Exosomes Derived From Lipotoxic Hepatocytes In Inducing LSEC Capillarization And Promoting The Progression Of Nonalcoholic Fatty Liver Disease

Objective:Non-alcoholic fatty liver disease(NAFLD)is a worldwide high incidence of liver disease,and its incidence rate continues to rise.At present,there is a lack of ideal NAFLD drugs.The changes of hepatic sinusoidal capillarization caused by the loss of fenestra and dysfunction of hepatic sinusoidal endothelial cell(LSEC)are independent risk factors for the occurrence and development of NAFLD,but the specific mechanism of LSEC capillarization is not clear.Hepatocyte exosomes play an important role in the occurrence and development of chronic liver diseases such as NAFLD.The purpose of this study is to explore the effect and mechanism of lipotoxic hepatocytes derived exosome(LTH-ex)regulating LSEC capillarization and promoting NAFLD,so as to provide a new target for the treatment of NAFLD.Methods:(1)Human hepatocyte line LO2 were isolated and cultured.LO2 were co cultured with oleic acid and palmitic acid mixture(OPA)for 24 hours,the steatosis of LO2 was observed by Nile red staining to prepare lipotoxic hepatocytes(LTH).(2)LO2derived exosome(LO2-ex)and lipotoxic hepatocytes derived exosome(LTH-ex)were extracted by ultracentrifugation.The particle size and number of LO2-ex and LTH-ex were detected by nano particle tracer analyzer(NTA),and the structure and morphology of LO2-ex and LTH-ex were observed by transmission electron microscope(TEM).The expression of LO2-ex and LTH-ex exosome marker proteins CD9,CD63,Alix,TSG101,CD81 and Calnexin were detected by Western blot and flow cytometry.(3)LO2-ex and LTH-ex were co cultured with LSEC for 24 hours to observe the effects of LO2-ex and LTH-ex on the capillarization of LSEC,the expression of capillarization markers,endothelial cell relaxation dysfunction,endothelial cell angiogenesis and the loss of endothelial cell fenestra were detected.(4)The protein expression level of Olinked N-Acetylglucosamine transferase(OGT)in LO2 induced by lipotoxicity was detected by Western blot,real-time fluorescence quantitative PCR and cellular immunofluorescence staining.LO2-ex and LTH-ex were labeled with CM-DIL,and the distribution of LO2-ex and LTH-ex in LSEC was observed by laser confocal microscope.Western blot was used to detect the protein expression level of OGT in LO2-ex and LTH-ex.The OGT protein expression and O-Linked N-Acetylglucosamine(O-Glc NAc)modification of LSEC treated with LO2-ex and LTH-ex were detected by Western blot,and the co localization of O-Glc NAc and angiopoietin 2(Ang-2)was detected by cellular immunofluorescence staining.(5)The O-Glc NAc modification of LSEC was induced.The effect of elevated O-Glc NAc modification on the capillarization of hepatic sinusoidal endothelial cells was verified by Western blot,cellular immunofluorescence staining and angiogenesis experiments.(6)NAFLD mouse model induced by high fat diet(HFD)was constructed,and LO2-ex and LTHex were injected into caudal vein.HE staining,oil red O staining,Sirius red staining,immunohistochemical fluorescence staining and enzyme-linked immunosorbent assay were used to evaluate the effect of liver tissue repair and injury,and to clarify the role of LO2-ex and LTH-ex in the occurrence and development of hepatic sinusoidal capillarization and NAFLD.Results:(1)The release of hepatocyte exosomes increased after lipotoxic injury.LO2-ex and LTH-ex have typical characteristics of exosomes.(2)LO2-ex can inhibit LSEC tube forming and maintain LSEC window opening,while the tube forming capacity of LSEC increased and the number of window holes decreased after LTH-ex.(3)After LTH-ex intervention,NAFLD mice decreased liver index,increased body weight,fat droplets and collagen deposition,increased serum Ang-2 level,decreased angiopoietin1(Ang-1)level and increased the ratio of Ang-2 to Ang-1.(4)The expression of OGT in hepatocytes injured by lipotoxicity increased and OGT was enriched in LTH-ex.LTH-ex could promote the expression of OGT and the level of O-Glc NAc modification in LSEC.(5)Inducing O-Glc NAc modification can promote Ang-2 expression in LSEC and angiogenesis in vitro.Conclusion: The exosomes released by hepatocytes with lipotoxic injury may promote the O-Glc NAc modification of LSEC and enhance the capillarization of LSEC and the progress of NAFLD by transporting OGT.