Activation Of NADPH Oxidase Mediates NGF Downregulation,Resulting In Cardiomyocyte Apoptosis

Cardiovascular disease（CVD）occurs frequently in middle-aged and elderly people.Among them,heart failure（HF）is the potential end stage of most cardiovascular diseases.Cardiac sympathetic nerve terminal function decreases in chronic heart failure,and the alteration is correlated with downregulation of nerve growth factor（NGF）in cardiac myocytes.HF is associated with hypoxia and increased oxidative stress.Oxidative stress mediates cardiac sympathetic nerve terminal abnormalities,β-adrenergic receptor downregulation and cardiomyocyte apoptosis.NADPH oxidase is a major source of reactive oxygen species（ROS）production.However,it remains unclear about the mechanism of NGF downregulation in cardiomyocytes and the effect of NGF dowregulation on cardiomyocyte apoptosis.In the present study,we investigated whether the activation of NADPH oxidase mediates the downregulation of NGF that results in cardiomyocyte apoptosis and its underlying mechanism.In cultured H9C2cardiomyocytes in vitro,we first determined whether hypoxia and ROS mediate the downregulation of NGF;second determined whether the activation of NADPH oxidase mediates hypoxia-induced the downregulation of NGF and apoptosis using the NADPH oxidase inhibitor apocynin;finally investigated whether NADPH oxidase-derived ROS mediate apoptosis through NGF receptor Trk A signaling pathway using the NGF receptor Tyrosine receptor kinase A（Trk A）inhibitor K252a.This project includes three parts:Part Ⅰ:Hypoxia and reactive oxygen species mediate the downregulation of NGF expression in cardiomyocytesObjective:To observe whether reactive oxygen species（ROS）mediate the downregulation of NGF in cardiomyocytes during hypoxia.Methods:H9C2 cardiomyocytes were subjected to hypoxia for 6 hours（h）.Group 1:（1）Control,（2）Hypoxia.NGF protein expression was detected by Western blot;Group 2:（1）Control,（2）H₂O₂（final concentration of 100uM）,（3）H₂O₂（final concentration of500uM）,NGF protein expression was measured by Western blot.Results:Compared with the Control group,NGF protein expression was decreased in the Hypoxia group.Compared with the Control group,H₂O₂at the concentration of 100uM markedly induced the decrease of NGF protein expression.Part Ⅱ:NADPH oxidase mediates hypoxia-induced NGF downregulation,resulting in cardiomyocyte apoptosis.Objective:To investigate whether NADPH oxidase mediates hypoxia-induced NGF downregulation that results in cardiomyocyte apoptosis.Methods:H9C2 cardiomyocytes was subjected to hypoxia for 6 h.Group 1:（1）Control,（2）Hypoxia,（3）Hypoxia+NADPH oxidase inhibitor apocynin（Hypoxia+apocynin）,NADPH oxidase activity and the ratio of reduced to oxidized glutathione（GSH/GSSG）were measured.Group2:（1）Control,（2）Hypoxia,（3）Hypoxia+NADPH oxidase inhibitor apocynin（Hypoxia+apocynin）,NGF,Caspase-3,Cleaved caspase3,Bax and Bcl-2 protein expression was measured by Western blot.Results:Compared with the Control group,NADPH oxidase activity was increased and the GSH/GSSG ratio was decreased in the Hypoxia group,indicating increased oxidative stress.The NADPH oxidase inhibitor apocynin attenuated hypoxia-induced the increase in NADPH oxidase activity and the decrease in the GSH/GSSG ratio.Compared with Control group,NGF protein expression was decreased in the Hypoxia group and the decrease was attenuated by the treatment of the NADPH oxidase inhibitor apocynin,suggesting that the activation of NADPH oxidase mediates NGF downregulation.Furthermore,in comparison with the control group,cleaved caspase-3 protein,a marker of apoptosis,Bax protein and the ratio of Bax/Bcl-2 proteins were increased and Bcl-2protein tended to decrease in the Hypoxia group.Apocynin treatment attenuated hypoxia-induced increases in cleaved caspase-3,Bax and the Bax/Bcl-2 ratio and a decrease in Bcl-2 protein expression.These results suggest that the activation of NADPH oxidase mediates NGF downregulation that is associated with increased cardiomyocyte apoptosis.Part Ⅲ:NADPH oxidase-derived reactive oxygen species mediate cardiomyocyte apoptosis through the NGF receptor Trk A signaling pathway.Objective:To examine whether NADPH oxidase-derived reactive oxygen species mediate cardiomyocyte apoptosis through the NGF receptor Trk A signaling pathway.Methods:H9C2 cardiomyocytes was subjected to hypoxia for 6 h.Group 1:（1）Control,（2）Hypoxia,（3）Hypoxia+NADPH oxidase inhibitor apocynin（Hypoxia+apocynin）,Trk A protein expression was measured by Western blot;Group 2:（1）Control,（2）Hypoxia,（3）Hypoxia+NADPH oxidase inhibitor apocynin（Hypoxia+apocynin）and（4）Hypoxia+apocynin+Trk A inhibitor K252a（Hypoxia+apocynin+K252a）.Quantitation of apoptosis and necrosis was determined by flow cytometry.Results:Compared with the Control group,Trk A protein expression was decreased in the Hypoxia group,and the NADPH oxidase inhibitor apocynin inhibited the decrease of Trk A in the Hypoxia group.Furthermore,compared with the Control group,apoptosis and necrosis were markedly increased in the Hypoxia group.The NADPH oxidase inhibitor apocynin inhibited hypoxia-induced apoptosis and necrosis in the Hypoxia group,and the NGF receptor Tr KA inhibitor K252a abolished the protective effect of apocynin.The results suggest that NADPH oxidase-derived ROS mediate cardiomyocyte apoptosis through the NGF receptor Trk A signaling pathway.Conclusion:Hypoxia and ROS mediated the reduction of NGF expression in cardiomyocytes.The activation of NADPH oxidase mediated hypoxia-induced NGF downregulation that was associated with increased cardiomyocyte apoptosis.NADPH oxidase-derived ROS mediated cardiomyocyte apoptosis through the NGF receptor Trk A signaling pathway.