基于调控“孙络-微血管”稳态的microRNA研究内毒素性急性肺损伤的分子机制及川芎嗪的干预

ALI is characterized by injury of the pulmonary microvascular endothelial cells (PMVEC). Tetramethylpyrazine (TMP) can reduce the disorder, which is based on the theory of Collateral diseases and confirmed by bedside-to-bench study. The protective effects might be attributed to the regulation of gene expression of MVECs remodeling. Nowadays, the microRNAs(miRs) were found to regulate the gene expression on post-transcriptional level. Our group found TMP can suppress the up-regulation of miR-193b-5p induced by LPS in HUVECs. On the basis of our original research, we will select and identify the target genes regulated by those miRs using bioinformatics methods and dual fluorescent protein reporter assay system. We will observe the effects of regulations and the changes of "minute collaterals/microvascular" homeostasis (cytoskeleton remodeling, vascular permeability and so on). We propose to clarify whether "minute collaterals/microvascular" homeostasis can recover by TMP protection via regulating the miRs which targets the gene expression of MVECs remodeling. This research will further elaborate the protective mechanisms of TMP and provide a theoretical and experimental basis for the discovery of new targets for the clinical treatment of ALI.

肺微血管内皮细胞(PMVEC)损伤是急性肺损伤(ALI)发病的中心环节。基于“病证结合，以通为法”的络病理论，结合临床观察和实验研究发现，川芎嗪对此具有保护作用，其机制可能与干预微血管重构相关基因的表达有关，而近年发现的microRNA(miR)可在转录后水平调控血管重构相关基因。本课题组也发现，川芎嗪能抑制LPS在HUVEC诱导的ALI相关miR-193b-5p上调。本项目在原有的研究基础上，采用生物信息学方法和双荧光蛋白报告基因分析系统，从血管重构相关基因中筛选、确认相关miR调控的靶基因；分别在细胞和整体肺脏水平上调/下调miR，观察其对相应靶基因表达的调节作用及对内皮细胞骨架重构和血管通透性等“孙络-微血管”稳态相关指标的影响，阐明川芎嗪是否通过调控miR干预相关靶基因的表达以恢复“孙络-微血管”稳态。本研究将进一步揭示川芎嗪的保护机制，为临床防治ALI提供新的思路和治疗靶点。

肺微血管内皮细胞(PMVEC)损伤是急性肺损伤(ALI)发病的中心环节。基于“病证结合，以通为法”的络病理论，结合临床观察和实验研究发现，川芎嗪对此具有保护作用，其机制可能与干预微血管重构相关基因的表达有关，而近年发现的microRNA(miR)可在转录后水平调控血管重构相关基因。本项目在原有的研究基础上，采用生物信息学方法和双荧光蛋白报告基因分析系统，从血管重构相关基因中筛选、确认相关miR调控的靶基因；分别在细胞和整体肺脏水平上调/下调miR，观察其对相应靶基因表达的调节作用及对内皮细胞骨架重构和血管通透性等“孙络-微血管 ”稳态相关指标的影响，阐明川芎嗪是否通过调控miR干预相关靶基因的表达以恢复“孙络-微血管”稳态。重要结果：①川芎嗪可以有效抑制脂多糖诱导的HUVECs通透性升高；②川芎嗪减轻脂多糖诱导的HUVECs细胞骨架损伤的机制可能与其下调Rac1/LIMK1信号通路中Rac1-GTPase活性蛋白、LIMK1磷酸化蛋白表达有关； ③LPS能够降低内皮细胞中miR-143-3p、miR-194-5p的表达； ④川芎嗪可能通过调控miR-194-5p、miR-143-3p影响LPS诱导的内皮细胞中Rac1、LIMK1蛋白表达变化。本研究将进一步揭示川芎嗪的保护机制，为临床防治ALI提供新的思路和治疗靶点。