Faecalibacterium prausnitzii-derived microbial anti-inflammatory molecule regulates intestinal integrity in diabetes mellitus mice via modulating tight junction protein expression
普拉梭菌活性产物-微生物抗炎分子通过调控细胞紧密连接蛋白表达修复糖尿病小鼠肠道屏障
Funding information: National Natural Science Foundation of China, Grant/Award Number: 81370475; Natural Science Foundation of Guangdong Province, Grant/Award Number: 2017A030313647
Abstract
enBackground
Impaired intestinal barrier structure and function have been validated as an important pathogenic process in type 2 diabetes mellitus (T2DM). Gut dysbiosis is thought to be the critical factor in diabetic intestinal pathogenesis. As the most abundant commensal bacteria, Faecalibacterium prausnitzii (F. prausnitzii) play important roles in gut homeostasis. The microbial anti-inflammatory molecule (MAM), an F. prausnitzii metabolite, has anti-inflammatory potential in inflammatory bowel disease (IBD). Thus, we aimed to explore the function and mechanism of MAM on the diabetic intestinal epithelium.
Methods
16S high-throughput sequencing was used to analyze the gut microbiota of db/db mice (T2DM mouse model). We transfected a FLAG-tagged MAM plasmid into human colonic cells to explore the protein-protein interactions and observe cell monolayer permeability. For in vivo experiments, db/db mice were supplemented with recombinant His-tagged MAM protein from E. coli BL21 (DE3).
Results
The abundance of F. prausnitzii was downregulated in the gut microbiota of db/db mice. Immunoprecipitation (IP) and mass spectroscopy (MS) analyses revealed that MAM potentially interacts with proteins in the tight junction pathway, including zona occludens 1 (ZO-1). FLAG-tagged MAM plasmid transfection stabilized the cell permeability and increased ZO-1 expression in NCM460, Caco2, and HT-29 cells. The db/db mice supplemented with recombinant His-tagged MAM protein showed restored intestinal barrier function and elevated ZO-1 expression.
Conclusions
Our study shows that MAM from F. prausnitzii can restore the intestinal barrier structure and function in DM conditions via the regulation of the tight junction pathway and ZO-1 expression.
摘要
zh背景
肠道屏障结构和功能异常是 2 型糖尿病的重要病理改变之一。 而肠道菌群失调被认为是诱发糖尿病肠道病变的关键因素。 普拉梭菌作为肠道中最为常见的共生菌, 在维持肠道内环境稳态中扮演着重要的角色。前沿研究发现, 源于普拉梭菌的活性产物:微生物抗炎分子(MAM)对炎症性肠病具有炎症抑制潜能。因此, 我们在本研究中探索 MAM 在糖尿病肠道上皮中的功能及机制。
方法
我们运用 16S 高通量测序分析 db/db 小鼠(2 型糖尿病小鼠模型)的肠道菌群。其次, 通过合成 FLAG 标记的 MAM 质粒, 并进行结肠上皮细胞转染, 以探索蛋白-蛋白互作机制并观测细胞间通透性改变情况。在体内实验中, 我们构建起 E.coli BL21(DE3)细菌蛋白表达系统用于合成 His 标签重组 MAM 蛋白。运用所合成提纯的重组 MAM 蛋白对 db/db 小鼠进行肠道干预, 观察体内干预后肠道上皮屏障结构和功能改善情况。
结果
db/db 小鼠肠道内普拉梭菌丰度显著下降。免疫沉淀联合蛋白质谱分析提示 MAM
可能与细胞紧密连接通路相关蛋白相互作用, 其中一个可能的靶点为 ZO-1。经过 FLAG 标记的MAM 质粒转染后, NCM460、Caco2 及 HT-29 肠道细胞的肠道屏障功能增强, 伴随着 ZO-1表达量的上调。与之相对应的, db/db 小鼠经过 His 标签 MAM 重组蛋白肠道干预后, 显示其肠道屏障功能得到改善, 并且 ZO-1 的表达量也有明显提升。
结论
我们的研究表明来源于普拉梭菌的 MAM 可通过调控紧密连接通路及ZO-1表达, 修复糖尿病状态下受损的肠道屏障及功能。
