Relationship Between PEMT And Mitophagy During The Progression Of NAFLD And The Mechanism Of Nuciferine Intervention

ObjectiveRecently,the morbidity of non-alcoholic fatty liver disease(NAFLD)increases year by year.NAFLD has become the major chronic liver disease worldwide.NAFLD can develop into liver fibrosis,liver cirrhosis and even hepatocellular carcinoma and is a serious threat to human health.Till now,the pathogenesis of NAFLD is still unclear.Deeply elucidating the pathogenesis of NAFLD can further enhance the understanding of this disease.Hepatic steatosis is the major pathological feature of NAFLD.Mitochondria play crucial role in maintaining the homeostasis of cellular lipid metabolism and are always the major topic in NAFLD studies.Many factors such as diet can cause the accumulation of lipid contents in hepatocytes and further induce the damage of mitochondria.These damaged mitochondria can aggravate hepatic steatosis and contribute to the progression of NAFLD when they cannot be eliminated timely.PINK1/Parkin-mediated mitophagy is a classic ubiquitin-dependent mitophagy and is one of the major approaches for eliminating damaged mitochondria in cell.Studies have found that the level of PINK1/Parkin-mediated mitophagy is reduced in hepatocytes in NAFLD model and this could trigger the accumulation of damaged mitochondria.Elucidating the modulatory mechanisms for initiating PINK1/Parkinmediated mitophagy provides new thoughts for developing drugs in the treatment of NAFLD.As the major lipid contents of cell membrane,glycerophospholipid serves as an important content during the formation of autophagosome membrane.The change in glycerophospholipid metabolism is an important influence factor in initiating autophagy.Phosphatidylethanolamine N-methyltransferase(PEMT),a key enzyme in modulating glycerophospholipid metabolism,can catalyze the transformation from phosphatidylethanolamine(PE)to phosphatidylcholine(PC).The change in PEMT expression is closely related to the progression of NAFLD.Currently,some studies have found that PEMT may modulate the initiation of PINK 1/Parkin-mediated mitophagy.However,the detailed relationship between PEMT and PINK1/Parkinmediated mitophagy in NAFLD has not been studied.Therefore,clinical,animal and cell experiments were conducted in current study,in order to deeply elucidate the detailed relationship between PEMT and PINK 1/Parkin-mediated mitophagy in NAFLD and to enhance the understanding of metabolic regulatory mechanisms on autophagy for researchers.Natural products have been popular in drug development.Nuciferine is a aporphine alkaloid derived from lotus leaf.Many studies have reported the lipidlowering effects of nuciferine and some studies have demonstrated the therapeutic effect of nuciferine on NAFLD.Moreover,nuciferine can activate autophagy.However,what are the metabolic regulatory mechanisms of nuciferine on NAFLD?Can nuciferine activate autophagy to improve NAFLD through affecting host metabolism?These questions are still need to be solved.Therefore,our current study aims to explore the mechanism of nuciferine on NAFLD at metabolic and signaling transduction levels based on elucidating the role of PEMT and PINK1/Parkin-mediated mitophagy in NAFLD.Our study can provide molecular biological evidences for nuciferine in the treatment of NAFLD and can enrich the theoretical basis for popularization and application of nuciferine in the treatment of NAFLD.Research Contents(1)Changes in PEMT and PINK1/Parkin-Mediated Mitophagy Levels in Liver in NAFLD PatientsLiver tissues were collected via liver biopsy in normal control(hepatic cyst and hepatic hemangioma)and NAFLD patients.The clinical indices were also collected.Immunohistochemistry and immunofluorescence were used to detect the expression of PEMT and factors related to PINK1/Parkin-mediated mitophagy in liver.The correlations between clinical indices and PEMT and factors related to PINK1/Parkinmediated mitophagy expression were also analyzed.