Abstract
Hypoxia/reoxygenation (H/R) accelerates the process of cardiomyocyte apoptosis during ischemia-reperfusion. Excessive reactive oxygen species (ROS) are a critical driver of oxidative stress injury. Cyclophilin A (CyPA) is a major ROS-induced factor in atherosclerosis. There is a positive feedback mechanism between CyPA and ROS, which enables the oxidative stress response to continue and expand. However, it is unclear whether this positive feedback mechanism exists in cardiomyocytes. Through western blotting and flow cytometric assays and TUNEL assay, we found that CyPA inhibited the apoptosis of H9c2 cardiomyocytes under H/R conditions. By dihydroethidium (DHE) staining and electron spin resonance (ESR) assays, we demonstrated that CyPA reduced ROS production and suppressed O2− production dependent on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. By western blotting, we showed that CyPA inhibited the expression of NADPH oxidase 2 (Nox2) protein by the AKT pathway. Through confocal microscopy assay, we found that CyPA reduced the expression of Nox2 membrane-bound subunits. The current study shows that a positive feedback mechanism does not exist in H9c2 cardiomyoblasts. CyPA protects H9c2 cardiomyoblasts against H/R-induced apoptosis via the AKT/Nox2 pathway. This could be a potential target for ischemia-reperfusion injury therapy.
Highlights
In ischemia-reperfusion injury, the main cause of injury to the tissue is not the ischemia itself; the injury occurs after the recovery of the blood supply and is caused at least in part by excessive reactive oxygen species (ROS) attacking the cells in the tissue that regains its blood supply
We found that the protein levels of BAX, caspase-3, and NADPH oxidase 2 (Nox2) were remarkably reduced in the H/R+Cyclophilin A (CyPA) group and the H/R+GSK2795039 group (Figures 7(a), 7(b), 7(d), and 7(e)), and the expression of Bcl-2 was significantly increased in both the two groups (Figure 7(c))
Studies have shown that these dual effects of CyPA on endothelial cells (ECs) may be dependent on CyPA concentration [24]
Summary
In ischemia-reperfusion injury, the main cause of injury to the tissue is not the ischemia itself; the injury occurs after the recovery of the blood supply and is caused at least in part by excessive reactive oxygen species (ROS) attacking the cells in the tissue that regains its blood supply. CyPA was the first member of the cyclophilin family to be discovered and is mainly located in the cytoplasm. It is widely distributed in almost all tissues [2, 3]. CyPA is classified into a variety of proteins termed foldases due to its enzymatic properties, its role in protein folding, and its cellular localization [4]. It is necessary for protein folding because of its enzymatic peptidyl prolyl cis-/
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