IP3R1/MCOLN1/VAMP8介导的血小板mtDNA释放在血瘀证中的作用及薯蓣皂苷的干预机制

Cardiovascular disease induced by blood stasis syndrome is the leading cause of human death. Platelet activation is a key factor in the development of blood stasis syndrome. However, the mechanism of platelet activation leading to blood stasis syndrome is unknown. Clinical studies demonstrated that Xinxuekang, Dunyeganxinning, the Chinese patent medicines of diosgenin，significantly improve the symptoms of blood stasis, but the mechanism is unclear. Our previous study demonstrated that the IP3R1 and lysosomal MCOLN1 were activated, the expression levels of VAMP8 and extracellular DAMPs（mtDNA）were increased after platelet activation.And, diosgenin reduced the activation of these proteins, and improved blood stasis syndrome. Therefore, we hypothesize that the activation of platelets IP3R1 leads to an increase calcium released from the endoplasmic reticulum, resulting in overload of mitochondrial Ca2+, which in turn leads to ROS overload, whereas ROS loading leads to activation of MCOLN1 on lysosomes and increase VAMP8 expression level, this leading to enhanced mtDNA released from autophagolysosome, which in turn led to platelet activation and blood stasis syndrome, in addition, diosgenin improve blood stasis syndrome by decrease IP3R1/MCOLN1/VAMP8-mediated mtDNA release. We intend to use PCR technology, fluorescence co-localization technology, knockout mouse technology, patch clamp technology, Western blot and other techniques to confirm our hypothesis. We first time explored the role of IP3R1/MCOLN1/VAMP8 pathway-mediated mtDNA release in blood stasis. This project promotes the development and innovation of basic theory of traditional Chinese medicine, enrich the scientific connotation and research fields of blood stasis syndrome, and provide new treatment ideas and targets for treat blood stasis syndrome and drug development.

血瘀证诱发的心脑血管疾病是人类死亡的首要原因, 血小板激活是血瘀证发生的关键因素，但机制不明。临床研究表明，薯蓣皂苷活血化瘀中成药心血康、盾叶冠心宁治疗血瘀证疗效明确，但是作用机制不详。本团队前期研究发现，血小板活化后，IP3R1、MCOLN1和VAMP8的表达量增加，而薯蓣皂苷可以降低这些蛋白表达、抑制血瘀证。因此，我们推测血小板IP3R1激活可引发钙离子超载，导致ROS负荷，致使溶酶体上MCOLN1激活、VAMP8表达量增加，自噬流增强，DAMPs（mtDNA）释放增加，进一步激活血小板诱发血瘀证，并且薯蓣皂苷可通过调控此通路治疗血瘀证。我们拟用PCR、荧光共定位、基因敲除鼠、膜片钳和蛋白质印迹法等技术证实我们的假说。本课题首次探讨了IP3R1/MCOLN1/VAMP8通路介导的mtDNA释放在血瘀证中的作用及活血化瘀干预的作用机制，为血瘀证的诊疗提供了新的思路和靶点。

血瘀证是指各类致瘀因素，如气滞、痰凝、气虚等导致的脉道壅塞、血行不畅而引起的病证。由血瘀证诱发的心脑血管疾病是人类死亡的首要原因。血瘀证主要的表现形式为血液流变学的异常，其中血小板激活是血瘀证发生的关键因素。临床研究表明，薯蓣皂苷活血化瘀中成药心血康、盾叶冠心宁治疗血瘀证疗效明确，但是作用机制不详。我们证实了血小板内质网上IP3R1的激活，可诱发钙离子超载，钙离子超载可促进ROS负荷，ROS负荷可激活溶酶体MCOLN1，进而促进溶酶体自噬体衔接蛋白VAMP8的表达，增加溶酶体与自噬小体的融合，促进其自噬溶酶体水解，增加DAMPs（mtDNA）释放；而DN可通过抑制IP3R1/MCOLN1/VAMP8介导的血小板mtDNA释放，抑制血小板激活，最终降低血瘀证的发生。本研究从蛋白分子水平丰富血瘀证的病理机制及活血化瘀中药治疗的疗效机理。.在本项目研究期间，发表高水平SCI文章3篇，中文核心期刊1篇；申请国家发明专利3项（授权2项）；博士研究生3名，硕士研究生6名。本研究从分子蛋白、亚细胞器、细胞与动物水平探索血小板介导血瘀证的机理及中药干预机制，将为血瘀证的防治提供一种新的思路与靶点，具有十分重要的理论意义和应用价值。