环氧二十碳三烯酸通过VDAC1/IP3R1下调线粒体相关内质网膜(MAM)水平抗心肌缺血再灌注损伤

Myocardial ischemia reperfusion injury (MIRI) is the key factor influencing the prognosis of patients with myocardial infarction. Mitochondria associated endoplasmic reticulum (MAM) is widely involved in the metabolism of phospholipids, calcium signaling, and cell apoptosis. Increase MAM levels were closely associated to MIRI. But the dynamic process under MAM formation and dissociation and its regulation also needs to be elucidated. VDAC1/GRP75/IP3R1 constitutes Ca2+ efficient pathway from the endoplasmic reticulum to mitochondria. As metabolic products of arachidonic acid, epoxyeicosatrienoic acids (EETs) can effectively protect against MIRI. Our previous studies suggested that EETs can supress endoplasmic reticulum stress and MAM level, improve mitochondrialin function. Molecular docking demonstrates EETs can interact with VDAC1, GRP75, IP3R1, the hypothesis that EETs depresses MAM level through VDAC1/GRP75/IP3R1 to protect against MIRI. This project intends to investigate the effects of EETs on VDAC1/GRP75/IP3R1 complex, and changes in MAM constituents PACS-2, Mfn2, MCU, SERCA, Sig1R, etc. This study will provide the knowledge about MAM and prpose a novel therapeutic choice in MIRI.

心肌缺血再灌注损伤(MIRI)是影响心肌梗死病人预后的关键因素。线粒体相关内质网膜(MAM)广泛参与Ca2+转运和磷脂代谢, 影响线粒体功能, 调控细胞凋亡。MAM水平异常增加与MIRI密切相关。但MAM形成和解离的动态过程及其调控还亟待阐释。VDAC1/GRP75/IP3R1构成Ca2+从内质网至线粒体的高效通道。环氧二十碳三烯酸(EETs)具有显著抗MIRI作用。我们前期发现EETs抑制内质网应激和MAM形成，改善线粒体功能；分子对接显示EETs能与VDAC1、GRP75、IP3R1相互作用，提出假说：EETs通过VDAC1/GRP75/IP3R1下调MAM水平抗MIRI。本课题考察EETs对VDAC1/GRP75/IP3R1复合物的影响，观察其它MAM构成蛋白PACS-2、Mfn2、MCU、SERCA、Sig1R等的变化，提高对MAM动态及其调控的认识，为综合防治MIRI提供新思路。

心肌缺血再灌注损伤(MIRI)是影响心肌梗死病人预后的关键因素。线粒体相关内质网膜(MAM)广泛参与Ca2+转运和磷脂代谢，调控线粒体功能，影响细胞命运。MAM水平异常增加与MIRI密切相关。VDAC1/GRP75/IP3R1构成Ca2+从内质网至线粒体的高效转运通道。我们前期发现环氧二十碳三烯酸(EETs)具有显著抗MIRI作用。EETs抑制内质网应激和改善线粒体功能。通过本项目研究发现，给予14,15-EET可显著降低MIRI小鼠心肌梗死范围，减少心肌细胞凋亡，改善心功能。提取富集的MAM和线粒体组分检测发现，IP3R1、GRP75、VDAC1水平在MIRI小鼠有显著升高，而14,15-EET处理可以明显降低这些蛋白水平，并且降低IP3R1、GRP75、VDAC1的相互作用。在氧糖剥夺/复氧（OGD/R）处理乳鼠原代心肌细胞，14,15-EET干预升高心肌细胞的线粒体膜电位，改善线粒体呼吸功能，降低线粒体内ROS的生成，免疫荧光染色显示14,15-EET明显降低心肌细胞中内质网、线粒体的共定位，降低心肌细胞线粒体中钙离子浓度，明显抑制OGD/R处理造成的心肌细胞损伤。在sEH基因敲除小鼠（sEH-/-）进一步观察，敲除sEH基因导致内源性EET升高，降低心肌梗死指数，降低MIRI小鼠心脏总蛋白中MAM关键组分，不影响心肌纤维横截面积。在乳鼠原代心肌细胞高表达内质网膜-线粒体外膜连接子（Ad-Linker）可明显减弱14,15-EET的抗MIRI作用。通过siRNA敲低VDAC1蛋白或者IP3R1蛋白也明显减弱14,15-EET的抗MIRI作用。综上所述，EETs通过降低VDAC1/GRP75/IP3R1在内质网、线粒体彼此靠近处局部的分布含量，并抑制VDAC1/GRP75/IP3R1相互作用，从而下调MAM水平，减轻线粒体钙超载，改善线粒体功能，发挥抗MIRI作用。本课题的研究成果提高对MAM动态及其调控的认识，为综合防治MIRI提供新思路。