PERK Regulates The Mitochondria-associated ER Membranes To Convey Ferroptosis And The Protective Effect Of Quercetin In Alcoholic Liver Disease

Objectives:Considering the disorder of lipid metabolism and iron deposition in alcoholic liver disease(ALD),ferroptosis may play a vital role in its pathogenesis.The aim of this study was to determine whether mitochondria-associated endoplasmic reticulum membranes(MAMs)regulated by RNA-dependent protein kinase-like ER kinase(PERK)is associated with ferroptosis occurred in ALD and the protective effect of quercetin.Methods:1.Male C57BL/6J mice(n=32)were isocalorically pair-fed either a regular(CON)or ethanol-containing Lieber De Carli liquid diets(Et OH,28% energy replacement)supplemented with quercetin(EQ,100 mg/kg.bw)or not(Q)according to chronic and binge ethanol feeding protocol for 12 weeks.After 12 weeks,mice were executed,serum and liver tissue samples were collected for further biochemical assays.Serum aspartate aminotransferase(AST),alanine aminotransferase(ALT),hepatic total iron,liable iron pool(LIP),glutathione(GSH)and malondialdehyde(MDA)were detected.Hepatic pathological change and lipid deposition were observed with hematoxylin-eosin staining(H&E)and Oil-Red O staining.The ultrastructure of hepatocytes were observed by transmission electron microscope.Real-time quantitative(q RT-PCR)and western blot were used to detect ferroptosisrelated genes or proteins.Endoplasmic reticulum stress-related proteins were measures by western blot.MAMs in liver tissue were extracted and western blot was used to detect the levels of PERK and ACSL4 in MAMs.Proximity ligation assay(PLA)and immunofluorescence were used to measure the number of MAMs and PERK-regulated MAMs,respectively.2.Ethanol-exposing AML12 cells(Et OH,200 m M)were treated with the inhibitor of ferroptosis(ferrostatin-1,2 μM),necroptosis(Necrostatin-1s,50 μM)or apoptosis(Z-VAD-FMK,50 μM),respectively,and colorimetry was used to detected cellular lactate dehydrogenase(LDH)release.Ethanol-incubating AML12 cells were treated with different ferroptotic agonists(RSL3,1 μM;erastin,2.5 μM)or inhibitors(ferrostatin-1;DFO/deferoxamine,20 μM),quercetin(20 μM),PERK si RNA,plasmic encoding PERK-K618A(ΔK)or PERK-ΔC(a C-terminal deletion-mutant lacking amino acids 582-1081,ΔC).After treatment,lipid peroxidation was valued by C11-bodipy,ferroptosis-related proteins were were detected by western blot,colocalization analysis of immunofluorescence was used to observe the number of MAMs and PERK-regulated MAMs.Results:1.Compared with CON,ethanol consumption resulted in increased serum ALT and AST(P < 0.05),hepatic total iron,LIP and MDA elevated by 40.6%(P < 0.05),31.3%(P < 0.05)and 2.3 times(P < 0.05),respectively.The level of GSH reduced by48.6%(P < 0.05)in Et OH group compared to CON group.Together with the increased Acyl-Co A synthetase long-chain family member 4(ACSL4)(P < 0.05),decreased glutathione peroxidase 4(GPx4),x CT protein level(P < 0.05)and increased expression of prostaglandin endoperoxide synthase 2(Ptgs2)m RNA(P <0.05),these data indicated the occurrence of ferroptosis in ALD which all reversed by quercetin(P < 0.05).2.Increased LDH release was reversed by ferrostatin-1,necrostatin-1s and Z-VAD-FMK in ethanol-exposing AML12 cells(P < 0.05).Quercetin increased the levels of GPx4 and x CT,decreased ACSL4(P < 0.05)expression,LDH release(P <0.05)and lipid peroxidation(P < 0.05),which induced by ethanol or/and RSL3.Moreover,RSL3 and erastin further increased the expression of ACSL4 in ethanol-incubated cells(P < 0.05).RSL3 further increased the levels of lipid peroxidation(P < 0.05),LDH release(P < 0.05)and GPx4(P < 0.05)expression and erastin further decreased x CT expression(P < 0.05)in ethanol-exposing cells compared to Et OH group.Ferrostatin-1 and DFO increased the level of GPx4 and x CT(P < 0.05),decreased LDH release(P < 0.05)and lipid peroxidation(P < 0.05)in ethanol-treated cells.3.Quercetin decreased glucose regulated protein 78(GRP78),p-PERK,p-/eukaryotic initiation factor 2α(p-e IF2α/e IF2α),C/EBP-homologous protein(CHOP)and activating transcription factor 4(ATF4)expression and increased the level of p90-ATF6 in ethanol-fed mice compared to Et OH group(P < 0.05).Moreover,liver MAMs number(P < 0.05),the level of PERK and ACSL4 in MAMs(P < 0.05)and co-localization of PERK and voltage-dependent anion channel-1(VDAC1)all reversed by quercetin in ethanol-fed mice.4.In ethanol-incubated cells,RSL3 further increased inositol 1,4,5-trisphosphate receptor type(IP3R1)or PERK labeling overlapped with VDAC1,which decreased by the treatment of Fer-1.Moreover,quercetin intervention reversed the co-localization of IP3R1 or PERK and VDAC1 in ethanol or ethanol plus RSL3-treated cells.Compared to Et OH group,the expression of GPx4 and x CT were increased and the level of ACSL4 was decreased in the ethanol-exposing cells transfected with PERK si RNA or ΔC(P < 0.05).Moreover,si PERK transfection decreased LDH relerase(P < 0.05)in ethanol-incubating cells.ΔC transfection further decreased lipid peroxidation(P < 0.05)and upregulated LDH relerase(P < 0.05)in ethanol-exposing cells which inhibited by necrostatin-1s and Z-VAD-FMK(P < 0.05).ΔK transfection further decreased the expression of GPx4 and x CT(P < 0.05),increased LDH release(P < 0.05)in ethanol-exposing cells.Conclusion:Our results showed enhanced ferroptosis regulated by increased MAMs through PERK upregulation in ALD and C terminal of PERK as a functional terminal directly affected the formation of MAMs.Quercetin could decrease MAMs-PERK to inhibit the occurrence of ferroptosis in ALD.