Study On The Effect Of Targeting PDPK1 With Phillyrin On Alleviating Alcoholic Steatohepatitis

BACKGROUND: Excessive alcohol consumption poses a great threat to human health.The liver,as the main organ of alcohol metabolism,bears the brunt of excessive alcohol consumption.Liver damage caused by alcohol consumption can be classified according to the disease progression: Alcoholic steatohepatitis(ASH),liver fibrosis,cirrhosis,and liver cancer.Alcoholic steatohepatitis is a critical period in the progression of alcoholinduced disease to the refractory stage,where effective treatment measures can be taken to maximize recovery of liver function.However,the pathogenesis of alcoholic steatohepatitis and effective therapeutic agents remain to be explored.Autophagy can play a key role in ASH disease progression,and 3-phosphatidylinositol-dependent protein kinase 1(PDPK1,also known as PDK1)has the potential to regulate cellular autophagy levels.However,the role of PDPK1 in ASH disease is currently unknown.Few PDPK1-based targeted therapeutic agents have been reported.PURPOSE: The study focused on exploring the role of PDPK1 in ASH disease and related therapeutic drugs,and discovering new ideas for the treatment of ASH.METHODS: In vivo,an ASH mouse model was constructed using Gao-Binge protocol in C57BL/6J mice,and H&E staining,Oil Red O staining and mouse serological indicators(TG,T-CHO,ALT,AST)were used to indicate the successful construction of the ASH model,Transmission electron microscopy was used to observe autophagosome,and IHC and WB results were used to detect the expression of PDPK1.Mice in the ASH model group were given 1 mg/kg of the autophagy-inducing agent rapamycin for treatment to verify the effect of autophagy on ASH.Afterwards,mice were treated with the PDPK1-targeting drug Phillyrin(PHI)(low dose group: 5 mg/kg;medium dose group:15 mg/kg;high dose group: 45 mg/kg)to verify the therapeutic effect of Phillyrin on alcoholic steatohepatitis.In vitro,SPR and CETSA assays demonstrated that Phillyrin was a targeting agent for PDPK1.Molecular docking analysis of the effect of Phillyrin on PDPK1 function.Knockdown and overexpression of PDPK1 were performed on AML-12 cells to verify the function of PDPK1 in ASH.AML-12 cells stimulated with 200 m M ethanol were treated with PDPK1-targeting agent,Phillyrin(200 μg/m L),to verify the ameliorative effect of Phillyrin on ethanol-induced disorders of lipid metabolism.RESULTS: In in vivo experiments,PDPK1 was significantly elevated in the liver of mice in the model group;administration of rapamycin,an autophagy inducer in ASH mice,revealed that promoting autophagy could alleviate alcoholic steatohepatitis;and administration of Phillyrin,a PDPK1-targeting agent in ASH mice,significantly improved the level of liver autophagy,liver injury and lipid metabolism disorders caused by alcohol in mice.In in vitro experiments,SPR and CETSA results demonstrated that Phillyrin was a target drug for PDPK1.Molecular docking revealed that Phillyrin could occupy the activation pocket of PDPK1 and inhibit its phosphorylation.PDPK1-targeting drug Phillyrin inhibited the phosphorylation of PDPK1 ser241 site and promoted cellular autophagy,thereby ameliorating alcohol-induced disorders of lipid metabolism.