠黏膜上皮细胞结构的变化情况,ELISA检测各组血清内毒素、炎症因子TNF-α水平变化,免疫组化法检测结肠组织Toll样受体4(TLR4)及核因子-κB(NF-κB)的表达变化,Western blot检测各组ZO-1蛋白表达。结果与Model组相比,粪菌移植明显改善小鼠的DAI指数和结肠组织的病理损伤,结肠上皮细胞间隙增宽程度减轻,腺上皮细胞间连接较紧密,结肠黏膜上皮细胞微绒毛完整,排列整齐,内毒素、TNF-α的含量明显下降,TLR4及NF-κB在结肠组织的表达明显下降,ZO-1蛋白表达明显升高,促进结肠黏膜屏障的修复,差异具有统计学意义(t=7.9543,P<0.0001;t=3.7641,P=0.0010;t=4.5899,P=0.0020;t=13.2886,P<0.0001;t=4.9750,P=0.0010;t=6.9388,P<0.0001;t=8.3744,P<0.0001)。结论FMT可减少内毒素及炎症因子的产生,改善结肠炎症,TLR4-NF-κB信号通路可能是FMT修复结肠黏膜屏障功能的机制之一。

Effects of fecal microbiota transplantation on TLR4 signaling pathway and intestinal mucosal barrier in mouse model of experimental colitis

Objective To explore the effect and possible mechanisms of fecal microbiota transplantation(FMT)on intestinal mucosal barrier in mice with ulcerative colitis(UC).Methods Mice were given 2.0%DSS solution to construct mouse models of UC.50 adult male C57 BL/6 J mice were randomly selected to take 10 stool samples(these 10 did not participate in subsequent experiments),and the remaining 40 were weighed,numbered,and randomly divided into the blank control group(Con group),DSS model control group(Model group),mesalazine group(Model+5-ASA group),or fecal fluid group(Model+FMT group),receiving enema with 0.9%NaCl solution,0.9%NaCl solution,Mesalazine and fecal filtrate respectively.The disease activity index(DAI)and pathological conditions of colon tissue in each group were evaluated;the changes of colonic mucosal epithelial cell structure of the mice in each group were detected with transmission electron microscopy;ELISA was performed to test the changes in serum endotoxin and level of inflammatory factor TNF-αin each group;the expression of TLR4 and NF-κB in colon tissue was detected using histochemical method,and the expression of ZO-1 protein was detected using Western blot.Results Compared with the Model group,FMT significantly improved the DAI index of the mice and the pathological damage of the colon tissue;the width of the colonic epithelial cells was reduced,the connection between the glandular epithelial cells was tight,and the microvilli of the colonic mucosal epithelial cells were intact and arranged neatly;the contents of endotoxin and TNF-αdecreased significantly,the expression of TLR4 and NF-κB in colon tissue decreased significantly,and the expression of ZO-1 protein increased significantly,which promoted the repair of colonic mucosal barrier.The difference was statistically significant(t=7.9543,P<0.0001;t=3.7641,P=0.0010;t=4.5899,P=0.0020;t=13.2886,P<0.0001;t=4.9750,P=0.0010;t=6.9388,P<0.0001;t=8.3744,P<0.0001).Conclusion FMT can reduce the production of endotoxin and inflammatory factors and improve colon inflammation.TLR4-NF-κB signaling pathway may be one of the mechanisms for FMT to repair the function of colonic mucosal barrier.