Cyclophilin A Regulates Apoptosis Of Cardiomyocytes Induced By Hypoxia/reoxygenation Via AKT/Nox2 Pathway

Objective To explore whether Cyclophilin A regulates apoptosis of cardiomyocytes induced by hypoxia/reoxygenation via AKT/Nox2 pathway.Methods H9c2 cells were cultured in vitro.Protein expression levels of BAX,Bcl-2,Caspase 3,Nox1,Nox2,Nox4,AKT,p-AKT-Ser473,and ?-actin were analyzed by western blotting.Flow cytometry(FCM)and TUNEL assays were used to investigate apoptosis rate.The ROS production and suppressed O2-production dependent on reduced nicotinamide adenine dinucleotide phosphate(NADPH)oxidase were detected by dihydroethidium(DHE)staining and electron spin resonance(ESR)assays.The expression of AKT protein and Nox2 protein was inhibited by AKT inhibitor and NADPH oxidase 2(Nox2)inhibitor,and the inhibitory effect was detected by Western blot.The expression of Nox2 membrane-bound subunits was observed by confocal microscopy assay.Results(1)After hypoxia for 18 hours and reoxygenation for 4 hours,the expression of BAX and Caspase-3 in H9c2 cells decreased with the increase of CyPA-stimulated concentration gradient,and the expression of Bcl-2 protein increased with the concentration gradient of CyPA stimulation(p<0.05);CyPA(1000 ng/mL)significantly inhibited the expression of BAX and Caspase-3 in H9c2 cells under Hypoxia/Reoxygenation(H/R)conditions and promoted the expression of Bcl-2protein(p<0.05).CyPA significantly inhibited apoptosis of H9c2 cells under H/R conditions(p<0.05).(2)The fluorescence intensity of DHE staining of H9c2 cells in H/R + CyPA group was significantly lower than that in H/R group alone(p<0.05);The ESR spectral signal intensity of H9c2 cells in the H/R + CyPA group was significantly weakened compared to the H/R group alone(p<0.05).(3)Under H/R conditions,the level of Nox2 protein was increased compared with the control group(p<0.05);After CyPA intervention,the expression of Nox2 protein was significantly decreased compared to the H/R group(p<0.05);After CyPA intervention,the protein level of p-AKT was significantly increased(p<0.05);Nox1 protein and Nox4 protein were rarely expressed in H9c2 cells and they were not significantly increased under H/R conditions(p<0.05).(4)After inhibition of the AKT signaling pathway by the pan-AKT inhibitor GSK690693 targeting AKT1/2/3,the protein level of p-AKT in the GSK690693 intervention group was significantly reduced compared to the H/R + CyPA group,and The expression of Nox2 was significantly increased(p<0.05).(5)After inhibiting the activity of Nox2 by using GSK2795039(NADPH oxidase 2inhibitor),the protein levels of BAX,Caspase-3 and Nox2 were significantly decreased in the H/R + CyPA group and the H/R + GSK2795039 group(p<0.05),and the expression of Bcl-2 was significantly increased(p<0.05).Used confocal microscopy to observe the changes of Nox2 distribution in H9c2 cells.In normal state,Nox2 is distributed in cytoplasm of H9c2 cells.After H/R stimulation,the expression of Nox2 on plasma membrane increases,suggesting the role of Nox2 on apoptosis,which is modified by CyPA or GSK2795039 treatment(p<0.05).Conclusions(1)CyPA inhibits apoptosis of H9c2 cells under hypoxia/reoxygenation conditions.(2)CyPA decreased O2-production under hypoxia/reoxygenation conditions.(3)AKT/Nox2 pathway mediates the role of CyPA in opposing hypoxia/reoxygenation-induced apoptosis.(4)Inhibiting the AKT/Nox2 pathway partially abrogates CyPA-mediated protection under hypoxia/reoxygenation conditions.(5)CyPA reduced the expression of Nox2 membrane-bound subunits to protect cardiomyocytes against H/R-induced apoptosis via the AKT/Nox2 pathway.