1.Effect of serum deprivation on lipid metabolism in HepG2 cells through autophagolysosome pathway and its mechanisms
Weifang SONG ; Qiuyan JIANG ; Ruixin YAO ; Shengnan LI ; Ting SHI ; Ha-ijuan ZHANG ; Xiaofeng LIANG
Chinese Journal of Pathophysiology 2024;40(12):2295-2301
AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolyso-somes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L so-dium 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 expres-sion of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated pro-tein 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(P＜0.05).(4)Knockdown of TFEB did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,TC,TG,LDL-C,ALT and AST compared to the so-dium oleate group.(5)Inhibition of AMPK phosphorylation did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,and AMPK phosphorylation in the sodium oleate+serum deprivation group com-pared to the sodium oleate group.CONCLUSION:Serum deprivation improves sodium oleate-induced lipid metabolism damage in HepG2 cells through the autophagolysosome pathway mediated by AMPK-TFEB.
2.Effect of serum deprivation on lipid metabolism in HepG2 cells through autophagolysosome pathway and its mechanisms
Weifang SONG ; Qiuyan JIANG ; Ruixin YAO ; Shengnan LI ; Ting SHI ; Ha-ijuan ZHANG ; Xiaofeng LIANG
Chinese Journal of Pathophysiology 2024;40(12):2295-2301
AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolyso-somes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L so-dium 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 expres-sion of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated pro-tein 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(P＜0.05).(4)Knockdown of TFEB did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,TC,TG,LDL-C,ALT and AST compared to the so-dium oleate group.(5)Inhibition of AMPK phosphorylation did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,and AMPK phosphorylation in the sodium oleate+serum deprivation group com-pared to the sodium oleate group.CONCLUSION:Serum deprivation improves sodium oleate-induced lipid metabolism damage in HepG2 cells through the autophagolysosome pathway mediated by AMPK-TFEB.
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