LAMPs通过影响肝细胞稳态干预酒精性肝损伤进程及一贯煎的影响

With the great improvement of the material level, the liver injury caused by alcohol abusing and the incidence of alcoholic liver disease has being increased in recent years, However, there is no effective therapy to control alcoholic liver disease. Our previous studies have found that H2O2 induced LAMP1 down-regulation in hepatocyte, then influenced the function of lysosome and the maintenance of cell survival homeostasis. Other research reported that the expression of LAMP1/2 decreased in alcohol-induced pancreatitis, but the mechanism and the effect of LAMPs decreasing in alcohol liver disease have not been reported. We hypothesized that LAMPs down regulation, lysosomal membrane permeability increasing, lysosomal PH modifying and autophagy flux inhibition may be the potential mechanism of alcohol liver disease. In order to confirm the hypothesis, we replicated the alcohol liver injury model in vitro and in vivo, applied cellular and molecular biology techniques, observed the effect of LAMPs expression change either on the lysosomal function or on the maintenance of cell survival homeostasis, explored the molecular mechanism by which regulated LAMPs expression and the influence of Yiguanjian on this process. Our research is to elucidate the molecular mechanisms of alcoholic liver injury from a new perspective and provide a new target for the treatment of alcoholic liver injury.

随着物质水平的极大提高，过量饮酒引起的肝损伤日益增多，酒精性肝病的发病率逐年上升，但目前尚无有效控制和治疗的方法。本课题组前期研究发现肝细胞损伤后，溶酶体膜相关蛋白(Lysosomal membrane associated proteins, LAMPs)表达下降，进而影响溶酶体功能及细胞稳态的维持。有研究发现酒精诱导引起的胰腺炎中有LAMP1/2表达下降，但LAMPs表达变化的机制及在酒精性肝病中的作用尚未见报道。我们推测，酒精性肝损伤时，LAMPs表达下降、溶酶体膜通透性降低，PH值变化及自噬流阻滞可能是酒精性肝损伤的新机制。为证实这一假说，我们复制体内外酒精性肝损伤模型，采用细胞、分子生物学技术，观察LAMPs表达变化对溶酶体功能及细胞生存稳态的影响、探讨LAMPs表达变化的分子机制及一贯煎对这一进程的影响，从新的视角阐明酒精性肝损伤的分子机制，并为酒精性肝损伤的防治提供新靶点。

酒精性脂肪性肝病是长期过量饮酒而导致肝脏疾病的早期病变，目前已成为慢性肝病的最主要病因之一。自噬是对细胞内受损的细胞结构、衰老的细胞器及不需要的大分子物质等进行清除、降解的过程。自噬过程中产生的核苷酸、氨基酸和脂肪酸，可被细胞再循环利用，产生ATP和生成大分子物质，在维持肝细胞的生存稳态方面发挥重要作用。而这一过程是溶酶体依赖性的，LAMP1/2是位于溶酶体膜上的高度糖基化的蛋白质，对于维持溶酶体膜的完整性及正常的溶酶体功能至关重要。目前研究发现影响溶酶体膜蛋白LAMP1/2表达变化的分子机制主要涉及糖苷酶的剪切和组织蛋白酶的降解两种。本项目采用国际通用的慢性酒精喂养加急性酒精灌胃方法复制酒精性肝损伤模型，模拟人体酒精性肝损伤的真实过程，同时复制肝细胞体外酒精损伤模型。结果显示：酒精作用后，肝脏出现明显的炎症损伤及脂肪变，酒精代谢异常，乙醛生成增多、向乙酸的转化减少。维持溶酶体膜结构完整性的关键蛋白LAMP1/2表达显著下降，溶酶体膜通透性增加，溶酶体的功能受阻，大自噬异常，LC3-II/ LC3-I表达显著下降，P62表达显著升高。酒精诱导的LAMP1/2表达下降，分子机制主要与组织蛋白酶CTSB/CTSD的降解相关，糖苷酶的剪切在这一过程中并未发挥作用。. 一贯煎是养肝阴、疏肝气中医经典方剂。研究表明，一贯煎对酒精性肝病患者有良好的降脂和保肝作用，但一贯煎治疗酒精性肝病的具体分子机制尚未阐明。本项目结果显示一贯煎可通过减少组织蛋白酶CTSB/CTSD对LAMP1/2 的降解上调LAMP1/2 蛋白表达，稳定溶酶体膜的完整性和稳定性，上调LC3-II/ LC3-I表达，下调P62表达，恢复溶酶体功能，进而维持肝细胞的生存稳态，减轻高脂结合酒精诱导的慢性肝损伤，对酒精性脂肪肝的防治提供理论和实践基础。