Inhibitory Effect Of PXR On Ammonia-induced Hepatocyte Autophagy Via P53

Objective:Ammonia is a toxic by-product produced from the metabolism of nitrogenous in the body,and is also the main biotoxin that induces hepatic encephalopathy.Recent studies have found that autophagy can be induced by ammonia and improves hepatocyte urea cycle and energy metabolism system to facilitate cell survival.Pregnane X receptor(PXR)and other nuclear receptors are involved in a wide range of physiological activities as the transcription factor and are the main regulators of drug metabolism and drug resistance.But the involvement and the role of PXR in ammonia-induced autophagy has not been reported.This study explores the effect of PXR on ammonia-induced hepatocyte autophagy and its molecular mechanism,providing a valuable reference for clinical application of PXR activators.Methods:Stably transfected liver cell lines with PXR overexpression(PXR~+)and PXR knockdown(PXR~-)were constructed.The expression of autophagy biomarkers(LC3B and SQSTM1)were detected by western blot in cells treated with 3 mM,5mM,10 mM,and 15 mM ammonia concentrations and the formation of autophagosome/autophagic lysosome were observed by electron microscopy under the optimal ammonia concentration,to analyze the changes in hepatocyte autophagy with PXR~+and PXR~-and also investigate the effect of rifampicin,an exogenous PXR activator,on ammonia-induced autophagy in the hepatocytes.By detecting the phosphorylation level of P53 and AMPK subunits in PXR~+and PXR~-hepatocytes,the activation of P53/AMPK signal axis after ammonia induction and the effect of PXR on the P53/AMPK signal axis were analyzed.The full-length and truncated fragments of the AMPKβ1 promoter were constructed into the luciferase reporter plasmid,and the regulatory activity of the p53 transcription factor on AMPKβ1 was analyzed by dual-luciferase reporter assey system.The potential p53 binding site on the AMPKβ1promoter was analyzed using online transcription factor prediction software,and a mutation reporter vector targeting this site was constructed and co-transfected with p53 to detect the change in double fluorescein activity.ChIP assay was used to analyze the direct binding region of P53 in AMPKβ1 promoter in vivo.The AMPKβ1promoter was co-transfected P53 with or without PXR expression plasmids,and the effect of PXR on p53 transcriptional activity was analyzed using the dual-luciferase reporter assey.Finally,the endogenous protein-protein interaction between PXR and p53 was further analyzed by CoIP assay.Results:The level of autophagy in PXR~-liver cell line was increased after ammonia induction compare to wild type cell line,which appeared in up-regulated LC3B-II level,and increased degradation of SQSTM1,and the opposite effect was found in PXR~+cells.In addition,the PXR stable activator,rifampicin,elicits a similar effect as PXR~+cells.Under ammonia stimulation,the AMPKβ1 subunit and its phosphorylated Ser108(p-AMPKβ1)level were significantly higher in the PXR~-cell line than in the wild-type control cell line.The P53 total protein and phosphorylated protein increased after PXR knockdown.Mechanism analysis of the inhibitory effect showed that There are two adjacent P53 binding active sites in the AMPKβ1 promoter region(-253nt~-19nt),and simultaneous mutation of both two sites can significantly reduce P53-dependent AMPKβ1 transactivation.P53 can directly bind to AMPKβ1 in the(-253nt~-19nt)promoter region.The expression of PXR inhibits the transcriptional regulation of P53 on AMPKβ1 and there is a protein interaction between PXR and P53.Conclusions:The expression and activation of PXR inhibits ammonia-induced autophagy in hepatocytes.Ammonia specific activates the autophagy regulatory protein P53 and AMPKβ1 subunit,while PXR inhibits protein expression levels of both P53 and AMPKβ1.The P53 binding site exists in the AMPKβ1 promoter(-253nt~-19nt)region,which up-regulates the transcription level of AMPKβ1.PXR inhibits the transcriptional activity of P53 by interacting with P53 and decreased the expression of AMPKβ1 induced by ammonia at the transcriptional level.