组织蛋白酶CTSK在酒精性心肌病中对自噬溶酶体途径调控机制的研究

Alcohol abuse is a serious problem in China and results in the highest mortality rate in the world. Excessive drinking can cause severe alcoholic cardiomyopathy (ACM), and eventually lead to heart failure and death. At present, ethanol and its metabolite acetaldehyde are considered as the main causes of ACM although the molecular mechanism is still elusive. Therefore, to investigate the pathogenesis and therapeutic targets of ACM is imperative. Study from the applicant's group found that chronic drinking can induce autophagosome accumulation and mitochondrial dysfunction, as well as the upregulation of lysosomal cysteine cathepsin K (CTSK). Ablation of CTSK significantly improves cardiac dysfunction induced by metabolic disorder. However, the role of CTSK upregulation in alcohol-induced autophagosome accumulation and the regulation of late stage of autophagy remains unclear. In addition, whether CTSK-mediated autophagic impairment induces mitochondrial damage and metabolic disturbance has yet been discussed. Therefore, to further deepen the understanding of the pathogenesis of ACM, and provide theoretical basis for the development of new therapeutic targets, this study aims to elucidate the role of CTSK in the disorder of autophagic process in ACM, to explore the regulatory mechanism of CTSK on autophagy-lysosome pathway, and to examine whether CTSK triggers autophagosome accumulation and mitochondrial damage by regulating transcription factor TFEB.

我国是全球因饮酒致死最多的国家，过量饮酒会引起严重的酒精性心肌病（ACM），最终会发展为心脏衰竭而死亡。目前已知乙醇及其代谢产物乙醛是导致ACM的主要原因，但发病机制尚不明确，因此研究对ACM的致病机制和治疗靶标势在必行。申请人研究发现，慢性饮酒可导致心肌自噬体蓄积和线粒体功能障碍，同时引起了溶酶体半胱氨酸组织蛋白酶 cathepsin K（CTSK) 的上调，CTSK基因缺失可明显改善代谢异常引起的心功能失调。然而饮酒引起的CTSK升高对心肌自噬体蓄积以及对自噬晚期的调控机制尚不得知，该过程是否导致线粒体损伤及代谢异常也一直缺乏深入的探讨。本项目研究拟阐明慢性饮酒通过上调CTSK干扰正常的自噬过程，明确CTSK对自噬溶酶体途径的调控机制，验证CTSK是否对转录因子TFEB进行调控进而导致自噬体蓄积和线粒体损伤，进一步深化对ACM发病机制的认识，为新的基因治疗靶点的选择提供理论依据。

本项目主要通过体内外实验及临床血清样本研究了急慢性饮酒导致心肌损伤的发病机理，明确CTSK和TFEB对自噬溶酶体途径和线粒体损伤的调控机制。我们研究发现在酒精性心肌病小鼠模型中，cathepsin K 显著上调，并引起自噬损伤，提示酒精性心肌中病中 cathepsin K 对自噬可能存在调节作用。在临床水平，我们进一步发现在酒精性心肌病 (ACM) 患者中，Cathepsin K显著升高，同时我们筛选了组间差异代谢物，并验证了他们与心脏功能的关系。结果表明长期重度饮酒者左室收缩期末内径 (LVESD)、左室舒张末期内径(LVEDD)、左室质量指数、收缩期血压、血清甘油三酯和Cathepsin K水平显著升高，Cathepsin K与左心室质量指数（LV mass）呈显著正相关。在小鼠和细胞水平，急性酒精毒性可明显导致小鼠心肌收缩力下降和舒张功能障碍，心输出量减少，表现为心脏衰竭。同时在小鼠和人心肌细胞中发现TFEB和AMPK活性水平显著降低，溶酶体功能和线粒体功能受损。Cathepsin K 缺失或TFEB激活明显逆转了酒精毒性引起的心脏功能障碍、线粒体功能失调以及ROS的堆积。此外，我们还发现TFEB可通过调节炎性因子和线粒体功能减少细胞凋亡从而对脓毒症引起的心肌损伤起到保护作用，Cathepsin K敲低对非小细胞肺癌的发生发展也起到了一定的抑制作用。综上所述，通过加深对ACM发病机制以及Cathepsin K和TFEB的作用的认识，可以为选择新的治疗靶点提供理论依据。同时，通过对人血清样本中相关基因的筛选和分析，我们希望找到预防或诊断ACM或其他相关疾病的生物标志物。