Piezo蛋白介导机械刺激调控黏液屏障在IBS肠通透性改变中的作用研究

Our previous study confirmed that the increased intestinal permeability was the important pathophysiological mechanism of irritable bowel syndrome (IBS).Intestinal mucus barrier was the crucial component of intestinal barrier function. The maintenance of intestinal mucus barrier function was closely related to the biosynthesis, secretion and depolymerization of hydration of mucin from the intestinal goblet cells. The regulation of the latter was mainly dependent on the intestinal microenvironment encountered by the goblet cells. Previous study indicated that the mechanical microenvironment could regulate the secretion and synthesis of mucus by affecting the function of epithelial cells. Mechanical sensitive Piezo protein was the key ion channel to feeling the mechanical stimulation, we found it expressed in intestinal goblet cells and was closely related to the secretion of mucin. Therefore, whether the Piezo protein-mediated mechanical stimulation regulated the mucus barrier through affecting the synthesis, secretion and depolymerization of hydration of mucin from the intestinal goblet cells and the related mechanism were the main focus of this project. We will intervene in Piezo protein and the downstream key signaling pathways such as Ras - MAPK/Erk - AP1 PLC - IP3 / DAG - Ca2 + - Syt2 and ATP - ADO - CFTR - HCO3 - by in vitro and in vivo methods to confirm the pivotal role of Piezo protein mediated mechanical stimulation in the regulation of mucus barrier. Our study will further clarity the core mechanism piezo protein participates in the changes of intestinal permeability by regulating the mucus barrier, and provide new therapeutic target for IBS.

我们前期发现肠通透性增加是IBS重要病理生理机制。肠黏液屏障是肠屏障功能重要组成部分，其功能的维持与肠杯状细胞中黏蛋白合成、分泌和水合解聚密切相关，而后者的调节主要依赖杯状细胞所处肠道微环境。研究提示机械微环境可通过影响上皮细胞功能来调节黏液分泌和合成。机械敏感Piezo蛋白是感受机械刺激的关键离子通道，我们发现Piezo蛋白在肠杯状细胞中表达，与黏蛋白分泌有关。因此，Piezo蛋白介导的机械刺激是否通过影响杯状细胞黏蛋白合成、分泌和水合解聚来调控黏液屏障以及机制是本课题研究重点。我们通过离体和在体干预Piezo以及下游关键信号通路Ras-MAPK/Erk-AP1、PLC-IP3/DAG-Ca2+-Syt2和ATP-ADO-CFTR-HCO3-，证实Piezo介导的机械刺激在黏液屏障调控中的关键作用,明确Piezo通过调控黏液屏障参与IBS肠通透性改变的核心机制，为IBS治疗提供新的靶点。

研究背景：肠粘液层在保护肠道免受机械、化学和生物攻击方面起着至关重要的作用，它有助于维持肠道稳态。WAS模型小鼠结肠粘液表达的减少伴随着肠蠕动异常。但其具体机制仍不明确，Mucin2是结肠粘液屏障的核心成分，主要由肠道杯状细胞合成和分泌。先前的研究表明，这一过程受到多种因素调节，如神经递质和免疫信号。但肠道微环境中丰富的机械因素对杯状细胞的影响尚不清楚。Piezo1是一类能感知机械刺激的机械敏感性离子通道并在杯状细胞中高表达。因此推测Piezo1可能通过影响杯状细胞黏液分泌功能从而参与黏液屏障的调节。因此本项目探究Piezo1是否以及如何通过调控肠黏液屏障参与IBS的发生发展。研究方法和内容：利用LS174T细胞在体外构建肠黏液屏障模型，采用慢病毒低表达/过表达Piezo1基因，测定黏液屏障功能、黏蛋白Mucin2和相关信号通路分子表达。利用Piezo1通道抑制剂/激动剂作用于体内外肠黏液屏障，检测黏液屏障功能等指标。探究急性炎症等模型下Piezo1对黏液屏障的调节。研究结果：Piezo1在肠道杯状细胞的细胞膜上高表达，而Piezo2几乎不表达，因此Piezo1更可能直接参与调控黏液屏障。在体外LS174T细胞中，Piezo1通道活性可调节肠黏液屏障功能。Piezo1蛋白表达与LS174T细胞黏液屏障功能和黏蛋白Mucin2的表达量成正相关。体外给予剪切力和静水压可促进杯状细胞表达Mucin2。Piezo1正向调节Erk/Ca 2+和负向调节H3K9me3分子表达，从而影响Mucin2的表达。因此，Piezo1感知机械刺激，并介导其影响Mucin2的表达。在体模型中，肠杯状细胞Piezo1通道活性直接正向调控黏液屏障功能。我们成功构建了Piezo1肠道杯状细胞特异性敲除小鼠，模型鼠表现为杯状细胞数目下降、黏液层厚度下降、黏液屏障的通透性增加和对DSS诱导的结肠炎更易感。研究结论：Piezo1在肠杯状细胞中高表达，介导机械刺激调控Mucin2的表达，从而影响肠黏液屏障。该调控机制与Erk/Ca 2+和H3K9me3通路密切相关。本研究的科学意义是：揭示了Piezo1参与肠道黏液屏障功能破坏的机制，使得当前对生理和疾病状态下肠黏液屏障的调节的研究具有更加深入的意义。为将来研究Piezo1蛋白在疾病损伤环境下对肠道黏液屏障修复的影响及应用打下坚实的基础。