Aerobic Exercise Stimulates Endogenous Irisin Secretion And Inhibits ALCAT1 Expression To Improve Myocardial Infarction-induced Renal Injury And Its Molecular Mechanism

Objective:Cardiorenal Syndrome ?(CRS ?)is induced by cardiac diseases such as myocardial infarction,and is highly correlated with an increased risk of death.Therefore,safe and effective prevention and treatment methods are very important in slowing down the renal dysfunction induced by myocardial infarction.Pathological remodeling of ALCAT1 in the renal mitochondria after myocardial infarction causes excessive ROS production and induces apoptosis.FNDC5/Irisin improves energy consumption,inhibiting inflammation,oxidative stress and apoptosis in multiple tissues and organs.As an exercise factor,Irisin is significantly increased by aerobic exercise,which circulates into target organ tissues and participates in the inhibition of oxidative stress and apoptosis of tissue cells.However,whether aerobic exercise can improve renal function injury induced by myocardial infarction and the specific mechanism of Irisin have not been reported in literature.The purpose of this study is to investigate aerobic exercise stimulates endogenous Irisin expression inhibits ALCATI level,and improves myocardial infarction induced renal function damage and its molecular mechanism.Methods:1 Animal experiments:(1)Animal grouping:C57/BL6 wild mice and alcatl-/-mice were randomly divided into sham myocardial infarction group(S),myocardial infarction group(MI),MI and exercise group(ME),with 8 mice in each group.After 1 wk,mice in the ME group were given moderate intensity aerobic exercise intervention through treadmill training for 6 wk.Echocardiography was performed to evaluate the cardiac function.The blood,the heart and kidney were quickly taken from the low-temperature environment.The heart was preserved in neutral formaldehyde for paraffin section staining.Three kidneys in each group were stored in formaldehyde for morphological experiments,and the remaining six were transferred to liquid nitrogen for WB,fluorescence PCR and kit detection experiments.(2)Test indicators:Cardiac function of mice was detected by echocardiography in vivo;The volume percentage of myocardial collagen(CVF%),renal morphology and degree of fibrosis were detected by paraffin section staining.Serum Irisin concentration was detected by ELISA.Fluorescent PCR and kit were used to detect renal function,oxidative stress index and antioxidant kinase.The number of apoptotic positive particles in renal slices was determined by TUNEL kit.Western blotting analysis of FNDC5,p-AMPK/AMPK,Sirtl,PGC-la,p-AKT/AKT,SOD2,ALCAT1,Bax/Bcl-2,Caspase3 and Cytochrome C in mice kidney.2.Cell experiment:(1)Cell intervention and grouping:NRK(Rat Kidney Cells)cells were treated with recombinant protein,drug and lentivirus intervention,including recombinant human Irisin protein(rhIRISIN,100 ng/ml,24 h),and AMPK agonist(AICAR,100 nm,24 h),AMPK inhibitors(Compound C,4 nm,24 h),Sirtl inhibitors(EX527,1 nm,24 h),Sirtl agonist(Resveratrol,10 nm,8 h),alcatl highly expressed lentivirus(ALCAT1 OE,MOI=1),hydrogen peroxide(H2O2,100 nm,1 h).NRK cells were divided into normal control group(group C),H2O2+AICAR group,H2O2+rhIRISIN group,H2O2+Compound C group,H2O2+Compound C+rhIRISIN group,H2O2+EX527 group,H2O2+EX527+rhIRISIN group,Control group,ALCAT1 OE group,ALCAT1 OE+rhIRISIN group,sh-ALCAT1+Resveratrol group,ALCAT1 OE+Resveratrol+rhIRISIN group.