The Role Of Vascular Peroxidase In Myocardial Ischemia/reperfusion Injury

Ischemia - reperfusion injury refers to a phenomenon that the cardiac structure and function are not improved but aggravated when blood supply returns to the heart after a period of ischemia. In clinic, ischemia -reperfusion injury usually occurs in patients with therapy of reperfusion, vascular bypass or thrombolysis after myocardial infarction, resulting in increased infarct size and deteriorated cardiac function. It seriously compromises the therapeutic efficacy. There is plenty of evidence that the increased level of oxidative stress is one of the major mechanisms that accounts for ischemia - reperfusion injury.Oxidative stress refers to the oxidative damage due to the generation of reactive oxygen species (ROS) in a system exceeding the system's ability to neutralize and eliminate them. NADPH Oxidase (Nox) and peroxidase are the most important enzymes in the body to generate ROS. Peroxidase is a class of heme containing enzyme, which oxidizes a variety of chemical substances in vivo through converting H₂O₂ (weak oxidant) into H0C1 (strong oxidant). Vascular peroxidase (VPO), a novel protein, has been recently found in the cardiovascular system. It has been supposed as a member of peroxidase family because it shares similar biologic properties with myeloperoxidase (MPO). For example, it is able to convert H₂O₂ into H0C1 in the in vitro experiment. Therefore, it is speculated that VPO may play a key role in oxidative stress.It had been shown that the Nox enzymes are the major source of ROS generated by the heart. The main product of Nox is O-2.-, which can be converted into H₂O₂ by superoxide dismutase (SOD) and becomes the substrate of VPO. We thus hypothesize that VPO and Nox may exert synergy role in oxidative stress through the mediator of H₂O₂.The purpose of the present study was to explore whether VPO participates in the oxidative stress induced by ischemia-reperfusion and to determine whether VPO and Nox have synergic role in promoting oxidative damage of the myocardium.METHODSThe left main coronary artery of male SD rat hearts was subjected to 1-h occlusion followed by 3-h reperfusion. Apocynin (50 mg/kg, i.p.) was administrated 30 min before reperfusion. Animals were divided into normal control group, sham group, ischemia-reperfusion group and ischemia-reperfusion + apocynin group. The mRNA expression of VPO1 and Nox2 was measured by RT-PCR. The protein expression of VP01 and Caspase-3 was examined by immunohistochemistry. Infarct size was determined by TTC staining. Plasma creatine kinase (CK) and the activity of Nox and caspase-3 in hearts were measured by spectrophotometry. Myocardial cell apoptosis was detected by TUNEL assay.H9c2 cardiac cells were cultured in low-nutrition medium with 95% N₂ and 5% CO₂ for 24 h, and then were cultured for 12 h under the regular conditions (normal-nutrition medium, 95% air and 5% CO₂). Apocynin (400μM) or phloroglucinol (600μM) were given prior to reoxygenation. The cells were divided into normoxic group, vehicle group, hypoxia-reoxygenation group, hypoxia-reoxygenation + apocynin group and hypoxia - reoxygenation + phloroglucinol group. Apoptosis were analyzed by Hochest staining.RESULTSCompared with the sham group, the myocardial infarct size, cardiomyocyte apoptosis, plasma CK, Nox and Caspase-3 activity were significantly increased in ischemia - reperfusion group accompanied by the increased myocardial VPO1, Nox2 mRNA and protein expression. These effects were reversed by pre-treating the rats with apocynin. It is of note that apocynin treatment did not show significant effect on Nox2 mRNA expression but it inhibited the activity of Nox. There were not significant difference in all measured parameters between sham and normal control group.Compared with normoxia group, the percentage of cell apoptosis was significantly increased in hypoxia - reoxygenation, which was reversed in the presence of apocynin or phloroglucinol. The vehicle of apocynin or phloroglucinol did not affect the apoptosis of cardiomyocyte.CONCLUSIONVPO paticipated in the oxidative stress induced by myocardail ischemia-reperfusion. VPO and Nox have synergic role in promoting oxidative damage of the myocardium.