Piezo介导机械信号调控肠上皮通透性在IBS发病中的作用

Our previous studies confirmed that increased intestinal epithelial permeability was the important pathogenesis of irritable bowel syndrome. Dysfunction of intracellular junction was proved a key to induce the increase of intestinal permeability. However, the mechanism remains unclear. Studies showed that intestinal mechanical microenvironment including strain, shear and intraintestinal pressure significantly influence intracellular junction, which played an important role in regulation of intracellular junction and intestinal epithelial permeability. Piezo was proved a vital mechanical sensitive ion channel, which was of vital importance to regulate intracellular junction. Our team firstly discovered piezo expressed in intestinal epithelium cells. Our studies also indicated the piezo protein of the intestinal epithelium increased in the irritable bowel syndrome (IBS) animal model, which was positive correlation with the increased intestinal epithelial permeability and negative correlation with tight junction proteins. Therefore, we presumed that piezo protein took part in regulation of intestinal epithelial permeability by regulation of intracellular junction. Therefore, whether and how piezo protein participated in regulation of intestinal epithelium permeability was the topic aim in this study. We will perform scientific experiments in vitro and in vivo by specific intervention of piezo and related molecular pathway ( RhoA/ROCK-MLCK- actinRas- Erk/ MAPK) to prove the function and mechanism of piezo in regulation of intestinal permeability. Which will give us an advanced understanding of mechanism of increased intestinal epithelial permeability in pathogenesis of IBS, and will present therapeutic target of IBS.

我们发现肠上皮通透性增加是IBS重要病理生理机制，并证实肠上皮细胞连接障碍导致肠上皮通透性增加，但其机制仍不明确。研究发现肠道机械微环境如张应力、剪应力和肠腔内压力明显影响肠上皮细胞间连接，在肠上皮通透性调控中至关重要。Piezo是感受机械刺激关键机械敏感离子通道，在调节细胞间连接中起核心作用。我们首次发现Piezo在肠上皮细胞中表达，IBS模型鼠中肠上皮Piezo表达增加，与肠上皮通透性增加正相关，与肠上皮紧密连接蛋白表达负相关，我们推测Piezo可能通过影响肠上皮细胞间连接来调控肠上皮通透性。因此，Piezo是否以及如何调控肠上皮通透性是本课题研究重点。我们将通过离体和在体干预Piezo以及下游关键信号通路RhoA/ROCK-MLCK-Actin、Ras-Erk/MAPK，证实其在肠上皮通透性调控中的关键作用，明确IBS肠上皮通透性增加的机制，为IBS治疗提供新的靶点。

研究背景：广泛研究报道，肠上皮通透性增加是IBS重要病理生理过程，但其具体机制仍不明确。Piezo是肠上皮细胞中感受机械刺激关键机械敏感离子通道，此前已发现Piezo在IBS模型鼠肠道中表达增加，与肠上皮通透性增加正相关，与肠上皮紧密连接蛋白表达负相关，因此推测Piezo可能通过影响肠上皮细胞间连接来调控肠上皮通透性。因此本项目探究Piezo是否以及如何调控肠上皮通透性，以及在IBS发生中作用。研究方法：利用Caco-2细胞在体外构建肠上皮屏障模型，采用慢病毒低表达/过表达Piezo1基因，测定屏障功能、紧密连接蛋白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上皮屏障，检测屏障功能、细胞增殖修复等指标。探究急性炎症等模型下Piezo1对上皮屏障的调节。研究结果：Piezo1在肠道上皮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肠上皮通透性。在体外Caco-2细胞上皮模型中，Piezo1通道活性可调节肠上皮屏障功能。Piezo1蛋白表达与Caco-2细胞上皮的屏障功能负相关，与紧密连接蛋白claudin-1的表达量成负相关。Piezo1负向调节ROCK1/2通路分子表达，从而影响claudin-1的表达。而Piezo1对肠上皮细胞Erk通路无明显调节作用。此外Piezo1负向影响上皮细胞增殖，但正向促进细胞迁移修复。Piezo1感知机械刺激，并介导其影响claudin-1和occludin蛋白的表达。在体模型中， Piezo1通道活性直接负向调控上皮屏障功能。在DSS炎性模型中，Piezo1抑制剂 GsMTx4可部分缓解炎症相关指标。我们成功构建了Piezo1肠道特异性敲除小鼠，在DSS结肠炎时的炎症反应较轻。研究结论： Piezo1在肠上皮细胞中，介导机械刺激调控紧密连接蛋白claudin-1表达，从而影响肠上皮通透性。该调控机制与ROCK1/2通路密切相关。此外Piezo1可调节上皮细胞增殖迁移。Piezo1活性还可快速影响肠上皮通透性，其抑制剂可显著缓解肠道炎症改变。本研究的科学意义是：揭示了Piezo1参与肠道上皮屏障功能破坏的机制，使得当前对生理和疾病状态下肠粘膜上皮屏障的调节的研究具有更加深入的意义。为将来研究Piezo1在疾病损伤环境下对肠道上皮修复的影响打下坚实的基础。