血清剥夺通过自噬溶酶体途径对HepG2细胞脂代谢的影响及其机制  

Effect of serum deprivation on lipid metabolism in HepG2 cells through autophagolysosome pathway and its mechanisms

作　　者：宋维芳[1] 蒋秋艳 要睿昕 李胜男 师婷[1] 张海娟[1] 梁晓峰 SONG Weifang;JIANG Qiuyan;YAO Ruixin;LI Shengnan;SHI Ting;ZHANG Ha-ijuan;LIANG Xiaofeng(Fenyang College,Shanxi Medical University,Fenyang 032200,China;Jinan University,Guangzhou 510632,China)

机构地区：[1]山西医科大学汾阳学院,山西汾阳032200 [2]暨南大学,广东广州510632

出　　处：《中国病理生理杂志》2024年第12期2295-2301,共7页Chinese Journal of Pathophysiology

基　　金：山西医科大学汾阳学院院级科研项目(No.2020B06);吕梁市重点研发项目(No.2021SHFZ-2-96)。

摘　　要：目的:探讨血清剥夺通过自噬溶酶体途径对肝癌细胞脂代谢的影响及作用机制。方法:用1mmol/L的油酸钠干预HepG2细胞复制细胞高脂模型,用转录因子EB小干扰转染试剂(TFEB-siRNA)敲低HepG2细胞TFEB的基因表达,用AMP活化蛋白激酶(AMPK)抑制剂化合物C(CC)干预HepG2细胞抑制AMPK磷酸化的表达。通过Western blot实验分析各组核内外TFEB、溶酶体相关膜蛋白1(LAMP1)、微管相关蛋白轻链3(LC3)、细胞自噬接头蛋白(p62)、AMPK和p-AMPK蛋白的表达情况,使用总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、谷丙转氨酶(ALT)和谷草转氨酶(AST)试剂盒分析各组的脂质合成和肝功能损伤的情况,通过油红O染色分析各组细胞脂滴积累情况。结果:(1)油酸钠干预呈浓度梯度显著降低LAMP1、LC3-II/LC3-I及核内TFEB的蛋白表达水平,呈浓度梯度显著升高p62的蛋白表达水平(P<0.01)。(2)与NC组相比,油酸钠组中LAMP1、LC3-II/LC3-I和AMPK磷酸化水平及核内TFEB表达显著降低,p62表达水平显著升高;与油酸钠组相比,油酸钠+血清剥夺联合干预组中核内TFEB、LAMP1、LC3-II/LC3-I和AMPK磷酸化水平显著升高,p62表达水平降低(P<0.05)。(3)与NC组相比,油酸钠组中TC、TG、LDL-C、ALT和AST水平升高;与油酸钠组相比,血清剥夺减少了油酸钠诱导的HepG2细胞中的脂滴数量以及降低了TC、TG、LDL-C、ALT和AST的水平(P<0.05)。(4)TFEB被敲低以后,与油酸钠组相比,油酸钠+血清剥夺组中核内TFEB、LAMP1、LC3-II/LC3-I、p62、TC、TG、LDL-C、ALT和AST表达水平均无显著差异。(5)AMPK磷酸化水平被抑制后,与油酸钠组相比,油酸钠+血清剥夺组中核内TFEB、LAMP1、LC3-II/LC3-I、p62和AMPK磷酸化水平均无显著差异。结论:血清剥夺可通过AMPK-TFEB介导的自噬溶酶体途径减轻油酸钠对HepG2细胞的脂代谢损害。AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolysosomes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L sodium oleate to create a cell high-fat model.The gene expression of transcription factor EB(TFEB)in HepG2 cells was knocked down using TFEB small interfering RNA(TFEB-siRNA)transfection reagent.AMP-activated protein kinase(AMPK)inhibitor compound C(CC)was used to inhibit AMPK phosphorylation expression in HepG2 cells.The expression of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated protein light chain 3(LC3),autophagy adaptor protein(p62),AMPK,and p-AMPK proteins in each group was analyzed through Western blot experiments.Lipid metabolism and liver function damage in each group were analyzed using total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)assay kits.The accumulation of lipid droplets in each group of cells was analyzed through oil red O staining.RESULTS:(1)Sodium oleate intervention resulted in a concentration-dependent decrease in the protein expression levels of LAMP1,LC3-II/LC3-I,and nuclear TFEB,while increasing the protein expression level of p62(P<0.01).(2)Compared to the NC group,the sodium oleate group showed decreased expression of LAMP1,LC3-II/LC3-I,nuclear TFEB,and AMPK phosphorylation levels,with an increase in p62 expression.Compared to the sodium oleate group, the sodium oleate + serum deprivation combined intervention group showed increased nuclear TFEB, LAMP1, LC3-II/LC3-I, AMPK phosphorylation levels, and decreased p62 expression levels (P<0. 05). (3) The levels of TC, TG, LDL-C, ALT, and AST were increased in the sodium oleate group compared to the NC group. Serum deprivation reduced the number of lipid droplets induced by sodium oleate in HepG2 cells and decreased the levels of TC, TG, LDL-C, ALT, and AST compared to the sodium oleate group

关 键 词：血清剥夺 自噬溶酶体 HEPG2细胞 脂代谢 AMPK/TFEB信号通路 

分 类 号：R735.7[医药卫生—肿瘤] R329.21[医药卫生—临床医学] R363