The Role And Mechanism Of Phospholipase D1-mediated Autophagy In Non-alcoholic Fatty Liver Disease

Background and aims: Non-alcoholic fatty liver disease(NAFLD),is a type of liver disease associated with metabolic dysfunction.The global prevalence of NAFLD is as high as 25%.In China,NAFLD has surpassed viral hepatitis and became the primary cause of chronic liver disease and abnormal liver biological indicators in healthy physical examinations.A sedentary lifestyle,lack of physical activity,high-calorie meals,nutritionally unbalanced and unhealthy eating habits are closely associated with the increasing incidence of NAFLD.At present,lifestyle modification is still the main means of prevention and treatment of NAFLD,and there is no effective drug treatment in clinical practice.It is necessary to explore the pathogenesis and key regulatory molecules of NAFLD.It has important scientific value and positive clinical significance to explore specific drugs to achieve more precise intervention for improving the quality of life of NAFLD patients.Numerous studies have shown that phospholipase D1(Phosphlipase D1,PLD1)is related to metabolic diseases.Our previous study found that the expression and activity of PLD1 was increased in the NAFLD mouse model constructed with a high-fat diet,and that PLD1 was involved in the activation of hepatic stellate cells by regulating autophagy through m TOR,and injury of autophagy activity played an important role in NAFLD.Based on the above results,we hypothesized that PLD1 mediated autophagic activity plays an important role in NAFLD.Therefore,in-depth study of the specific regulatory relationship between PLD1 and NAFLD,and to explore the role and mechanism of PLD1-mediated autophagy in NAFLD,can provide an experimental basis for PLD1 to become a new drug target for NAFLD prevention and treatment,which has important scientific value.Materials and methods: In this study,firstly,we collected MAFALD patient specimens;utilized 16-week high-fat diet,16-week high-fat and high-cholesterol diet,and 8-week methionine-choline-deficient diet to construct mice model of NAFLD respectively;used FFA to stimulate Hep G2 cells to construct NAFLD in vitro Model.Then the histomorphological analysis was used to detect lipid deposition,liver enzyme detection kit to detect liver injury indicators,q PCR,WB,and IHC to detect the expression and activity of PLD1,as well as autophagy-related indicators and fatty acid synthesis,β-oxidation,secretion and transport related genes.Then,we used PLD1 overexpression plasmid and PLD1 small interfering RNA to transfect Hep G2 for gain-of-function and loss-of-function experiments to explore the regulation mechanism of PLD1 and NAFLD.Finally,we screened out the PLD1-specific small molecule inhibitor Vu0359595 by molecular screening and virtual docking and verified its effect on autophagy activity and lipid metabolism genes in in vitro and mouse models of NAFLD.Results: In NAFLD patients,animal models,and in vitro cell models,the expression and activity of PLD1 were increased,the expression of autophagy activity indicators LC3II/I and Beclin-1 were decreased,the expression of autophagy substrate p62 was increased,and autophagy activity was impaired.At the same time,the NAFLD models showed abnormal expression of lipid metabolism genes,and increased lipid deposition.Overexpression of PLD1 aggravated the injury of autophagy caused by high-fat sitimulation and promoted lipid deposition in Hep G2cells;knockdown of PLD1 reduced the injury of autophagy caused by high-fat stimulation and reduced lipid deposition in Hep G2 cells.The specific small molecule inhibitor of PLD1 Vu0359595 was screened by molecular screening and virtual docking.Vu0359595 can improve lipid deposition by reducing autophagy damage and influence the expression of lipid metabolism genes in vitro and animal NAFLD models.Conclusion: Altogether,our results indicated that the expression and activity of PLD1 were increased in the pathogenesis and development of NAFLD.The increased PLD1 inhibited autophagy maturation,which further changed the expression of Acc1 and Cpt1α,then promoted lipid accumulation in the progression of NAFLD.The PLD1 inhibitor Vu0359595 could neutralize the inhibitor of autophagy,decrease the lipid accumulation in NAFLD.Therefore,PLD1 is a key regulator in the occurrence and development of NAFLD,and may be a potential target for NAFLD treatment.PLD1-specific small molecule inhibitor may become a new drug for the treatment of NAFLD.