Lamp2介导脂噬双重调控信号参与PBC高脂血症的机制研究

Correction of lipid metabolism disorders in PBC patients with hyperlipidemia has good clinical efficacy, so it is of great significance to discuss the regulation of lipid metabolism in PBC. Recent studies have found that lysosome dependent lipophagy can directly internalize, encapsulate, and degrade lipid droplets. It is an important way for fat lipolysis, but the mechanism is unclear. In the previous study, we found abnormal expression and distribution of Lamp2 in liver cells and autophagy disorders in PBC patients. In addition, serum Lamp2 levels were positively correlated with cholesterol and triglyceride levels. Lamp2 knockout leads to autophagy abnormality and lipid disaggregation. Furthermore, Lamp2 autophagy has subtype specificity, and lipid droplet degradation regulation protein Plin5 can be degraded by Lamp2A mediated CMA. Therefore, we hypothesize that Lamp2 regulates lipophagy of lipid droplets through both macroautophagy and CMA autophagy, and the A and B subtypes of Lamp2 play a synergistic role. In this study, we are going to explore the regulatory mechanism of lipophagy mediated by Lamp2 using molecular biology, live cell transportation, ultrastructure analysis, and co-immunoprecipitation methods combined with model animals that are liver-specific conditional knockout for Lamp2 subtypes.. This study may provide both theoretical and experimental basis for understanding PBC pathological process and finding new target point for treatment.

PBC合并高脂血症患者中纠正其脂代谢紊乱可显著改善临床症状，在PBC发病进程中脂代谢调控意义重大。新近研究发现，溶酶体依赖的自噬脂解可对脂滴进行内化、包裹和降解，是脂解的重要方式，但机制不清。本课题组发现：PBC患者肝细胞Lamp2表达及分布异常，自噬障碍；血清Lamp2水平与胆固醇、甘油三酯水平成正相关；Lamp2敲除自噬异常，脂滴分解障碍。Lamp2自噬具有亚型特异性，脂滴蛋白Plin5可被Lamp2A介导的CMA所降解。因此，我们认为Lamp2通过大自噬和CMA自噬双重信号调控了脂滴的自噬脂解，其A、B亚型扮演了协同作用。本课题拟以脂滴自噬脂解为切入点，利用分子生物学、活细胞运输、超微结构分析、免疫共沉淀等方法，结合肝细胞Lamp2亚型特异性敲除的模式动物和病理标本验证，探讨Lamp2对脂滴自噬脂解的调控作用及机制，为深入理解PBC病理过程、发现治疗新靶点提供理论依据和实验基础。

背景与目的:NAFLD的病理特征是肝脏脂质稳态失衡，其中发生了脂噬起着至关重要的作用。然而，潜在的分子机制尚不清楚。我们研究了伴侣介导的自噬(CMA)在NAFLD的发病中的作用。方法:在NAFLD患者和高脂饮食(HFD)小鼠的肝组织中评估CMA活性。使用肝脏特异性LAMP2A敲除小鼠和缺乏LAMP2A [L2A(-)细胞]的HepG2细胞研究CMA对肝细胞脂肪分解的影响。Plin5，脂滴(LD)相关蛋白的表达也在人和小鼠肝组织及[L2A(-)]细胞中进行了评估。结果:在这里，我们发现NAFLD患者和动物模型的肝脏中CMA功能紊乱，表现出LAMP2A的明显减少，同时CMA正向调控因子升高。在高脂肪胁迫下，肝脏特异性lamp2a敲除小鼠和L2A(-)细胞累积了更多的脂滴和更高的血清甘油三酯。同时，敲除LAMP2A会阻碍LD的分解，但不会增加LD的形成。此外，LD相关蛋白Plin5是CMA底物，其通过CMA降解是LD分解所必需的。结论:我们认为CMA诱导的Plin5降解的紊乱可能导致了NAFLD的脂滴分解障碍，从而解释了NAFLD的脂质稳态失衡。