Related Mechanisms Of FoxO3 On Cardiac Oxidative Damage And Aging In Mice

Objectives:Paraquat(PQ)promotes cell senescence in brain tissue,which contributes to Parkinson's disease.Furthermore,PQ induces heart failure and oxidative damage,but it remains unknown whether and how PQ induces cardiac aging.Here,we demonstrate that PQ induces phenotypes associated with senescence of cardiomyocyte cell lines and results in cardiac aging-associated phenotypes including cardiac remodeling and dysfunction in vivo.Moreover,PQ inhibits the activation of FoxO3,an important longevity factor,both in vitro and in vivo.Cardiovascular disease has become the main factor affecting the health of China's population,and aging is an important inducing factor leading to the high incidence of cardiovascular disease.Therefore,the exploration of cardiac aging and targeted intervention therapy is the current research hotspot and frontier in the field of aging.In this study,we explored the role of FoxO3 in cardiac aging and the possible mechanisms involved through H₂O₂induction of myocardial cell senescence,as well as natural young mice(3 months),naturally aged mice(24 months),knockout of FoxO3 young mice(3 months)and knockout of FoxO3 aged mice(23 months).Through transcriptome sequencing,the molecular and cytological mechanisms mediating cardiac aging were further elucidated to find effective ways to alleviate oxidative damage of myocardium and effectively interfere with senescence related cardiovascular diseases.Methods:Part?:(1)QPCR and WB were used to detect the effect of PQ on FoxO3 mRNA and protein,and to detect the change of Akt/FoxO3 pathway related protein expression.(2)FoxO3was interfered with by si RNA,ROS content in H9c2 cells was detected by DCH2DCF,mitochondrial membrane potential was detected by JC-1 staining,apoptosis was detected by FITC-PI staining,and cell cycle was detected by PI staining.(3)FoxO3 gene silencing stable cell line(sh FoxO3)and control group(sh NC)of H9c2 cells were screened and established by lentivirus system,and WB was used to detect the effects of PQ on SOD2,Catalase,aging markers p53,p16 and apoptosis-related protein Bax/Bcl2.Part?:(1)To construct a mouse phenotype(FoxO3^-/-)with cardiac specific knockout target gene,labeled as CKO.Animal experiments were divided into three groups:the control group was injected with PBS,WT group with PQ and CKO group with PQ once a week for four weeks.QPCR and WB were used to detect the effect of PQ on FoxO3 mRNA and protein in cardiac tissue,and to detect the change of Akt/FoxO3 pathway related protein expression.The nuclear expression of FoxO3 was detected by immunofluorescence assay.(2)ROS content in cardiac tissue was detected by DHE immunofluorescence,and lipid oxidation degree in cardiac tissue was detected by MDA.TUNEL staining was used to analyze the apoptosis,and the changes of8-OHd G were detected by immunohistochemistry.WB was used to detect the changes of SOD2,Catalase,p53,p16,p21,p27 and Bax/BCL2 proteins.(3)Overexpression vectors SOD2 and Catalase were constructed and transfected into H9c2 cells.Ch IP-PCR showed that FoxO3 was bound to SOD2 and Catalase promoter.The FoxO3 and Catalase adeno-associated virus vector system(AAV9)were constructed separately,to analysis of cardiac overexpression of FoxO3 and Catalase alleviated PQ-induced oxidative damage.Part?:(1)H₂O₂induced H9c2 cell senescence,WB was used to detect the effects of senescence induced by H₂O₂on FoxO3 protein,and to detect the change of Akt/FoxO3 pathway related protein expression.(2)FoxO3 was interfered with by si RNA,and ROS content in H9c2 cells was detected by DCH2DCF,and apoptosis was detected by FITC-PI staining.(3)FoxO3 gene silencing stable cell line(sh FoxO3)and control group(sh NC)of H9c2 cells were screened and established by lentivirus system,and WB was used to detect the SOD2,aging markers p53,p16,p21,p27,and apoptosis-related Cleaved-Caspase3,Caspase3,Cleaved-Caspase9,Caspase9,Bax/Bcl2 protein,and telomere length changes in senescence H9c2 cells.Part?:(1)To construct a mouse phenotype(FoxO3^-/-)with whole body knockout target gene,labeled as EKO,and verified the silencing efficiency of FoxO3 at the protein level.Animal experiments were divided into four groups:natural young mice(3 months),naturally aged mice(24 months),knockout of FoxO3 young mice(3 months)and knockout of FoxO3 age mice(23months).QPCR and WB were used to detect the FoxO3 mRNA and protein in natural aged mice,and to detect the change of Akt/FoxO3 pathway related protein expression.The nuclear expression of FoxO3 was detected by immunofluorescence assay.