Trpv4基因编辑对小鼠肠道屏障及菌群-代谢微环境的影响

Effects of Trpv4 editing on intestinal barrier and flora-metabolic microenvironment in mice

摘要:

目的 瞬时受体电位香草酸4型(transient receptor potential vanilloid 4, TRPV4)是由Trpv4编码的非选择性阳离子通道，深度参与多器官系统的生理病理调控，但其突变对动物肠道及肠道菌群的综合影响机制尚未明确。本研究旨在探究Trpv4 exon 8 c.1491+1G>A突变对小鼠肠道屏障完整性及菌群-代谢微环境的调控作用，为解析宿主基因与肠道菌群的互作机制提供实验依据。方法 以实验室前期构建的Trpv4 exon 8 c.1491+1G>A基因编辑小鼠为研究对象，采用实时荧光定量PCR (quantitative real-time PCR, qPCR)和蛋白质印迹法(Western blotting, WB)技术检测肠组织中Trpv4 mRNA及TRPV4蛋白的表达水平；通过病理切片观察肠道组织结构变化；结合16S rRNA基因高通量测序分析肠道菌群结构差异；利用LC-MS非靶代谢组学技术检测粪便代谢物变化，并分析菌群与代谢物的相关性。结果 Trpv4基因编辑导致小鼠肠组织Trpv4 mRNA转录及TRPV4蛋白表达异常，引发肠道组织结构异常及肠屏障功能损伤。肠道菌群分析显示，基因编辑小鼠肠道菌群稳态失衡，拟杆菌门(Bacteroidota)相对丰度显著升高，芽孢杆菌门(Bacillota)与拟杆菌门比例(F/B)显著下降，葡萄球菌属(Staphylococcus)等常见共生菌丰度降低。代谢组学分析表明，Trpv4基因编辑小鼠存在脂质代谢紊乱及免疫相关代谢物异常，其中拟杆菌门相关类群与脂质相关代谢物呈正相关，而脱硫弧菌属(Desulfovibrio)、肠杆菌属(Enterobacter)等则与部分脂质代谢物及免疫相关代谢物呈负相关。结论 Trpv4 exon 8 c.1491+1G>A基因编辑小鼠肠道屏障受损，肠道菌群结构与代谢微环境发生改变。本研究为解析特定基因突变与肠道菌群的互作提供了基础数据，也为Trpv4相关疾病的诊断与治疗策略开发提供了理论支撑。

Abstract:

Objective Transient receptor potential vanilloid 4 (TRPV4), a non-selective cation channel, is deeply involved in the physiological and pathological regulation of multiple organ systems, while the comprehensive influencing mechanism of its mutation on animal intestines and intestinal flora is not clear. This study explored the regulatory effects of Trpv4 exon 8 c.1491+1G>A mutation on intestinal barrier integrity and flora-metabolic microenvironment in mice, aiming to provide an experimental basis for analyzing the interaction mechanisms between host genes and intestinal flora.Methods Trpv4 exon 8 c.1491+1G>A gene-edited mice previously constructed in our laboratory were taken as the research objects, and the expression levels of Trpv4 and TRPV4 in the intestinal tissue were determined by qPCR and Western blotting, respectively. Pathological sections were prepared for observation of the structural changes of the intestinal tissue. The 16S rRNA gene high-throughput sequencing was conducted to reveal the structural differences of intestinal flora. Non-targeted metabolomics based on LC-MS was employed to examine the changes of fecal metabolites, and the correlations between flora and metabolites were analyzed.Results Trpv4 editing led to the abnormal expression of Trpv4 and TRPV4 in the intestinal tissue of mice, which resulted in the structural abnormality of the intestinal tissue and the impairment of intestinal barrier function. In addition, the gene-edited mice exhibited an imbalance in intestinal flora, with significantly increased relative abundance of Bacteroidota, a significantly decreased Bacillota/Bacteroidota (F/B) ratio, and reduced abundance of common commensal bacteria such as Staphylococcus. Metabolomic analysis indicated that the gene-edited mice presented disordered lipid metabolism and abnormalities in immune-related metabolites. The abundance of Bacteroidota was positively correlated with lipid metabolites, while that of Desulfovibrio and Enterobacter was negatively correlated with lipid and immune metabolites.Conclusion Trpv4 exon 8 c.1491+1G>A gene-edited mice exhibited impaired intestinal barrier function, along with alterations in intestinal flora structure and the metabolic microenvironment. This study provides basic data for elucidating the interactions between specific gene mutations and the gut microbiota and offers theoretical support for the development of diagnostic and therapeutic strategies for Trpv4-related diseases.