基于肠道菌群研究健脾清肠法调控TLRs/NF-κB通路抑制化疗致肠黏膜炎作用

Intestinal microflora and TLRs/NF-κ B signal pathway have become research hotspots in the pathogenesis of gastrointestinal mucositis, while regulated targets between the concrete changes of Intestinal microflora with gastrointestinal mucositis are not known yet. Past studies have confirmed that the methods of invigorating spleen and cleaning intestine can significantly relieve the intestinal mucosa injury and down-regulate the expression of NF-κ B in intestinal tissue. So we combine it with the mechanism that basing on the intestinal epithelial cell damage pathologically, causing intestinal dysbacteriosis, then changing TLRs/NF-κB signal pathway, and transporting by competing expression with inflammatory cytokine. Then we put forward the hypothesis which the methods of invigorating spleen and cleaning intestine could regulating intestinal microflora and TLRs/NF-κB signal pathway-mediating inflammatory effect. By using high-throughput sequencing, inflammasome, morphology and molecular biology technology to test intestinal microflora, intestinal mucosal barrier, signaling pathways, then both in vivo and vitro, we make sure that the TLRs/NF- B signal pathway is regulated by the methods of invigorating spleen and cleaning intestine and discuss the relationship between the regulation with changes of intestinal microflora. This research further reveal the mechanism of the methods of invigorating spleen and cleaning intestine and provide Chinese medicine treatment targets for gastrointestinal mucositis induced by chemotherapy.

肠道菌群和TLRs/NF-κ B已成为肠黏膜炎发病机制的研究热点，然而肠道菌群的种类变化与调控黏膜炎的作用靶点尚未明确。既往研究证实健脾清肠法能显著改善肠黏膜损伤，调节肠道菌群，下调NF-κ B表达，结合“化疗引起肠上皮细胞损伤和肠道菌群失调，激活TLRs/NF-κB信号通路，导致促炎细胞因子和抗炎细胞因子的竞争性应答，引起肠黏膜炎症。”机制，提出健脾清肠法能“调节肠道菌群和调控TLRs/NF-κB信号通路，抑制炎症效应”的假说。鉴此，本项目采用高通量测序、炎症复合体检测、组织形态学等技术对肠黏膜炎大鼠从肠道菌群、肠黏膜屏障、信号通路等方面，明确健脾清肠法对TLRs/NF-κB信号通路的调控，探讨该作用与肠道菌群变化的关系。并在体外模拟上述应激条件，阐述健脾清肠法对TLRs/NF-κB信号通路主要靶点调控的作用机制，为化疗致肠黏膜炎提供中药治疗靶点。

目的：构建肠黏膜炎大鼠模型及体外炎症模型，从肠道菌群、肠黏膜屏障等方面，明确健脾清肠法对TLRs/NF-κB信号通路的调控，探讨该作用与肠道菌群变化的关系，并阐述健脾清肠法对TLRs/NF-κB信号通路主要靶点调控的作用机制，为化疗致肠黏膜炎提供中药治疗靶点。.方法：采用第二代高通量测序技术分析脾清肠法对化疗致肠黏膜炎大鼠肠道菌群中相关有益菌和致病菌的分布变化；用ELISA和组织病理学、分子生物学技术进行肠屏障损害指标检测；免疫组化检测小肠组织中紧密连接蛋白Occludin蛋白的表达，ELISA法检测各组大鼠血清TNF-α、IL-10表达，Western blot法检测肠组织中MyD88、p65与TLRs含量。采用Caco-2肠上皮屏障功能损害的炎症模型，给予健脾解毒方调节，观察体外模型的TLRs、NF-κB、MyD88、IL-6、TNF-α表达。.结果：肠黏膜炎模型体内试验显示，与空白组比较，模型组Occludin蛋白表达量减少（P＜0.01）；与模型组比较，健脾解毒方各剂量组Occludin蛋白表达量均增加，其中以健脾解毒方高剂量组增加最显著（P＜0.01）；模型组大鼠血清TNF-α表达高于空白组（P＜0.05），各治疗组的TNF-α均低于模型组，且健脾解毒方中、高剂量组的TNF-α低于健脾解毒方低剂量组（P＜0.05）；模型组大鼠的IL-10低于空白组（P＜0.05），健脾解毒方高剂量组、培菲康组高于模型组（P＜0.05）；模型组MyD88、p65、TLR4蛋白表达高于空白组（P＜0.01），健脾解毒方中、高剂量组、培菲康组低于模型组，且健脾解毒方中、高剂量组的蛋白表达低于健脾解毒方低剂量组（P＜0.01）。肠黏膜炎模型体外试验显示，健脾解毒组与模型组相比，NF-κB、TLR4、IL-6、TNF-α、p-65mRNA蛋白表达低于模型组（P＜0.01），且健脾解毒方组与模型组相比上皮细胞骨架结构更为完整。.结论：健脾解毒方能够抑制TLRs/NF-κB信号通路，调节炎性相关因子，减轻5-FU所致大鼠肠道黏膜炎症，健脾清肠法能调节肠道菌群和调控 TLRs/NF-κB 信号通路，抑制炎症效应。