PLCδ-TRPC-HDAC3信号通路在阿片类药物后处理减轻心肌缺血再灌注损伤中的作用机制研究

Acetylation modification plays a pivotal role in the epigenetic regulation of cardiac ischemia reperfusion injury (IRI). Our previous data and pre-experiments showed that the nuclear translocation of HDAC3 was involved in the opioid postconditioning (OPoC)-induced cardioprotection, but the underlying mechanisms are still unclear. The nuclear translocation of HDAC3 is associated with the activation of Ca2+ signaling pathway in the cell. Classical transient receptor potential (TRPC) channel was wildly expressed in the myocardial and was the key factor to regulate the concentration of Ca2+. Phospholipase Cδ (PLCδ) is involved in the regulation of TRPC. We speculate that the nuclear translocation of HDAC3 plays a vital role in OPoC induced myocardial protection, which is regulated by Ca2+ mediated by PLCδ-TRPC signal pathway. We established OPoC model in H9c2 cell line, primary cardiomyocyte and rat heart. Then by western blot, immunofluorescence, specific inhibitor, RNAi and calcium imaging, we will elucidate PLCδ-TRPC-HDAC3 signaling pathway in OPoC induced cardioprotection in vitro and in vivo. The results of this study can enrich the theory of OPoC induced myocardial protection, and also provide more molecular targets for the development of new myocardial protection drugs.

乙酰化修饰是心肌缺血再灌注损伤（IRI）的关键表观遗传机制。课题组已发表文章及预实验表明：组蛋白去乙酰化酶3（HDAC3）入核转运参与阿片类药物后处理（OPoC）心肌保护，但分子机制未明。既往研究表明：HDAC3入核转运与细胞内Ca2+浓度密切相关；经典型瞬时感受器电位（TRPC)通道在心肌细胞中广泛表达，是调控细胞内Ca2+浓度的关键因子；磷脂酶Cδ（PLCδ）可调控TRPC。据此我们推测：PLCδ-TRPC信号通路可通过调节胞内Ca2+浓度调控HDAC3入核转运，介导OPoC心肌保护。本项目拟在H9c2细胞系、原代心肌细胞及大鼠心脏建立OPoC模型，通过免疫印迹、免疫荧光、小RNA干扰、钙成像等技术，于离体、在体水平阐明PLCδ-TRPC-HDAC3信号通路在OPoC心肌保护中的作用机制。本研究可以丰富OPoC心肌保护理论，为开发新型心肌保护药物提供分子靶点。

心血管疾病是世界范围内的主要死亡原因，主要由缺血性心脏病引起，缺血性心脏病会造成不可逆的心肌损害，最终导致心力衰竭。及时的血管再通是拯救缺血心肌、提高临床疗效最有效的方法，而血管再通会引起再灌注损伤。既往研究发现，阿片类药物瑞芬太尼后处理能够有效保护缺血心肌，减轻缺血再灌注损伤。本团队前期研究发现组蛋白去乙酰化酶3在瑞芬太尼后处理的心肌保护作用中起重要作用，但其下游机制仍有待进一步研究。既往研究已证实缺血后处理在再灌注早期可激活自噬并促进受损的自噬流，发挥抗心肌缺血再灌注损伤的心肌保护作用。由于瑞芬太尼后处理可模拟缺血后处理减轻心肌缺血再灌注损伤，本项目分别以蛋白去乙酰化酶3、自噬为切入点，研究了阿片类药物瑞芬太尼对心肌缺血再灌注损伤的保护作用及其部分机制。研究发现，瑞芬太尼后处理通过抑制HDAC3的表达，从而促进GSK-3β 9位点的磷酸化，保护H9C2心肌细胞对抗缺氧复氧损伤。瑞芬太尼后处理在心肌缺血再灌注早期可激活自噬并促进自噬体与溶酶体的融合；瑞芬太尼后处理可上调LAMP2蛋白的表达，恢复溶酶体的功能并促进自噬体与溶酶体的融合，增强I/R损伤后受损的自噬流；自噬参与瑞芬太尼后处理介导的抗I/R损伤的心肌保护作用。该项目通过基础研究，揭示了HDAC3和自噬在瑞芬太尼后处理中的重要作用，为防治心肌缺血再灌注损伤提供了潜在靶点。