LASS2通过诱导mtROS激活自噬抑制胆固醇合成改善非酒精性脂肪肝脂代谢紊乱

LASS2 improves lipid metabolism of non-alcoholic fatty liver disease by inducing mtROS to activate autophagy and suppress cholesterol synthesis

摘要

目的 明确人源长寿保障基因（LASS2）过表达或敲低在非酒精性脂肪性肝病（NAFLD）中的作用，并初步探讨其分子机制。方法 采用高脂饮食（HFD）喂养Ｃ57ＢＬ／6Ｊ小鼠建立NAFLD动物模型，游离脂肪酸（FFAs）诱导小鼠原代肝细胞（MPHs）脂肪变性。通过腺病毒载体在体内外构建LASS2过表达或敲低模型，检测各组小鼠肝重、肝脏脂肪含量、血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、甘油三酯（TG）、总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）及肝脏TG、TC水平。采用Western blot检测胆固醇代谢及自噬通路相关蛋白表达水平；利用MitoSOX^TM Ｒｅｄ探针标记活细胞成像检测线粒体活性氧（mtROS）水平。结果 与对照组相比，HFD诱导后小鼠肝重、血清ALT、AST、TG、TC、HDL-C、LDL-C及肝脏TG、TC水平均显著升高，而LASS2过表达可明显降低上述指标（P<0.05）。RT-qPCR及Western blot结果显示，LASS2过表达显著抑制SREBP2及HMGCR的mRNA和蛋白表达水平（P<0.05）。活细胞成像显示，LASS2过表达诱导mtROS生成增加（P<0.05）。Western blot结果显示，LASS2过表达显著上调LC3-II/LC3-I比值及Beclin-1、ATG3、ATG5、ATG7蛋白表达水平，同时下调ｐ62蛋白表达（P<0.05）。LASS2敲低则产生相反的效应。结论 LASS2可能通过诱导mtROS生成激活自噬，进而抑制胆固醇合成，改善脂代谢紊乱，为NAFLD的治疗提供潜在新靶点。

Abstract

Objective To determine the role of longevity assurance homolog 2 (LASS2) overexpression or knockdown in non-alcoholic fatty liver disease (NAFLD) and to explore the underlying molecular mechanisms. Methods NAFLD was induced in C57BL/6J mice by feeding a high-fat diet (HFD). Mouse primary hepatocytes (MPHs) were treated with free fatty acids (FFAs) to establish steatosis, LASS2 was overexpressed or knocked down both in vivo and in vitro using adenovirus vectors. Liver weight, hepatic fat content, and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), as well as hepatic TG and TC levels were measured in each group. Western blot was used to detect protein levels related to cholesterol metabolism and the autophagic pathway. Imaging of live cells labelled with the MitoSOX^TM Red probe to detect levels of mitochondrial reactive oxygen species (mtROS). Results Compared with controls, HFD-fed mice showed significant increases in liver weight, and serum levels of ALT, AST, TG, TC, HDL-C, and LDL-C, as well as hepatic TG and TC levels (P<0.05). Overexpression of LASS2 markedly reduced all these parameters (P<0.05). RT-qPCR and Western blot revealed that LASS2 overexpression strongly suppressed SREBP2 and HMGCR at both mRNA and protein levels (P<0.05). Live-cell imaging showed that LASS2 overexpression increased mtROS production (P<0.05). Western blot further demonstrated that LASS2 overexpression raised the LC3-II/LC3-I ratio and the protein levels of Beclin-1, ATG3, ATG5, and ATG7, while decreasing p62 expression (P<0.05). Knockdown of LASS2 produced the opposite effects. Conclusion LASS2 may activate autophagy by promoting mtROS production, thereby suppressing cholesterol synthesis and improving lipid metabolism disorders. These findings suggest LASS2 as a potential new therapeutic target for NAFLD.

关键词

人源长寿保障基因 / 非酒精性脂肪性肝病 / 胆固醇代谢 / 自噬 / 活性氧

Key words

longevity assurance homolog 2 / non-alcoholic fatty liver disease / cholesterol metabolism / autophagy / reactive oxygen species

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基金资助

国家自然科学基金资助项目(81960494)