(2)Test indicators:CCK-8 kit was used to detect cell activity and cell mortality;The function of electron transfer chain in mitochondrial membrane was measured by mitochondrial function tester.DCFH-DA green fluorescent probe was used to detect ROS levels in living cells.The TUNEL kit detected apoptotic positive particles.Western blotting was the same as animal experiments.Results:(1)Aerobic exercise intervention can significantly improve the degree of brush-like margin detachment and tubulointerstitial fibrosis of myocardial infarction mice,reduce the level of renal injury markers,and improve renal function.Masson and PAS staining results showed that compared with the MI group,the renal tubular interstitial collagen volume percentage(CVF%)and renal tubular injury scores in the ME group were significantly reduced(p<0.01),and blood urea nitrogen and blood creatinine were significantly reduced in renal function indicators(P<0.01).(2)Aerobic exercise intervention significantly reduced ALCAT1 protein expression,increased FNDC5/Irisin expression in myocardial infarct mice,and ALCAT1 inhibited FNDC5 activation.Compared with the MI group,the expression of FNDC5/Irisin in the ME group was significantly increased(p<0.01),and the expression of ALCAT1 was significantly reduced(p<0.05).(3)Aerobic exercise intervention significantly reduced oxidative stress and apoptosis in the kidney of myocardial infarction mice.In terms of oxidative stress,compared with MI,the oxidative stress indicators H2O2 and MDA in the ME group were significantly reduced(p<0.01),the antioxidant enzymes GSH-Px and CAT activity,and SOD2 protein expression were significantly increased(p<0.01,p<0.05).In terms of apoptosis,compared with the MI group,the expression of Casapse3 protein and Bax/Bcl-2 ratio in the kidney of the ME group were significantly reduced(p<0.01).(4)Aerobic exercise intervention may play a protective role in myocardial infarction by activating the FNDC5/Irisin-AMPK-Sirt1-PGC-1 a/AKT signaling pathway.Compared with the MI group,the expression of Sirtl and PGC-la protein and the ratios of p-AMPK/AMPK and p-AKT/AKT in the ME group were significantly.increased(p<0.01,p<0.05).(5)Knockout of ALCAT 1 significantly improved renal function damage in myocardial infarction mice.Compared with the S group,the serum Scr level in the KS group was significantly reduced(p<0.05);compared with the MI group,the serum Scr level in the KMI group was significantly reduced(p<0.01);compared with the ME group,the BUN level in the KME group was significantly reduced(p<0.05)0.01).(6)Knockout ALCAT1 regulates the expression of FNDC5/Irisin in the kidney of myocardial infarction mice.Compared with the S group,the expression of irisin mRNA and FNDC5 protein in the KS group was significantly increased(p<0.01);compared with the MI group,the irisin mRNA was significantly increased in the KMI group(p<0.01);compared with the ME group,the FNDC5 protein expression was increased in the KME group Significantly increased(p<0.01).(7)Knockout of ALCAT1 significantly improved oxidative stress in the kidney of myocardial infarction mice.Compared with the S group,H2O2 and NQO1 mRNAs were significantly reduced in the KS group(p<0.01);compared with the MI group,MDA and NOX4 and NQO1 mRNAs were significantly reduced in the KMI group(p<0.01),and SOD2 was significantly increased(p<0.01);Compared with the ME group,CAT and SOD2 were significantly increased in the KME group(p<0.01),and NQO1 mRNA was significantly decreased(p<0.01).(8)Knockout of ALCAT1 significantly improved renal cell apoptosis in myocardial infarction mice.Compared with the S group,the ratio of Bax/Bcl-2 in the KS group was significantly reduced(p<0.01),and the expression of Caspase3 protein was significantly increased(p<0.01);compared with the MI group,the number of apoptotic cells,Bax/Bcl-2 in the KMI group The ratio and Caspase3 protein expression were significantly reduced(p<0.01);compared with the ME group,the number of apoptotic cells and Caspase3 protein expression in the KME group were significantly reduced(p<0.01).(9)Deletion of ALCAT1 promotes the activation of FNDC5/Irisin-AMPK-SirtlPGC-1?/AKT signaling pathway.Compared with S group,Sirtl and PGC-1? protein expression and p-AKT/AKT ratio were significantly increased in KS group(p<0.01);compared with MI group,Sirtl and PGC-1? protein expression and p-AMPK/AMPK in KMI group,P-AKT/AKT ratio increased significantly(p<0.01);compared with the ME group,Sirtl and PGC-1? protein expression and p-AMPK/AMPK,p-AKT/AKT ratio significantly increased in the KME group(p<0.01)0.01).