TRPM7通道介导的Ca2+信号调控糖尿病血管内皮细胞凋亡及机制研究

Recent researches show that apoptosis of endothelial cells induced by hyperglycemia during diabetes plays crucial role in diabetic vasculopathy, however, the underlying mechanisms remain elusive. Our pre-experiment showed that expression and activity of TRPM7 channel were upregulated in vascular endothelial cells in mice with type 2 diabetes mellitus. TRPM7 blocker, NS8593, significantly inhibited endothelial cell apoptosis in diabetic mice. Genetic silencing of TRPM7 suppressed mitochondrial calcium uniporter (MCU) activity and alleviated mitochondrial calcium overload. It suggested that the Ca2+ signal induced by TRPM7 channel was involved in the process of apoptosis in endothelial cells during diabetes mellitus. We proposed that Ca2+/MCU/mitochondrial calcium overload induced by TRPM7 upregulation was one of the critical pathophysiological basis of diabetic vasculopathy. Based on the studies before, we intend to investigate the effects of TRPM7 channel on high glucose-induced endothelial apoptosis in endothelial cells, TRPM7 endothelial-specific knockout mice and TRPM7 blocker NS8593-treated mice in vivo and in vitro. This study will demonstrate the mechanisms of endothelial cell apoptosis induced by TRPM7 and evaluate whether TRPM7 as a novel therapeutic target for diabetic vasculopathy treatment.

高糖诱导的血管内皮细胞凋亡在糖尿病过程中起关键性作用，但凋亡产生的原因及具体途径尚不清楚。预实验结果发现糖尿病过程中,血管内皮细胞TRPM7通道表达及活性增强，TRPM7抑制剂可改善血管内皮细胞凋亡，TRPM7基因沉默抑制内皮细胞MCU活性和线粒体Ca2+超载，提示TRPM7介导的Ca2+信号作用至关重要。因此提出假设，TRPM7介导Ca2+/MCU/线粒体Ca2+超载促进内皮细胞凋亡而致糖尿病血管病变发展。在上述基础上，本项目拟：明确TRPM7参与高糖诱导的内皮细胞凋亡以及TRPM7通过介导Ca2+/MCU/线粒体Ca2+超载信号通路调控此过程，在内皮特异性TRPM7敲除小鼠中进一步确认上述机制，并使用TRPM7抑制剂观察其对糖尿病血管内皮损伤的保护作用。本研究将从TRPM7介导的Ca2+信号这一新的角度阐明糖尿病血管内皮细胞凋亡的机制，并评估TRPM7是否为防治糖尿病血管病变的新靶点。

内皮功能异常是包括2型糖尿病、肥胖在内的多种代谢紊乱过程中血管功能障碍的主要诱发因素，然而，内皮细胞功能异常产生的原因及具体途径仍不清楚。本研究采用膜片钳、分子生物学、转基因动物模型等实验手段对此进行深入研究，动物方面，我们构建了TRPM7血管内皮特异性敲除小鼠，并采用高脂喂养诱导的2型糖尿病模型，细胞方面，我们使用原代分离培养的小鼠血管内皮细胞。本课题组研究结果显示，（1）高糖诱导下，血管内皮细胞TRPM7表达及活性均明显升高，TRPM7敲除或过表达可以减轻或者增加高糖诱导的内皮细胞凋亡；（2）高糖诱导下，TRPM7介导的Ca+内流通过降低线粒体内膜Ca2+摄取单向转运体MCU与MCU调节蛋白MICU的结合水平，减弱MICU对MCU的调控能力，从而调控线粒体Ca2+超载，最终促进高糖诱导的内皮细胞凋亡；（3）内皮特异性敲除TRPM7可缓解糖尿病过程中血管内皮损伤并保护血管舒张功能；（4）糖尿病动物模型上验证TRPM7通过胞浆及线粒体Ca2+超载从而促进内皮细胞凋亡；（5）在体使用TRPM7抑制剂NS8593可以保护糖尿病过程中血管舒张功能；（6）游离脂肪酸（FFA）可增加内皮细胞氧化应激水平从而激活TRPM2通道；（7）TRPM2介导的Ca2+内流可通过PERK/ATF4/TRB3通路促进肥胖过程血管舒张功能障碍的发生；（8）血管紧张素II（Ang II）刺激下，TRPM2-Ca2+信号激活Ca2+敏感激酶CaMKII诱导脂肪细胞胰岛素抵抗；（9）TRPM2抑制剂可以缓解高血压过程中伴发的血糖紊乱及胰岛素抵抗。该项目从TRPM7以及TRPM2介导的Ca2+信号角度，探讨了TRPM7-Ca2+通道以及TRPM2-Ca2+通道在代谢紊乱过程中血管功能障碍的作用，为评估二者是否可以作为防治代谢紊乱过程中血管功能障碍的新靶点提供实验室依据。