p120/p66Shc/PINK1通路双向调节肺血管内皮细胞线粒体衰老与自噬对机械通气肺损伤的影响

Mitochondria plays important role in maintaining the structure and function of cells. The increase of pulmonary vascular permeability is a key step of ventilator induced lung injury (VILI). However, the damage and mitophagy of mitochondrial which is important to maintain the integrity of pulmonary membrane structure and function is not elucidated in VILI. Preliminary experiments suggested that mechanical stretch could active p66Shc and inhibit PINK1, which induced the senescence and decreased the mitophagy of mitochondria. And p120 had a dual-directional regulating effect in this process although the mechanism was not clear. Therefore, we hypothesized that p120 negative regulated RhoA kinase and PKC kinase, and then inhibited the phosphorylation of p66Shc and PINK1, which could prevent the senescence of mitochondria and promoted the mitophagy. We used HLMVEC cells, VILI model, PINK1 knock out mice and p120 conditional knock out mice. Techniques including siRNA/cDNA transfection, immunofluorescence, methods for detecting mitophagy and mitochondria function were applied in this research to explore the mitochondrial damage and the changes of mitophagy as well as the dual-directional regulating effect of p120 on mitochondrial. This project will provide theoretical basis and new insight for the clinical treatment of VILI from the perspective of energy balance.

线粒体对维持细胞结构和功能完整具有重要作用，肺血管通透性增加是机械通气肺损伤（VILI）的主要环节，维持肺泡膜结构及功能完整的细胞线粒体在VILI中损伤与自噬修复机制尚未明确。预实验发现机械牵张肺血管内皮细胞（HLMVEC）激活p66Shc，诱发线粒体衰老与损伤，并降低PINK1活性，抑制线粒体自噬；同时发现p120可双向调节此过程，但其机制未明。故假设机械通气时p120 负性调控RhoA及PKC激酶，抑制p66Shc/PINK1磷酸化，防止线粒体衰老损伤并促进其自噬，减轻肺水肿。通过牵张离体HLMVEC、在体VILI模型、PINK1基因敲除及p120条件性基因敲除小鼠，采用siRNA、CO-IP、confocal及线粒体损伤、自噬、功能测定等技术，探讨VILI过程中细胞线粒体衰老损伤与自噬变化及p120对其双向调节的作用与机制，从能量平衡角度为VILI临床防治提供理论基础和新思路。

线粒体对维持细胞结构和功能完整具有重要作用，机械通气肺损伤（VILI）中线粒体衰老损伤、自噬清除机制及与肺水肿的关系尚未明确。预实验结果发现：机械牵张人肺微血管内皮细胞（HLMVEC）激活p66Shc，诱发细胞衰老、线粒体损伤；并降低PINK1活性，抑制线粒体自噬。p120可双向调节其衰老损伤及自噬，机制并未明确。故我们假设：机械通气时p120负性调控RhoA及PKC激酶，抑制p66Shc及PINK1磷酸化，防止线粒体损伤并促进线粒体自噬，减轻肺水肿。该研究通过离体HLMVEC机械牵张2h及4h进行相应机制探讨，并在活体小鼠建立呼吸机相关性肺损伤模型，通过体外注射小干扰RNA敲除部分小鼠体内的PINK1、p120，然后进行机械通气，收集各组肺组织及肺泡灌洗液和血样，采用siRNA、CO-IP、confocal及线粒体损伤、自噬、功能测定等技术，探讨VILI过程中细胞线粒体衰老损伤及自噬清除机制，明确p120双向调节细胞线粒体衰老及自噬在VILI中的保护作用，前期结果表明机械牵张可促使p66shc磷酸化及抑制PINK1磷酸化，进一步加重细胞衰老及线粒体自噬，而低表达p66sch或PINK1后可加重细胞衰老及线粒体自噬，为VILI肺水肿的基础研究及临床防治提供新靶点。