The Effect Of TFE3 To Hepatocyte Steatosis And The Mechanism Study

BackgroundThe incidence of non-alcoholic fatty liver disease(NAFLD)is increasing year by year,which greatly affects the human health,but up to now,less effective therapeutic strategies has been discoverd.NAFLD is characterized by abundant triglyceride(TG)accumulation in liver cells,which induces hepatocyte steatosis,and may eventually leads to the incidence of liver cirrhosis and hepatocellular carcinoma.Enhancing the degradation ability of TG in early stage may be of guiding significance on the treatment of NAFLD.The latest studies have shown that autophagy may be involved in the degradation of TG,in which TG is sequestered in autophagosomes and delivery to lysosomes to be degraded into fatty acids.The increased autophagy can simultaneously promote the ?-oxidation of fatty acids,thereby to reduce liver lipid accumulation.The transcription factor E3(TFE3)has been reported as an important gene involved in the regulation of lysosome biogenesis and autophagy,but whether TFE3 can affect NAFLD through the regulation of autophagy has not been reported yet.ObjectiveThis study was designed to observe the effect of TFE3 on lysosome biogenesis,occurrence of autophagy,and ?-oxidation function during the process of hepatocyte steatosis,and to deeply explore the effect and the mechanism of TFE3 on hepatocyte steatosis,providing new ideas for the prevention and treatment of NAFLD.Methods1.The mouse NAFLD model was established with high-fat diet for 16 weeks;1 mM FFA(oleic acid : palmitic acid = 2:1)was used to stimulate hepatic L02 cells for 24 hours to establish the hepatocyte steatosis model;Western blot were used to detect theexpression of autophagy-related genes in the two models.2.Stably transfected cell lines with overexpression of TFE3 or silencing of TFE3 were constructed through the lentivirus-mediated gene regulation technology,respectively;in hepatocyte steatosis model,the expressions of genes related to lysosome biogenesis and autophagy were detected in both stably transfected cell lines by using real time PCR and Western blot;lysosome biogenesis and autophagy were observed by the transfection of pEGFP-LC3 plasmid and Lyso-Tracker Red staining of lysosomes.3.The effect of TFE3 on lipid deposition was observed by oil red O staining,and the difference of the contents of TG in L02 cells was measured.Autophagy occurrence was blocked by using Atg5 siRNA and then the role of autophagy on the hepatic steatosis regulated by TFE3 was studied.4.The expressions of fatty acid ?-oxidation-related genes were detected by real-time PCR and Western Blot,respectively.The expression of PGC1? was silenced by PGC1? siRNA and then the role of PGC1? on the fatty acid ?-oxidation regulated by TFE3 was studied.5 Transcriptional regulation of TFE3 on PGC1? was performed by chromatin immunoprecipitation and dual-luciferase reporter assay system.Results 1.In both mouse NAFLD model and L02 hepatocyte steatosis model,expressions of autophagy markers LC3-II and Atg5 were decreased,and the expressions of CTSL and VPS11,representatives of lysosomal function,were also decreased.2.Over-expression of TFE3(LV-TFE3)significantly increased the expressions of genes related to lysosome biogenesis and autophagy occurrence;on the contrary,silencing of TFE3(LV-shTFE3)decreased the expressions of genes related to lysosome formation and autophagy.3.LV-TFE3 significantly alleviated hepatocyte steatosis,whereas LV-shTFE3 enhanced it.After the autophagy was blocked,the mitigative effect of LV-TFE3 on hepatocyte steatosis was almost disappeared.4.LV-TFE3 significantly improved the respiratory function of mitochondria and promote the fatty acid ?-oxidation.After the expression of PGC1? was interfered,the promotion effect of LV-TFE3 on fatty acid ?-oxidation was decreased significantly and the hepatocyte steatosis was also increased.5.TFE3 could bind to the 2 E-box sequences within the promoter region of PGC1?thus directly regulate the expression of PGC1? at a transcriptional level.Conclusions 1.In the process of hepatocyte steatosis,cell lysosomal function decreases and autophagy is obviously insufficient.2.The expression of TFE3 is decreased in hepatocyte steatosis,while overexpression of TFE3 may increase the biogenesis of lysosomes and the occurrence of autophagy.3.With the overexpression of TFE3,the increased autophagy promotes the decomposition of TG,and the subsequent PGC1?-mediated fatty acid ?-oxidation is enhanced.Therefore the fatty acids derived from the autophagy-mediated decomposition of TG are degraded,thus the hepatocyte steatosis is significantly alleviated.4.TFE3 can directly regulate the expression of PGC1? at a transcriptional level thus regulate hepatocyte steatosis.