(2)q PCR was used to detect changes in senescence-associated secretory factor SASP:TNF?,MMP3,IL-8,IL-6 and IL-1?.The changes of mitochondrial copy number in Cox?/?-globion,Cytb/H19,PGC1?and mitochondria were detected by natural aging and EKO in elderly mice.The changes of mitochondrial morphology in myocardial tissue were detected by transmission electron microscopy.(3)Immunofluorescence was used to detect the changes of gamma?H2AX and Lamin B in WT young,WT old,EKO young and EKO old mice.(4)WB was used to detect the changes of aging-related proteins p53,p16,p21,p27,apoptosis-related proteins Cleaved-Caspase3,Cleaved-Caspase9,Bax/BCL2,autophagy-related protein LC3B and proliferation-related protein PCNA.and the changes of P-m TOR,P-AMPK,P-Erk,P-MAPK and P-I?B proteins in WT young,WT old,EKO young,and EKO old mice.(5)High throughput sequencing was used to analyze the changes of differentially expressed genes and related signaling pathways in WT young,WT old,EKO young,and EKO old mice.And the changes of the target genes Myh7,Aldob,Mapk9,ACADSB,PTGES3,HADHB,Tcf7L2,and NADK2 were verified at the mRNA level.Results:Part?:(1)PQ induced significantly inhibited the expression of FoxO3 mRNA and protein,and inhibited the activity of Akt/FoxO3.(2)PQ induced ROS accumulation,apoptosis,mitochondrial membrane potential decline,inhibited cell cycle,promoted cell senescence,knocked out FoxO3,further aggravated the above phenotypes.Part?:(1)PQ induced cardiac ventricular remodeling in mice,and WGA showed that PQ induced cardiomyocyte hypertrophy;up-regulated the expression of hypertrophic related genes,induced accumulation of ROS in mouse heart tissue,and promoted the production of lipid oxide malondialdehyde(MDA).Cardiac-specific knockout of FoxO3 further exacerbates the above phenotype.(2)Overexpression of SOD2 and Catalase significantly promoted cell proliferation and inhibited PQ-induced apoptosis.Ch IP-PCR demonstrated that FoxO3 binds to SOD2,a Catalase promoter region,and positively regulates the expression of SOD2 and Catalase.Overexpression of FoxO3 and Catalase in the heart indicates that FoxO3 and Catalase alleviate PQ-induced ventricular remodeling.Part?:(1)H₂O₂induced senescence of H9c2 cells,significantly inhibited the expression of FoxO3 protein,and activation of Akt/FoxO3.(2)WB showed that si RNA effectively silenced FoxO3,H₂O₂induced ROS accumulation in H9c2 cells,apoptosis,promoted cell senescence,knocked out FoxO3,further aggravated the above phenotype.Part ?:(1)Natural aging decreased the expression of FoxO3 mRNA and protein in mouse heart tissue and inhibits the activity of Akt/FoxO3 pathway.(2)SASP results showed that natural aging up-regulated TNF?,MMP3,IL-6 and IL-1?cytokines,knocked out FoxO3 systemically,promoted the expression of IL-6 and IL-1?,and inhibited the expression of TNF?.(3)Aging heart tissue inhibits energy metabolism and destroys mitochondrial morphology and structure.Systemic knockdown of FoxO3 further leads to mitochondrial structural disorder.(4)Aging heart tissue promoted?H2AX expression and inhibited Lamin B expression.EKO older mice further promoted?H2AX expression and inhibited Lamin B expression.(5)High-throughput sequencing of WT young mice,WT old mice,EKO young mice,and EKO old mice showed that 37differential genes were overlapping.According to go and KEGG analysis,37 differential genes were mainly concentrated in mitochondrial oxidative phosphorylation and energy metabolism pathways.ACADSB and PTGES3 were further studied as target genes.Conclusion:(1)PQ and H2O2 induced senescence phenotype of H9c2 cells,inhibited proliferation,promoted apoptosis,increased?-gal activity,and p53,p16 protein expression.In vivo experiments showed that FoxO3 knockout aggravated PQ-induced and natural aging of cardiac hypertrophy,fibrosis,apoptosis,and oxidative damage.(2)In vivo experiments confirmed that FoxO3 binds to the cardiac Cat and SOD2 gene promoters by chromatin immunoprecipitation,and overexpression of Cat or SOD2 attenuates the PQ-induced FoxO3knockout cardiomyocyte senescence phenotype.Overexpression of FoxO3 and Cat in the heart significantly inhibits PQ-induced cardiac damage and aging-associated phenotype.(3)FoxO3was involved in m TOR,AMPK,and autophagy pathways to regulate aging.High-throughput sequencing had again shown that FoxO3 was involved in mitochondrial biosynthesis and energy metabolism,thereby regulated mammalian heart aging.