LaminB1-dependent Nucleophagy Regulates Lipid Metabolism To Mediate Hepatocyte Steatosis

Objective: To explore the changes of nucleophagy and LaminB1 in hepatocyte steatosis induced by oleic acid(OA),to study the mechanism of LaminB1-dependent nucleophagy in hepatocyte steatosis,and to verify whether LaminB1 can reduce liver lipid deposition by regulating nucleophagy-mediated lipid metabolism pathway,which provides a new theoretical basis for using LaminB1 and nucleophagy as therapeutic targets for hepatocyte steatosis.Methods:1.Cultured LO2 cells in cell incubator divided into control group(0h group)and OA-induction group(3h,6h,12 h,24h).They were treated with0.5Mm OA to induce LO2 cells steatosis,the changes of TG in each group were detected,and oil red “O” staining observed the changes of lipid droplets at different induction time points,to evaluate the degree of lipid degeneration in LO2 cells.2.Nuclear and Cytoplasmic Extraction kit separates the cytoplasmic proteins and nuclear proteins.Western Blot separately detected the expression of cytoplasmic LC3Ⅱ/Ⅰ and LaminB1 in cytoplasmic and nuclear.3.According to the purpose of the experiment,LO2 cells cultured in cell incubator were divided into control,OA,OA+SiRNA-LMNB1,OA+pEGFP-h-LMNB1 and OA+NC group.the expression of LC3Ⅱ/Ⅰ and p62 in cytoplasm and nucleus,the degree of hepatocyte steatosis and the expression of lipid metabolism related proteins SREBP1 c,FASN and ATGL were detected.Results:1.The results of hepatocyte steatosis showed that the TG and lipid droplets in LO2 hepatocytes increased with the induction time after 0.5m M OA induction.2.Changes of nucleophagy-associated proteins after OA-induced steatosis.Compared to 0h,the lever of LaminB1 in nucleus decreased and LC3Ⅱ/Ⅰ in nucleus increased at 3 hours,with the prolongation of OAinducted time,nuclear LaminB1 accumulated and the ratio of nuclear LC3Ⅱ/Ⅰ showed a downward trend;In the cytoplasm,LaminB1 expression was not detected,and the change of LC3Ⅱ/Ⅰ was not statistically significant at 3 hours,and then the ratio of cytoplasmic LC3Ⅱ/Ⅰ decreased.3.The effect of LaminB1 on the expression of nucleophagy-related proteins showed that SiRNA-LMNB1 increased nuclear LC3Ⅱ/Ⅰ ratio and down-regulated nuclear p62 expression.P-EGFP-h-LMNB1 decreased nuclear LC3Ⅱ/Ⅰ ratio and upregulated nuclear p62 expression.4.The effect of LaminB1 on hepatocyte steatosis showed that SiRNALMNB1 decreased the content of intracellular TG and lipid droplets,and p-EGFP-h-LMNB1 promoted the increase of intracellular TG content and the accumulation of lipid droplets.5.The effect of LaminB1 on the lipid metabolism related proteins showed that SiRNA-LMNB1 promoted the expression of ATGL,p-EGFPh-LMNB1 inhibited the ATGL expression compared to OA group.Both had no effect on the expression of SERBP1 c and FASN.Conclusion:1.In the process of hepatocyte steatosis induced by OA,LaminB1 degradation was inhibited,resulting in LaminB1 accumulation,and nucleophagy were inhibited.2.Silencing LMNB1 could induce nucleophagy to promote the lipolytic protein ATGL expression,thus alleviating hepatocyte steatosis.And did not affect the lipid generating proteins SERBP1 c and FASN expression.