黄芪多糖通过线粒体钠钙交换蛋白调节自噬与炎症延缓内皮细胞衰老的机制研究

Endothelial dysfunction is the starting factor of vascular aging. Effectively preventing vascular endothelial injury can delay the occurrence and development of atherosclerosis. We cloned a new Na+/Ca2+ exchanger protein, NCEX ( NCLX ), which was highly expressed in aortic endothelial cells. NCLX inhibited autophagy, inflammation and oxidative stress reaction by adjusting mitochondrial function, thus reducing endothelial injury. Our previous studies showed that NCLX expressed in DM rat aortic endothelia regulated mitochondrial function. Suppression of NCLX expression induced by hyperglycemia blocked mitochondrial Ca2+ efflux, caused mitochondria function injury, increased ROS production, generated autophagy-related NLRP3 and inflammatory cascade. Astragalus polysaccharides (APS) upregulated the expression of NCLX gene and protein in DM rat aortic endothelia, and alleviated atherosclerotic lesion. It is suggested that APS play a protective role for endothelial cells, and its mechanism may be related to NCLX. Our preliminary experiments showed that APS upregulated NCLX expression in natural aging endothelial cells, regulated autophagy, and delayed the aging of endothelial cells. But the specific pathways and key factors remain unclear. To investigate the protective effect of APS on endothelial cells through NCLX-mediated autophagy and inflammatory response is expected to further clarify the function of astragalus polysaccharide and its mechanism. This study is also expected to find new safe and effective prevention and treatment of endothelial cell injury and early vasculopathy, and to provide theoretical basis for retarding vascular aging.

内皮细胞功能紊乱是血管衰老的启动因素，预防血管内皮损伤可延缓动脉粥样硬化的发生、发展。我们克隆到一种在主动脉内皮细胞中高表达的新型钠钙交换蛋白NCEX（NCLX），发现NCLX通过调节线粒体功能，抑制自噬、炎症和氧化应激反应，减少血管内皮细胞的损伤。前期研究示糖尿病大鼠主动脉内皮表达的NCLX可调节线粒体功能。高糖下调NCLX继而阻碍线粒体钙外排，呼吸链产生ROS增多，线粒体损伤积累，NLRP3炎性小体激活，诱发炎症瀑布反应。黄芪多糖治疗的糖尿病大鼠主动脉组织NCLX基因和蛋白表达均上调，主动脉粥样硬化病变减轻，提示黄芪多糖可能是通过NCLX途径发挥其对内皮细胞的保护作用。预实验显示黄芪多糖也可以上调自然衰老的内皮细胞NCLX的表达，调控自噬，延缓内皮细胞的衰老，但具体作用通路和关键因子尚不清楚，对该机制的探讨可深化对黄芪多糖作用机制的认识，有望为延缓血管衰老进程提供新的药物治疗靶点。

背景：衰老是动脉粥样硬化发展的最重要的危险因素之一，构成血管系统的血管内皮细胞是血管稳态的关键调节因子，随年龄增长其数量和功能均会下降。前期研究发现黄芪多糖（APS）治疗的糖尿病大鼠主动脉组织线粒体钠钙交换蛋白NCLX表达上调，主动脉粥样硬化病变减轻，提示APS可能是通过NCLX途径发挥保护内皮细胞的作用，但具体作用机制尚不明确。主要研究内容：应用不同浓度APS干预高糖或H2O2诱导衰老的大鼠主动脉内皮细胞（RAECs），证实APS具有抗炎、降低氧化应激、增强自噬、抗衰老的作用；深入研究相关机制，发现APS通过线粒体NCLX 途径，产生抗炎、改善线粒体功能、抗衰老的作用。APS干预自然衰老的大鼠，体内证实APS改善氧化应激，对主动脉内皮具有抗衰老作用。重要结果、关键数据：细胞实验：高糖或H2O2建立RAECs衰老模型，应用不同浓度APS干预，发现①200μg/mL和800 μg/mL APS可显著降低高糖条件下衰老相关β-Gal阳性细胞比例，降低衰老相关蛋白p16、p21和p53表达；②APS增强自噬相关蛋白LC3B-II/LC3B-I、Atg7表达，同时降低自噬负相关蛋白p62/SQSTM1表达，透射电镜显示自噬小体数量增加，线粒体破坏得到改善；③APS改善H2O2诱导的线粒体损伤，提升抗氧化能力；④APS抑制炎症小体NLRP3。进一步应用siRNA技术敲低NCLX表达，证实APS可通过增强NCLX表达抑制RAECs炎症小体NLRP3及IL-1β释放、改善线粒体功能，延缓衰老。动物实验：APS减少23月龄大鼠主动脉壁厚度，修复血管弹性组织；减轻老年大鼠氧化应激反应，延缓主动脉内皮衰老，提高血清及组织SIRT-1水平。科学意义：深化对APS抗衰老作用机制的认识，探寻内皮细胞损伤和早期血管病变的有效防治措施，为治疗衰老相关心血管病变提供新的药物靶点。本项目基本按原定计划开展了研究，取得预期的研究成果，已发表论著5篇，其中SCI收录论文3篇，Medline收录论文1篇；核心论文1篇；培养博士研究生3名。