Perilipin 5 Induces Apoptosis In Activated Hepatic Stellate Cells Via AMPK Signaling Pathway

ObjectivesActivation of hepatic stellate cells(HSCs)are considered as the main events during the progression of hepatic fibrogenesis.Induction of apoptosis in activated HSCs contributes to the resolution of liver fibrosis.HSCs contain rich lipid droplets in the cytoplasm at the quiescent stage.Perilipin 5(Plin 5),a coated protein on the surface of lipid droplets,plays a critical role in lipid homeostasis in mammalian cells.However,little is known about Plin 5 in HSC activation.In the current report,rat primary HSCs were hired to understand the role of Plin 5 in liver fibrosis.We found that Plin 5 enriched in quiescent HSCs and almost disappeared in activated HSCs.The Lentiviral system was used to produce viral particles(pFLRu-PLIN5)by transfecting 293 T cells.To investigate the effect of Plin 5 on the proliferation of activated HSCs and whether AMPK is involved in Plin 5 regulation of apoptosis,autophagy and mitochondrial dysfunction.It provides new insights into the mechanism of Plin 5-induced apoptosis in activated HSCs.Targeting Plin 5 might be a potential to fight liver fibrosis.Methods(1)Liver tissue,visceral fat and primary HSCs were isolated from healthy rats,C57 BL / 6J mice and NAFLD model C57 BL / 6J mice,respectively;rat HSC-T6 and mouse HSC-GRX were cultured in vitro;RT-PCR and immunofluorescence assays were used to detect Plin 5,Plin 1,Plin 2 Plin 3 and HIG 2 levels in various tissues and cells.(2)rHSC-primary was transduced with pFLRu-PLIN5 for in vitro culture.The proliferation state was observed by fluorescence microscopy and MTS assay;?-SMA and collagen were determined by Western blot,and the levels of Cyclin D1 and p21 were determined by western blot.(3)Plin5 overexpressing rHSC-primary was treated with or without ICEprotease inhibitor,and Bcl-2 and Bax,caspase cascade enzyme and caspase-3 activities were detected by western blot.(4)Treatment of rHSC-primary with different concentrations of AICAR for 24 hours.Western blot was used to detect the phosphorylation of AMPK and the phosphorylation levels of autophagy-related genes ULK1,and LC3.(5)The rHSC-primary transduced with pFLRu-PLIN5 was treated with Compound C and chloroquine,respectively.Proliferation was measured by MTS;caspase-3 activity,AMPK phosphorylation,Bcl-2 and Bax,Cyclin D1 and p21 levels were evaluated by western blot.(6)pFLRu-PLIN5 transducing rHSC-primary was co-transfected with pEGFP-LC3.The percentage of LC3 fluorescent puncta was observed under a fluorescence microscope.(7)pFLRu-PLIN5 transducing rHSC-primary was pretreated with or without methyl pyruvate and ICE-like protease inhibitor.The level of PGC-1? was detected by RT-PCR;ATP level was determined by ATP assay kit;Caspase-3 activity,AMPK level,Bcl-2 and Bax,Cyclin D1 and p21 levels were analyzed by western blot.Results(1)Plin 5 mRNA levels in visceral fat,quiescent HSCs,healthy liver,D7-HSCs showed a downward trend(P<0.05).Compared with quiescent HSCs,Plin 2 levels in activated HSCs decreased by more than 50%,while mRNA levels of Plin1,Plin 3 and HIG2 were not significantly different between HSCs(P>0.05).Compared with the normal diet group,the Plin 5 and Plin 2 of HSCs in the high-fat diet group decreased by 90% and 40%,respectively(P < 0.05).(2)Microscopical and MTS assays showed that the ectopic expression of Plin 5 reduced the activity of rHSC-primary,inhibited the production of ?-SMA and collagenase,down-regulated Cyclin D1 protein,up-regulated p21 protein(P<0.05),and inhibited cell proliferation.(3)Overexpression of Plin 5 in rHSC-primary down-regulated Bcl-2,up-regulated Bax,enhanced PARP,caspase-3 and caspase-9 levels(P<0.05),which were reversed by protease inhibitors(P<0.05).(4)Plin 5 overexpressed rHSC-primary and AICAR-treated rHSC-primary,AMPK phosphorylation levels were increased,and AICAR dose-dependently increased LC3-II / LC3-I ratio(P < 0.05).(5)rHSC-primary transducted with pFLRu-PLIN5 was pretreated with compound C.It was found to abolish the cell cycle arrest and apoptosis of HSCs induced by Plin 5 overexpression(P<0.05).(6)After transfection of rHSC-primary with pFLRu-PLIN5,the levels of p-AMPK,p-ULK1 and LC3-II / LC3-I were significantly increased(P<0.05),which can be eliminated by chloroquine(all P < 0.05).(7)rHSC-primary transduced with pFLRu-PLIN5 was transfected with pEGFP-LC3 increased LC3 puncta(P<0.05).(8)Plin 5 overexpressed rHSC-primary,p-AMPK,p21 levels increased,mitochondrial function regulatory factors PGC-1? mRNA,ATP,Cyclin D1 and Bcl-2 levels decreased(P < 0.05).These effects can be restored after supplementation with endogenous ATP levels(P < 0.05).ConclusionPlin 5 impairs mitochondrial biosynthesis,reduces ATP levels,leads to AMPK activation and induces apoptosis in HSCs.