Research Towards The Effects And Mechanisms Of Nobiletin On NAFLD Via Lipophagy-Inflammation Pathway And Lipidomic Study

ObjectiveNon-alcoholic Fatty Liver Disease(NAFLD)was characterized as excess lipid deposition in liver without excessively drinking alcohol and other clear causes.Nobiletin(NOB),a citrus flavonoid,was considered as a promising candidate for NAFLD treatment while there is limited research towards its exact mechanism.This study was performed to figure out whether the anti-NAFLD effect of NOB was based on lipophagy and inflammatory response as well as lipid alternation.Method and materials1.The animal model of NAFLD was built by feeding APOE-/-mice with HFD for 24 weeks.In the meantime,50,100,200 mg/kg/d NOB were used as the the administration dosages of L-NOB,M-NOB,H-NOB,respectively.The lipid deposition in mice model of NAFLD were observed by H&E staining,Oil red O staining and biochemical tests.And the expression levels of autophagy,NLRP3 inflammasome and M1/M2 macrophages markers of liver macrophages were determined by western blot and immunohistochemistry.2.The cell model of NAFLD was built by treating HepG2 cell with FFA for 24 hours.In the meantime,20,30,40μg/mL NOB were used as the the administration dosages of L-NOB,M-NOB,H-NOB,respectively.The intracellular lipid deposition in cell model of NAFLD were observed by Oil red O staining and BODIPY staining.And the expression levels of autophagy,NLRP3 inflammasome were detected by western blot and cell fluorescence staining.Using the autophagy inhibitor chloroquine to detect the association between autophagy and lipid clearance.Fluorescence technique was used to study the lipophagy level in hepatocytes and the nuclear translocation of the autophagy regulator TFEB under NOB administration.In addition,RAW264.7 cells were treated with FFA and DHA to induce M1 and M2 macrophages,respectively.And the expression levels of M1/M2 macrophages markers of liver macrophages were determined by QPCR technique.3.The role of lipid alternation in animal model of NAFLD was determined by lipidomic technique based on LC-MS.PCA and PLS-DA were employed to screen out the significantly different lipid.ROC curve was used to identify the potential lipid biomarkers.The association among lipid biomarkers were displayed by correlation analysis.Results1.NOB ameliorated the weight gaining of APOE-/-mice and decreased the lipid deposition and hepatocellular injury.The serum and hepatic triglycerides(TG)and total cholesterol(TC)as well as hepatic low density lipoprotein cholesterol(LDLC)were also decreased by NOB treatment.Besides,NOB restrained the enlargement of adipocytes caused by HFD.2.NOB enhanced the expression of the markers of autophagy(LC3 and Beclinl)and the positive regulators of autophagy(TFEB,RAB7,LAMP1)in HFD-fed APOE/-mice.In FFA-treated HepG2 cells,the protein level of LC3 was elevated by NOB,and MDC staining presented more autophagosomes in NOB administration group than that in model group.Furthermore,CQ pretreatment increased the expression of LC3II and the production of autophagosomes,but caused more lipid droplets(LDs)than other groups.Besides,co-localization of LDs and lysosomes was enhanced in the presence of NOB.Additionally,NOB treatment increased nuclear translocation of TFEB and enhanced the number and function of lysosomes.3.The protein expression of NLRP3 and IL-1β were decreased by NOB in HFD-fed APOE-/-mice,and the protein level of NLRP3 and Pro-caspase-1 were ameliorated by NOB in FFA-treated HepG2 cells.Additionally,the liver macrophages polarization was modulated significantly by NOB,showing that the number of M1 macrophages were decreased while the percent of M2 macrophages were upregulated.4.Lipidomic analysis revealed significant lipid alternation among groups,mainly including GPs and GLs.There were 86,116,212 lipids altered after L-NOB,MNOB,and H-NOB administration comparing that in model group,respectively.And a total of 958 lipid metabolites were changed by HFD in model group comparing the control group.Further analysis revealed 54 lipid biomarkers,which were mainly made of GPs and GLs.Lipids in the same categories showed different change pattern but obvious and close correlation were displayed among them.ConclusionsNOB could significantly ameliorate NAFLD mainly via enhancing lipophagy,mitigating NLRP3 inflammasome,modulating macrophages polarization and hepatic lipid alternation.