基于TLR4/NF-κB分析“金汁”粪菌移植干预溃疡性结肠炎小鼠的机制

Based on TLR4/NF-κB Analyze the Mechanism of“Jinzhi”Fecal Microbiota Transplantation in Mice with Ulcerative Colitis

目的:基于TLR4/NF-κB探讨"金汁"粪菌移植(FMT)干预溃疡性结肠炎(UC)小鼠的机制。方法:选取29只野生型雄性清洁级C57BL/10小鼠为研究对象。随机选取7只作为对照组,其余22只大鼠采用葡聚糖酸钠诱导法建立野生型小鼠UC模型。将这22只大鼠分为造模对照组(n=12)和FMT干预组(n=10),分别给予PBS液、粪菌液灌肠干预处理。另选取5只野生型雄性清洁级C57BL/10小鼠作为粪菌移植供体来源。观察三组小鼠结肠组织TLR4、NF-κB(p65)表达水平,观察三组小鼠结肠组织中下游细胞因子表达水平,分析TLR4、NF-κB(p65)及下游各细胞因子水平与疾病活动指数(DAI)和组织病理评分(HPS)的关系。结果:干预前,对照组DAI低于造模对照组和FMT干预组(P<0.05),造模对照组与FMT干预组DAI比较,差异无统计学意义(P>0.05)。干预后,造模对照组DAI高于对照组和FMT干预组,FMT干预组高于对照组(P<0.05)。FMT干预组干预后DAI低于干预前(P<0.05)。造模对照组HPS高于对照组与FMT干预组,且FMT干预组高于对照组(P<0.05)。造模对照组结肠长度短于FMT干预组与对照组,FMT干预组短于对照组(P<0.05)。造模对照组TLR4、NF-κB(p65)、TNF-α、IL-6、IL-1、IL-8及Tfh均高于对照组和FMT干预组,FMT干预组均高于对照组(P<0.05)。FMT干预组Tfr水平高于造模对照组与对照组,造模对照组高于对照组(P<0.05);造模对照组Tfh/Tfr水平高于对照组与FMT干预组,对照组高于FMT干预组(P<0.05)。TLR4、NF-κB(p65)、TNF-α、IL-6、IL-1、IL-8、Tfh及Tfh/Tfr与DAI、HPS均呈正相关(P<0.05),Tfr与DAI、HPS均呈负相关(P<0.05)。结论:FMT可通过下调TLR4/NF-κB信号通路的表达,进而下调TNF-α、IL-6、IL-1、IL-8、Tfh及Tfh/Tfr表达水平,达到改善UC的临床症状。

Objective:To investigate the mechanism of“Jinzhi”fecal microbiota transplantation(FMI)in mice with ulcerative colitis(UC)based on TLR4/NF-κB.Method:A total of 29 wild-type male clean grade C57BL/10 mice were selected as the research objects.7 rats were randomly selected as the control group,and the other 22 rats were used to establish the wild-type mouse model of UC induced by dextran sulfate sodium.The 22 rats were divided into modeling control group(n=12)and FMT intervention group(n=10),and were treated with PBS solution and faecal bacteria solution enema,respectively.Another 5 wild-type male C57BL/10 mice were selected as the donor source of faecal bacteria transplantation.The expression levels of TLR4 and NF-κB(p65)in colon tissues of mice in the three groups were observed,and the expression levels of cytokines in the middle and downstream of colon tissues of mice in the three groups were observed,and the relationship between TLR4,NF-κB(p65),cytokines in the downstream and disease activity index(DAI)and hihiopathological score(HPS)was analyzed.Result:Before intervention,DAI in the control group was lower than those in the modeling control group and the FMT intervention group(P<0.05),and there was no significant difference in DAI between the modeling control group and the FMT intervention group(P>0.05).After intervention,DAI in modeling control group was higher than those of the control group and FMT intervention group,while FMT intervention group was higher than that of the control group(P<0.05).DAI in FMT intervention group after intervention was lower than that before intervention(P<0.05).The HPS of the modeling control group was higher than those of the control group and the FMT intervention group,and the FMT intervention group was higher than that of the control group(P<0.05).The colon length of the modeling control group was shorter than those of the FMT intervention group and control group,and that of the FMT intervention group was shorter than that of the control group(P<0.05).TLR4,NF-κB(p65),TNF-α,IL-6,IL-1,IL-8 and Tfh in modeling control group were higher than those in control group and FMT intervention group,while those in FMT intervention group were higher than those in control group(P<0.05).The Tfr level of FMT intervention group was higher than those of the modeling control group and control group,and that of the modeling control group was higher than that of the control group(P<0.05).The level of Tfh/Tfr in modeling control group was higher than those in control group and FMT intervention group,and that in control group was higher than that in FMT intervention group(P<0.05).TLR4,NF-κB(p65),TNF-α,IL-6,IL-1,IL-8,Tfh and Tfh/Tfr were positively correlated with DAI and HPS(P<0.05),while Tfr was negatively correlated with DAI and HPS(P<0.05).Conclusion:FMT can improve the clinical symptoms of UC by down-regulating the expression of TLR4/NF-κB signaling pathway,and then down-regulating the expression levels of TNF-α,IL-6,IL-1,IL-8,Tfh and Tfh/Tfr.