(10)Exercise or Irisin significantly inhibit H2O2-induced oxidative stress and apoptosis in NRK renal cells,which may exert cytoprotective effects by activating the FNDC5/Irisin-AMPK-Sirtl-PGC-1?/AKT signaling pathway.Compared with the H2O2 group,the ROS,Bax/Bcl-2 ratio and Caspase3 protein expression in the H2O2+AICAR group and the H2O2+rhIRISIN group were significantly reduced(p<0.01),and the SOD2 protein expression was significantly increased(p<0.01).FNDC5,Sirtl,and PGC-la and p-AMPK/AMPK,p-AKT/AKT ratios increased significantly(p<0.01,p<0.05).(11)The protective effect of Irisin on H2O2-induced oxidative stress and apoptosis in NRK cells is AMPK-dependent.Compared with the H2O2+Compound C group,the H2O2+Compound C+rhIrisin group significantly reduced ROS,Bax/Bcl-2 ratio,and Caspase3 protein expression(p<0.01),SOD2 protein expression significantly increased(p<0.05),Sirtl and PGC-1? and p-AMPK/AMPK,p-AKT/AKT ratios increased significantly(p<0.01).(12)The protective effect of Irisin on H2O2-induced oxidative stress and apoptosis in NRK cells is Sirtl-dependent.Compared with the H2O2+EX527 group,the H2O2+EX527+rhIrisin group significantly reduced ROS,Bax/Bcl-2 ratio,and Caspase3 protein expression(p<0.01,p<0.05),SOD2 protein expression significantly increased(p<0.01),Sirtl And PGC-1? and p-AKT/AKT ratios increased significantly(p<0.01,p<0.05).(13)The protective effect of Irisin on H2O2-induced NRK renal cell oxidative stress and apoptosis is mediated by ALCAT1-induced ROS.Compared with the ALCAT1 OE group,the ROS,Bax/Bcl-2 ratio and Caspase3 protein expression in the ALCAT1 OE+Resveratrol group and/or ALCAT1 OE+rhIRISIN group were significantly reduced(p<0.01,p<0.05),and the SOD2 protein expression was significant.Increased(p<0.05),Sirtl and PGC-1? and p-AMPK/AMPK and p-AKT/AKT ratios increased significantly(p<0.01)Conclusion:(1)Aerobic exercise significantly promotes the expression of FNDC5/Irisin in the kidneys of myocardial infarction mice,inhibits the level of renal oxidative stress and renal cell apoptosis induced by myocardial infarction,and improves renal function.(2)Exogenous rhIRISIN intervention significantly reduced oxidative stress and apoptosis of NRK renal cells induced by H2O2 and ALCAT1,and activated the FNDC5/Irisin-AMPK-Sirtl-PGC-1?/AKT signaling pathway.It shows that exercise activates NRK cells FNDC5/Irisin-AMPK-Sirtl-PGC-1?/AKT signaling pathway through FNDC5/Irisin,inhibits ALCAT1 and ROS levels,reduces renal cell oxidative stress and apoptosis.In summary,aerobic exercise reduces the levels of ALCAT1 and ROS,activates the renal FNDC5/Irisin-AMPK-Sirtl-PGC-1?/AKT signaling pathway,thereby reducing myocardial infarction-induced renal oxidative stress and cells apoptosis improves renal function injury induced by myocardial infarction.Innovation:(1)Aerobic exercise significantly increased the expression of FNDC5/Irisin in the kidneys of myocardial infarction mice and improved the renal function damage caused by myocardial infarction;knocking out alcatl to promote the protective effect of aerobic exercise on renal function damage in myocardial infarction mice.(2)Aerobic exercise significantly activates the FNDC5/Irisin-AMPK-SirtlPGC-1?/AKT pathway,and reduces ALCAT1-induced oxidative stress and apoptosis in NRK kidney cells.Shortcomings and outlook:This study lacks endogenous Irisin gene regulation at the animal and cell levels to verify its causal relationship.Due to the epidemic,Irisin knockout mice have reached the screening and modeling stage and cannot carry out experimental work.Irisin knockout will be used in the future.Modeling myocardial infarction and intervening with aerobic exercise to further verify the protective effect and molecular mechanism of Irisin on CRS ? kidney.