The Protection Of Phloroglucinol On Myocardial Ischemia-reperfusion Injury And The Underlying Mechanisms

Ischemia-reperfusion injury refers to the phenomenon that the damage of the tissue was not mitigated but aggravated after blood reperfusion under certain condition after ischemia, which was a common problem in the therapy of acute myocardial infarction. A lot of studies have shown that oxidative stress is one of the most important mechanisms of ischemia- reperfusion injury.Myeloperoxidase (MPO) is an extremely important enzyme in oxidative stress. MPO was secreted by the activated neutrophils, monocytes and macrophages, which plays a pivotal role in oxidative stress by catalyzing H2O2 to HOC1, a much stronger oxidizing substance. 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 HOC1 in the in vitro experiment. Therefore, it is speculated that VPO may play a key role in oxidative stress.It has been shown that phloroglucinol possesses anti-inflammatory and anti-oxidant properties. Since phloroglucinol is reported to inhibit the activity of peroxidase, and can increase the activity of catalase (CAT)an important enzyme that can hydrolyse H2O2, our study was conducted to explore the effect of phloroglucinol on myocardial ischemia-reperfusion injury and the underlying mechanisms.The rats were pretreated with phloroglucinol (15 or 30 mg/kg, i.g.) 30 min before the surgical operation, and then the left main coronary artery was subjected to 1 h occlusion followed by 3 h reperfusion. Animals were divided into sham group, ischemia-reperfusion group and ischemia-reperfusion + phloroglucinol (15 or 30 mg/kg, i.g.) group and vehichle group. Infarct size was determined by TTC staining. The protein expression of Caspase-3 was examined by immunohistochemistry. Myocardial cell apoptosis was detected by TUNEL assay. Plasma creatine kinase (CK) and the activity of MPO, CAT and caspase-3 in hearts and blood were measured by spectrophotometry. The protein content of MPO was measured by ELISA.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). Phloroglucinol (10-10 M or 10-9 M or 10-8 M) was given prior to reoxygenation. The cells were divided into normoxic group, hypoxia-reoxygenation group, hypoxia-reoxygenation + phloroglucinol (10-10 M or 10-9 M or 10-8 M) group and vehicle group, Apoptosis were analyzed by Hochest staining. The activity of VPO was measured by spectrophotometry. The mRNA expression of VPO1 was measured by RT-PCR.As the result showed, ischemia-reperfusion caused 43.5±3.7% necrosis in the area at risk and the increased myocardial apoptosis accompanied by the increased CK activity, MPO activity and protein content in blood and myocardium, and the decreased CAT activity. Pretreatment with phloroglucinol significantly reduced the myocardial necrosis and apoptosis accompanied by the decreased CK activity, MPO activity and content and the increased CAT activity.Compared with normoxia group, the cell apoptosis rate, VPO activity and VPO1 mRNA expression were significantly increased in hypoxia-reoxygenation, which were reversed by pretreatment with 10-9 M or 10-8 M phloroglucinol.These results suggest that phloroglucinol is able to protect the myocardium against ischemia-reperfusion injury in rats and the beneficial effects of phloroglucinol may be related to the inhibition of the activity of VPO1 and MPO while increasing the activity of CAT.