小窝蛋白通过NCOA4-FTH1途径调控铁稳态减缓MAFLD肝细胞衰老OA

Objective To investigate the role of caveolin-1(CAV1)in the progression of metabolic-associated fatty liver disease(MAFLD)and its potential mechanisms of action.Methods We identified differential CAV1 expressions in normal,obese,and non-alcoholic fatty liver disease individuals based on the human database GSE126848.A CAV1-knockout(KO)MAFLD mouse model was established by feeding with a high-fat diet for 16 weeks.CAV1 protein expression in the liver was determined by albumin and CAV1 co-localization,and CAV1 mRNA and protein levels were detected in primary hepatocytes.Lipid deposition and inflammation were assessed by hematoxylin-eosin,Oil Red O,and Nile Red staining.Mitochondrial damage was observed by transmission electron microscopy.Cellular senescence and iron metabolism changes were evaluated by immunohistochemistry for cyclin-dependent kinase inhibitor 1A(P21),dihydroethidium staining,and iron staining.We also constructed hepatocyte senescence models and divided them into blank control(Control),palmitic acid(PA),palmitic acid with CAV1 silencing control group(PA+Con-siRNA),palmitic acid with CAV1-small interfering RNA(PA+CAV1-siRNA),palmitic acid with CAV1 overexpression control group(PA+Con-GV107),palmitic acid with CAV1 overexpression groups(PA+CAV1-GV146),palmitic acid with CAV1 silencing and deferoxamine group(PA+CAV1-siRNA+DFO).Lipid deposition,senescence,and Fe2+levels were analyzed,and the effects of CAV1 on hepatocyte senescence and mitochondrial function were validated by Western blot,quantitative reverse transcription-polymerase chain reaction,and mitochondrial membrane potential detection(JC-1)assays.Results In vivo experiments showed that the compared with WT+HFD group,KO+HFD exacerbated lipid deposition,inflammation,and liver senescence,as evidenced by enhanced lipid staining,increased levels of senescence markers,including histone H2A variant X phosphorylation,cyclin-dependent kinase inhibitor 2A(P16),and P21(all P<0.01),decreased levels of the oxidative stress markers glutathione(P<0.05)and superoxide dismutase(P<0.01),increased reactive oxygen species(ROS)and malondialdehyde(P<0.001),and mitochondrial shrinkage with increased mitochondrial membrane density.CAV1 KO also decreased Fe3+(P<0.01)and increased Fe2+accumulation(P<0.001),associated with the nuclear receptor coactivator 4-ferritin heavy chain 1(NCOA4-FTH1)pathway.Compared with KO+HFD+CSD-CON group,supplementation with the CAV1 scaffolding domain significantly improved the reduction of Fe3+and the accumulation of Fe2+(both P<0.01).Compared with the PA+Con-siRNA group,the PA+CAV1-siRNA group showed accelerated lipid accumulation,mitochondrial damage,and cellular senescence,accompanied by elevated mitochondrial reactive oxygen species(mtROS)levels,Fe2+accumulation,increased NCOA4 expression(P<0.001),and decreased FTH1 expression(P<0.05),while CAV1 overexpression attenuated these effects(P<0.05,P<0.01).Immunofluorescence revealed that CAV1 silencing enhanced NCOA4 and FTH1 co-localization,and this effect was reversed by CAV1 overexpression.Notably,Compared with the PA+CAV1-siRNA group,treatment with deferoxamine(DFO)reduced mtROS levels and ameliorated the senescence induced by CAV1 silencing(all P<0.001).Collectively,these result indicate that CAV1 modulates liver senescence,potentially via the NCOA4-FTH1 pathway.Conclusions CAV1 can inhibit MAFLD hepatocyte senescence,possibly by regulating iron homeostasis via the NCOA4-FTH1 pathway.