线粒体自噬与线粒体未折叠蛋白反应参与小鼠运动改善代谢相关性脂肪性肝病的作用机制

关键词：代谢相关性脂肪性肝病  脂质沉积  自噬  线粒体自噬  线粒体未折叠蛋白反应

Keywords:metabolic associated fatty liver disease  hepatic lipid deposition  autophagy  mitophagy  mitochondrial unfolded protein response

摘要：

      摘要 目的：探讨运动缓解肝脏脂质沉积的机制，阐明线粒体自噬与UPRmt在运动调节肝脏脂质沉积中的作用。 方法：30只8周龄雄性C57BL/6J小鼠随机分为普通饮食组（NC组）、高脂肪果糖胆固醇饮食组（HFFC组）、高脂肪果糖胆固醇饮食+运动组（HFFC+EX组），每组10只。HFFC+EX组进行8周自主跑轮运动。采用HE、BODIPY染色观察肝组织病理；JC-1检测线粒体膜电位；ROS试剂盒检测线粒体ROS；氧电极法检测线粒体呼吸控制率；Western Blot及免疫荧光双标记检测LAMP-2A、LC3、P62、COXIV、Parkin、VDAC、LONP1、mtHSP60、mtHSP70蛋白表达。 结果：①与NC组比较，HFFC组肝细胞脂肪性变及脂滴显著增加（P<0.01）；与HFFC组比较，运动组肝脂肪性变减轻（P<0.01）。②与NC组比较，HFFC组线粒体膜电位下降、ROS升高（P<0.05），线粒体呼吸控制率降低（P<0.05）；与HFFC组比较，运动组线粒体膜电位恢复、ROS显著降低（P<0.01），线粒体呼吸控制率提升（P<0.05）。③与NC组比较，HFFC组LAMP-2A、P62、LC3共定位水平及LC3-Ⅱ/Ⅰ、LAMP-2A表达下降（P<0.01），P62表达升高（P<0.01）；运动后肝脏自噬水平显著提高（P<0.01）。④与NC组比较，HFFC组LONP1、mtHSP70、mtHSP60、ClpP表达降低（P＜0.05）；运动后UPRmt相关蛋白表达提高。⑤与NC组比较，HFFC组线粒体Parkin下降，胞浆Parkin、线粒体P62升高（P<0.01）；运动组线粒体Parkin招募增多、LC3-Ⅱ升高、P62降低，线粒体自噬水平显著提高。 结论：8周自主跑轮运动可提高MAFLD小鼠肝脏线粒体功能，上调UPRmt及线粒体自噬水平，降低线粒体ROS，有效缓解代谢相关性脂肪性肝病。

Abstract:

      Abstract Objective： To explore the mechanism by which exercise alleviates hepatic lipid deposition， and to clarify the roles of mitophagy and mitochondrial unfolded protein response （UPRmt） in the exercise-mediated regulation of hepatic lipid deposition. Method： Thirty 8-week-old male C57BL/6J mice were randomly divided into three groups： normal chow group （NC group）， high-fat， high-fructose， high-cholesterol diet group （HFFC group）， and high-fat， high-fructose， high-cholesterol diet plus exercise group （HFFC+EX group）， with 10 mice in each group. The HFFC+EX group underwent 8 weeks of voluntary wheel running. Hematoxylin-eosin (HE) and BODIPY staining were used to observe liver histopathology; the JC-1 assay was used to detect the mitochondrial membrane potential. A reactive oxygen species （ROS） assay kit was used to measure mitochondrial ROS. the oxygen electrode method was used to determine the mitochondrial respiratory control ratio; and Western blot and immunofluorescence double labeling were used to detect the expression of LAMP-2A， LC3， P62， COXIV， Parkin， VDAC， LONP1， mtHSP60， and mtHSP70. Result：①Compared with the NC group，the hepatocellular steatosis and lipid droplets were significantly increased in the HFFC group （P<0.01）； Compared with the HFFC group, hepatocellular steatosis was alleviated in the HFFC+EX group （P<0.01）.②Compared with the NC group，the HFFC group showed decreased mitochondrial membrane potential，increased ROS (P<0.05), and a reduced mitochondrial respiratory control ratio (P<0.05); Compared with the HFFC group，the HFFC+EX group exhibited a restored mitochondrial membrane potential，significantly decreased ROS（P<0.01）， and an elevated mitochondrial respiratory control ratio (P<0.05）.③Compared with the NC group，the HFFC group had lower colocalization levels of LAMP-2A， P62，and LC3，reduced LC3-II/I and LAMP-2A expression (P<0.01), and increased P62 expression (P<0.01); the hepatic autophagy level was significantly enhanced after exercise（P<0.01）.④Compared with the NC group，the expressions of LONP1，mtHSP70，mtHSP60，and ClpP were decreased in the HFFC group （P<0.05）； the expressions of UPRmt-related proteins were increased after exercise.⑤Compared with the NC group，the HFFC group showed decreased mitochondrial Parkin，and increased cytoplasmic Parkin and mitochondrial P62（P<0.01）； the HFFC+EX group had increased mitochondrial Parkin recruitment，elevated LC3-II，reduced P62，and a significantly improved mitophagy level. Conclusion： Eight weeks of voluntary wheel running can improve hepatic mitochondrial function， upregulate UPRmt and mitophagy, reduce mitochondrial ROS， and effectively alleviate metabolic associated fatty liver disease in mice.