VSOR氯离子通道在高糖诱导心肌细胞损伤中的作用及机制

Cardiovascular complications of diabetes have become the main cause of death. The main pathogenesis is that hyperglycemia-induced by diabetes activates RAAS system, the production of ROS, endoplasmic reticulum stress and inflammatory responses, which lead to the injury of cardiomyocytes and vascular endothelial cells. If these processes could be inhibited, cardiovascular complications may be alleviated. It has been reported that these factors above activate VSOR chloride channel, whereas inhibiting this channel could rescue injured cardiomyocytes. Does VSOR chloride channel also participate in the process of cardiomyocyte injury induced by diabetes? If so, what is the mechanism? So far, it has not been reported. To elucidate this hypothesis, high glucose is going to be used to establish cardiomyocyte injury model in this study. First, we will investigate the influence of high glucose to the VSOR chloride channels of cardiomyocyte. Next, VSOR chloride channel function will be changed by activating or inhibiting VSOR chloride channel. With this prerequisite, the cell injury induced by high glucose would be detected. In addition, biochemical and signal pathways including ROS, endoplasmic reticulum stress, apoptosis, inflammatory responses would aslo be tested. In this topic, we will study the regulation and mechanism of VSOR chloride channel and cardiomyocyte injury induced by high glucose for the first time. Moreover, we may provide new evidence and strategy to the prevention and treatment of diabetic cardiomyopathy

糖尿病的心血管并发症已成为糖尿病患者死亡的主要原因，其主要发病机制是高血糖激活了以ROS为核心的RAAS、内质网应激、炎性反应等网络介导的心肌及血管内皮细胞损伤。如何阻断该过程是降低心血管疾病并发症的重要手段。研究发现，ROS等损伤性因素的出现伴有容积敏感性（VSOR）氯离子通道的激活，阻断该通道能有效挽救受损的细胞，那么VSOR氯离子通道是否也参与了高糖诱导心肌损伤的发病过程？其机制如何？目前国内外尚未见报道。本课题拟在高糖诱导心肌细胞损伤模型上，一方面观察高糖刺激对心肌细胞膜VSOR氯通道的影响；另一方面，通过激活或阻断剂阻断VSOR氯通道，观察VSOR氯通道功能变化对高糖诱导心肌损伤的作用，以及核心信号分子ROS与内质网应激、炎性因子、细胞凋亡等指标及信号通路的调节。首次探讨高糖介导的心肌细胞损伤中氯离子通道的调控作用及分子机制，为预防和治疗糖尿病心肌病提供全新的依据和策略。

糖尿病是最常见、对人类危害最大的疾病之一，其心血管系统的并发症是致残、致死的主要因素。通过控制血糖水平、减轻高糖继发性损伤已成为治疗心血管疾病的重要靶点。本研究首次探讨了容积敏感性氯离子（VSOR）通道是否参与了高糖诱导的心肌细胞凋亡性损害以及调控机制。本研究构建用高糖诱导大鼠原代培养心肌细胞的心肌细胞凋亡的模型，通过对细胞存活率、凋亡、自噬、细胞信号通路及电生理离子电流的检测，结果发现： ①膜片钳电生理技术可记录到高糖诱导心肌细胞中有类似VSOR氯离子外流、应用阻断剂DIDS、DCPIB可有效阻断其电流的出现，通过氯离子荧光探针MQAE检测证实，高糖导致了细胞内氯离子水平的下降，上述二种方法证实高糖导致了VSOR氯离子通道的激活和氯离子的外流；② 应用MTT、流式细胞技术、TUNEL和caspase-3检测显示高糖能够诱导心肌细胞显著的凋亡发生，荧光探针证实，高糖激活了ROS的增加，使用氯离子通道阻断剂DIDS及DCPIB、以及ROS清除剂NAC可以有效抑制高糖诱导的ROS介导心肌细胞凋亡的发生；同时发现DIDS及DCPIB可抑制模拟ROS作用的H2O2刺激心肌细胞产生的VSOR氯电流，进一步提示高糖可能通过激活ROS水平，来介导调节VSOR氯离子通道功能；③高糖作用72小时，发生心肌细胞凋亡最为明显，检测细胞自噬水平的LC3II/LC3I、P62的表达显著下降，而应用DIDS/DCPIB作用后LC3II/LC3I、P62的表达显著增加，细胞凋亡减少，同时发现DIDS/DCPIB作用后心肌细胞Akt磷酸化增高、mTOR水平表达是减少，mTOR下游通路信号分子P-P70S6K/P70S6K, P-S6/S6也表达明显减少，应用Akt抑制剂（LY294002预处理后）后自噬标志物减少；自噬抑制剂3-MA逆转了DIDS及DCPIB的心肌细胞保护作用，提示氯离子通道阻断剂保护细胞可能通过Akt-mTOR信号通路调节自噬水平的变化。结论：VSOR氯离子通道参与了高糖致心肌细胞损伤过程，主要是通过ROS介导了Akt-mTOR通路发生心肌细胞的凋亡，氯离子通道阻断剂可能是通过Akt-mTOR这条通路激活自噬机制，达到保护心肌细胞及改善心脏功能的作用，具有潜在的治疗价值。