细胞色素P450酶诱导EET激活气道TRPV4通道参与哮喘发病的机制研究

The pathophysiological changes on airway epithelial and smooth muscle cell play central role in the development of asthma. However, the crosstalk between two cell types remains unclear. We previously show the upregulation of TRPV4 in the asthmatic epithelium, which is responsible for impaired airway barrier function and inflammation. Meanwhile, stimulation of allergen on epithelial cell results in elevated induction of EET by enhanced expression of cytochrome P450 epoxygenase (CYP). We further identify an epithelium dependent, TRPV4 sensitive migration and proliferation in smooth muscle, which might be regulated by epithelium derived EET. Thus, we propose the scientific hypothesis that allergen triggers EET release by increasing CYP to activate TRPV4 on both epithelial and smooth muscle cell, contributing to the development of asthma. We aim to evaluate the coupling mechanism between epithelial and smooth muscle cell, the expression mediating pathway in epithelial cell, along with the reaction system of channel complex in smooth muscle based on integrated-tissue-cellular-organelle dimensions using multiple human primary cells, targeted genetic deletion mice and various technologies, including patchy clamp, Ca2+ sparks, myograph and mice lung function test. This project would identify the pivotal role of airway TRPV4 in asthma, which might shed new insights for the prevention and treatment of asthma.

气道上皮与平滑肌功能的病理生理学改变是哮喘发病的核心环节，但二者协同偶联机制不明。我们前期发现哮喘患者气道上皮TRPV4表达增加并参与调控气道屏障及炎症。过敏原刺激可诱导上皮细胞表达细胞色素P450酶（CYP），增高其代谢产物EET分泌。进一步研究提示气道平滑肌存在哮喘上皮依赖、TRPV4敏感的迁移增殖活性，而上皮来源的EET可能介导了这一过程。我们提出本项目的科学假说：过敏原通过上调上皮关键酶CYP表达，增高EET分泌，偶联激活上皮细胞及平滑肌细胞的TRPV4通道，诱导哮喘各种病理生理学改变。本研究拟引入多种人原代细胞和靶向敲除动物，运用膜片钳、钙火花、Myograph及小鼠肺功能等技术，在整体-器官-细胞-亚细胞水平多维度探讨上皮与平滑肌通过CYP-EET的偶联机制、上皮TRPV4的调控机制以及下游平滑肌通道复合体的效应机制。本项目将为有效防治哮喘提供新思路，为开发新药提供有力依据。

气道上皮与平滑肌功能的病理生理学改变是哮喘发病的核心环节，但二者的协同偶联机制尚不明确。瞬时受体电位（TRP）通道是非选择性阳离子通道，可被多种理化因素激活。前期研究提示，TRPV4参与哮喘气道上皮钙离子调控与上皮通透性调控；进一步研究发现，细胞色素P450酶（CYP）诱导的EET可能同时偶联激活气道上皮细胞及平滑肌细胞的TRPV4通道，从而参与哮喘发病的相关病理生理变化。本研究引入多种人原代细胞和靶向敲除动物，运用膜片钳、钙火花、Myograph及小鼠肺功能等技术，从整体-器官-细胞-亚细胞水平多维度探讨气道上皮与平滑肌通过CYP-EET途径的偶联机制、上皮TRPV4的调控机制以及下游平滑肌通道的效应机制。研究发现，过敏原刺激能够上调哮喘气道上皮TRPV4表达与功能，以及上调CYP-EET表达，并增强共培养的平滑肌细胞的TRPV4的表达与功能。在气道上皮中，BMP4轴表达受过敏原刺激诱导的EET调控，并进一步调控TRPV4表达。在下游平滑肌中，CYP-EET-TRPV4轴参与慢性缺氧下5-羟色胺诱导的平滑肌收缩。综上所述，过敏原活化气道上皮CYP-EET途径，通过偶联激活气道上皮及平滑肌细胞的TRPV4通道，调节上皮通透性、气道炎症、平滑肌收缩和气道重塑，诱导哮喘发病。