(2)Changes in PEMT and PINK1/Parkin-Mediated Mitophagy Levels in Liver During the Progression of NAFLD in RatsHigh-fat diet(HFD)was used to induce NAFLD in rats and the changes in mitochondrial function,PC/PE ratio and levels of PEMT and factors related to PINK1/Parkin-mediated mitophagy during the progression of NAFLD were observed using several techniques such as RT-qPCR,western blot,immunohistochemistry and immunofluorescence.(3)Modulatory Effects of PEMT on PINK1/Parkin-Mediated Mitophagy in FFA-Induced Hepatic Steatosis Cell ModelFree fatty acid(FFA)was used to generate hepatic steatosis model in vitro and PEMT was over-expressed and knocked out in cell respectively.The effects of PEMT on hepatic steatosis,PC/PE ratio and PINK1/Parkin-mediated mitophagy were analyzed.(4)Effects of Nuciferine on NAFLD Model Rats and the Changes in Metabolite LevelsRats received HFD for 12 weeks to induce NAFLD model and nuciferine(20mg/kg)was orally treated to rats for 8 weeks after HFD feeding for 4 weeks.The therapeutic effects of nuciferine on NAFLD rats were firstly evaluated.Then,untargeted metabolomics was used to study the changes in serum metabolites in NAFLD rats after nuciferine treatment.The metabolic pathways related to the differential metabolites were enriched and the expression of key enzymes in metabolic pathways were validated.(5)Effects of Nuciferine on PEMT Expression and PINK1/Parkin-Mediated Mitophagy in NAFLD Model RatsIn order to study the effects of nuciferine on PEMT and PINK 1/Parkin-mediated mitophagy,the expression of PEMT and PINK1/Parkin-mediated mitophagy was detected in liver after nuciferine treatment based on the untargeted metabolomics data.(6)Mechanic Study of Nuciferine on FFA-Induced Hepatic Steatosis Cell Model Based on Upregulating PEMT and Inducing the Activation of PINK1/Parkin-Mediated MitophagyFFA was used to generate hepatic steatosis model in vitro and PEMT gene was knocked out in cell.The effects of nuciferine on hepatic steatosis,PEMT expression,and PINK 1/Parkin-mediated mitophagy before and after PEMT knocked out were evaluated.Results(1)The expression of PEMT was decreased and PINK1/Parkin-mediated mitophagy was inhibited in liver of NAFLD patients.PEMT and PINK 1/Parkinmediated mitophagy showed negative correlations with clinical indices in NAFLD patients and PEMT showed positive correlation with PINK 1/Parkin-mediated mitophagy.(2)In HFD-induced NAFLD model rats,the ratio of PC/PE,the levels of PEMT and PINK1/Parkin-mediated mitophagy were decreased with the extension of HFD feeding.(3)In FFA-induced hepatic steatosis cell model,the structures of mitochondria were damaged,the membrane potentials of mitochondria were reduced and the levels of PEMT and PINK1/Parkin-mediated mitophagy decreased with the extension of FFA intervention.(4)Hepatic steatosis was aggravated and the PINK1/Parkin-mediated mitophagy was further inhibited in hepatocytes after PEMT knocked out;overexpression of PEMT could alleviate hepatic steatosis and induce the initiation of PINK1/Parkin-mediated mitophagy.(5)Nuciferine exhibited therapeutic effects on NAFLD rat model.Besides,nuciferine could affect the levels of metabolites related to glycerophospholipid,linoleic acid,alpha-linolenic acid,arginine and proline metabolism processes.(6)Nuciferine treatment upregulated the expression of PEMT and factors related to PINK1/Parkin-mediated mitophagy in liver in NAFLD model rats.(7)Treatment of nuciferine alleviated the hepatic steatosis and upregulated the expression and induce the activation of PINK 1/Parkin-mediated mitophagy in vitro.The effects of nuciferine on hepatic steatosis and PINK1/Parkin-mediated mitophagy were totally abolished in vitro after PEMT knocked out.Conclusions(1)The decreased of PEMT expression could cause the dysfunction of glycerophospholipid metabolism,inhibit the activation of PINK1/Parkin-mediated mitophagy,aggravate hepatic steatosis and further contribute to the progression of NAFLD.(2)Nuciferine could modulate glycerophospholipid metabolism,induce the activation of PINK1/Parkin-mediated mitophagy and alleviate NAFLD through upregulating the expression of PEMT.