基于SIRT1/PGC-1α通路与Lon蛋白crosstalk探讨加味大柴胡汤对情志失调AS小鼠线粒体损伤的保护机制

Chronic psychological stress is one of the key factors to promote AS, and its mechanism is related to oxidative stress induced mitochondrial damage. The SIRT1/PGC-1α pathway plays an important role in antioxidative stress and promotion of mitochondrial synthesis.Study found that Lon protein affects mitochondrial biosynthesis and may have crosstalk with the SIRT1/PGC-1α pathway. The previous study of the research group found that modified Dachaihu Decoction can improve anxiety and depression, and had anti-oxidation and anti-AS effects, but the mechanism is not yet clear. In this study, hormone levels and mitochondrial function will measure before and after stress in mice,and examining the expression of SIRT1/PGC-1α and Lon protein signaling pathway-related factors, to investigate the effect of Modified DaChaiHu Decoction on the mitochondrial injury and signal pathway by chronic psychological stress.By regulating SIRT1,the downstream signaling factors and Lon protein pathways were observed to verify the existence of crosstalk.Using siRNA technology to verify the target of modified DaChaihu Decoction against mitochondrial damage, to explore its mechanism for regulating crosstalk on both channels. The research results of this project can provide evidence for the treatment of AS from modified DaChaihu Decoction from the theory of "qi and blood loss", and provide new ideas and methods for the development of prevention and treatment of AS in traditional Chinese medicine.

慢性心理应激是促发AS的关键因素之一，其机制与氧化应激诱导线粒体损伤有关。SIRT1/PGC-1α通路在抗氧化应激、促进线粒体合成方面具有重要作用。研究发现Lon蛋白影响着线粒体生物合成，可能与SIRT1/PGC-1α通路存在crosstalk。课题组前期研究发现加味大柴胡汤可改善焦虑抑郁状态，具有抗氧化、抗AS作用，但机制尚未明了。本课题通过观察小鼠应激前后激素水平、线粒体功能，检测SIRT1/PGC-1α、Lon蛋白通路相关因子的表达，探讨加味大柴胡汤对慢性心理应激线粒体损伤及信号通路的干预作用；通过调控SIRT1观察其下游信号因子和Lon蛋白通路的表达，验证其存在crosstalk；运用siRNA技术验证加味大柴胡汤抗线粒体损伤的作用靶点，阐明其调控两条通路crosstalk的机制。本课题研究结果可为本方从“气血失和”论治AS提供佐证，为开拓中医药防治AS提供新的思路和方法。

慢性心理应激是促发AS的关键因素之一，其机制与氧化应激诱导线粒体损伤有关。SIRT1/PGC-1α通路与Lon蛋白可能存在crosstalk，在抗氧化应激、促进线粒体合成方面具有重要作用。基于此，项目组采用了分子生物学、病理组织学、透射电镜、细胞培养、免疫荧光技术、慢病毒质粒转染、免疫共沉淀等方法，以线粒体功能及SIRT1/PGC-1α通路与Lon蛋白作为主要观察指标，从分子、细胞、组织水平研究加味大柴胡汤抗慢性心理应激AS线粒体损伤的作用机制。（1）慢性心理应激对动脉粥样硬化的影响以及发病机制研究发现：慢性心理应激可升高CRH、ACTH激素及CORT水平、导致HPA轴代谢亢进、降低5-HT以及BDNF水平、影响血脂代谢、降低NO浓度损伤血管内皮功能、加重线粒体功能损伤，从而促进动脉粥样硬化的发生发展。（2）加味大柴胡汤对慢性心理应激AS小鼠干预疗效及抗线粒体损伤作用机制研究发现：加味大柴胡汤能够有效缓解情志失调AS小鼠CUMS干预带来的应激激素紊乱和静默、少动、倦怠等应激行为学异常，调节血脂和血管内皮功能，降低线粒体ROS含量，增加线粒体ATP含量以及mtDNA表达、缓解线粒体损伤，上调SIRT1/PGC-1α信号通路以及 Lon蛋白表达，在一定程度上促进线粒体生物发生，发挥抗慢性心理应激AS的作用。（3）细胞实验研究表明：SIRT1/PGC-1α与Lon蛋白通路存在crosstalk，可以促进线粒体生物发生，改善线粒体功能，降低ROS含量，共同拮抗慢性心理应激促发AS过程中造成的线粒体损伤，且加味大柴胡汤具有调控SIRT1/PGC-1α与Lon蛋白crosstalk对抗慢性心理应激合并AS线粒体损伤的分子机制，基于慢病毒质粒转染技术验证了TFAM是加味大柴胡汤干预的潜在作用靶标之一。加味大柴胡汤从“气血失和”论治AS，可为临床中医药防治动脉粥样硬化合并焦虑抑郁等双心疾病提供理论基础和新的研究思路，对临床双心疾病的防治具有重要意义。