苓桂术甘汤调控miR-140-5p/TLR4/NF-κB信号通路对溃疡性结肠炎大鼠肠黏膜屏障及免疫平衡的影响

Effect of Linggui Zhugan decoction on intestinal mucosal barrier and immune balance in rats with ulcerative colitis by regulating the miR-140-5p/Toll-like receptor 4/nuclear factor-kappa B signaling pathway

摘要

目的 探讨苓桂术甘汤调控miR-140-5p/Toll样受体4（toll like receptor 4，TLR4）/核转录因子-κB（nuclear factor-kappa B，NF-κB）信号通路对溃疡性结肠炎（ulcerative colitis，UC）大鼠肠黏膜屏障及免疫平衡的影响。 方法 取SD大鼠用2，4，6-三硝基苯磺酸（2,4,6-trinitrobenzenesulfonic acid solution，TNBS）诱导建立UC模型，随机分为5组：模型组、苓桂术甘汤低剂量组、苓桂术甘汤高剂量组、NC-miR-140-5p（miR-140-5p阴性对照）组、苓桂术甘汤高剂量组+miR-140-5p antagomir（miR-140-5p抑制剂）组，每组10只，另取10只大鼠作对照组，分组干预后检测大鼠结肠黏膜损伤指数（colonic mucosal injury index，CMDI）评分与结肠长度；透射电镜观察各组大鼠结肠黏膜屏障超微结构；流式细胞实验检测各组大鼠外周血Treg、Th17细胞比例及Treg/Th17比值；ELISA检测大鼠血清免疫炎症相关因子白细胞介素（interleukin，IL）-6、IL-17、IL-10、转化生长因子β1（transforming growth factor-β1，TGF-β1）水平；采用实时荧光PCR及免疫印迹法检测大鼠结肠黏膜组织miR-140-5p/TLR4/NF-κB信号相关因子表达。 结果 与对照组比较，模型组大鼠结肠黏膜屏障超微结构损伤严重，CMDI、Th17细胞比例、血清IL-6与IL-17水平、结肠黏膜组织TLR4 mRNA、蛋白表达与p-NF-κB p65/NF-κB p65显著升高（P<0.05），结肠长度、Treg细胞比例、Treg/Th17比值、血清IL-10与TGF-β1水平、结肠黏膜组织claudin-1、claudin-4蛋白表达与miR-140-5p表达显著降低（P<0.05）。与模型组比较，苓桂术甘汤低剂量组、苓桂术甘汤高剂量组大鼠结肠黏膜屏障超微结构损伤均减轻，CMDI、Th17细胞比例、血清IL-6与IL-17水平、结肠黏膜组织TLR4 mRNA、蛋白表达与p-NF-κB p65/NF-κB p65均降低（P<0.05），结肠长度、Treg细胞比例、Treg/Th17比值、血清IL-10与TGF-β1水平、结肠黏膜组织claudin-1、claudin-4蛋白表达与miR-140-5p表达均升高（P<0.05）；NC-miR-140-5p组大鼠各指标差异无统计学意义（P>0.05）；高剂量苓桂术甘汤作用更强。下调miR-140-5p可减弱高剂量苓桂术甘汤对UC模型大鼠各指标的作用。 结论 苓桂术甘汤可通过上调miR-140-5p而抑制TLR4/NF-κB信号激活，进而促使Treg/Th17免疫平衡向Treg偏移，抑制免疫炎症，减轻UC大鼠肠黏膜屏障损伤。

Abstract

Objective To investigate the effect of Linggui Zhugan decoction on intestinal mucosal barrier and immune balance in rats with ulcerative colitis（UC） by regulating the miR-140-5p/Toll like receptor 4（TLR4）/nuclear factor-kappa B（NF-κB） signaling pathway. Methods Sprague-Dawley rats were induced with 2，4，6-trinitrobenzene sulfonic acid to establish a model of UC，and then the rats were randomly divided into model group，low-dose Linggui Zhugan decoction group，high-dose Linggui Zhugan decoction group，NC-miR-140-5p（miR-140-5p negative control） group，and high-dose Linggui Zhugan decoction group+miR-140-5p antagonist（miR-140-5p inhibitor） group，with 10 rats in each group. Another 10 rats were selected as control group. After grouping and intervention，colonic mucosal injury index（CMDI） score and colon length were measured for each group； transmission electron microscopy was used to observe the ultrastructure of intestinal mucosal barrier；flow cytometry was used to measure the percentages of Treg and Th17 cells in peripheral blood and Treg/Th17 ratio；ELISA was used to measure the serum levels of immune inflammatory factors such as interleukin-6（IL-6），interleukin-17（IL-17），interleukin-10（IL-10），and transforming growth factor-β1（TGF-β1） in rats； quantitative real-time PCR and Western blotting were used to measure the expression of the factors associated with the miR-140-5p/TLR4/NF-κB signaling pathway in the colonic mucosal tissue of rats. Results Compared with the control group，the model group had severe damage of the ultrastructure of colonic mucosal barrier and significant increases in CMDI，the percentage of Th17 cells，the serum levels of IL-6 and IL-17，the mRNA and protein expression levels of TLR4 in colonic mucosal tissue，and p-NF-κB p65/NF-κB p65（P<0.05），as well as significant reductions in colon length，the percentage of Treg cells，Treg/Th17 ratio，the serum levels of IL-10 and TGF-β1，the protein expression levels of claudin-1 and claudin-4，and the expression of miR-140-5p in colonic mucosal tissue（P<0.05）. Compared with the model group，the low- and high-dose Linggui Zhugan decoction groups had alleviation of the damaged ultrastructure of colonic mucosal barrier and significant reductions in CMDI，the percentage of Th17 cells，the serum levels of IL-6 and IL-17，the mRNA and protein expression levels of TLR4 in colonic mucosal tissue，and p-NF-κB p65/NF-κB p65（P<0.05），as well as significant increases in colon length，the percentage of Treg cells，Treg/Th17 ratio，the serum levels of IL-10 and TGF-β1，the protein expression levels of claudin-1 and claudin-4，and the expression of miR-140-5p in colonic mucosal tissue（P<0.05）. The NC-miR-140-5p group had no significant changes in these indicators（P>0.05），and high-dose Linggui Zhugan decoction had a stronger effect. Downregulation of miR-140-5p could weaken the effect of high-dose Linggui Zhugan decoction on various indicators in a rat model of UC. Conclusion Linggui Zhugan decoction can inhibit TLR4/NF-κB signal activation by upregulating miR-140-5p，thereby promoting the shift of Treg/Th17 immune balance to Treg，inhibiting immune inflammation，and alleviating intestinal mucosal barrier damage in UC rats.

关键词

苓桂术甘汤 / miR-140-5p/TLR4/NF-κB / 溃疡性结肠炎 / 肠黏膜屏障 / 免疫平衡

Key words

Linggui Zhugan decoction / miR-140-5p/Toll-like receptor 4/nuclear factor-kappa B / ulcerative colitis / intestinal mucosal barrier / immune balance

基金资助

国家自然科学基金资助项目(81573990)