Protective effects of metformin against myocardial ischemia-reperfusion injury via AMPK-dependent suppression of NOX4

Numerous studies have demonstrated that metformin can reduce the incidence of myocardial infarction and improve the prognosis of patients. However, its specific mechanism has not been determined. Using a rat model of myocardial ischemia-reperfusion injury (MIRI), it was observed that metformin significantly reduced infarct size, and decreased the levels of plasma lactate dehydrogenase and creatine kinase-MB form. A TTC-Evans blue staining was used to detect the infarct size and MTT assay was used to evaluate the cell viability. TUNEL assay was performed to evaluate apoptosis. Furthermore, 4-hydroxynonenal was detected by immunohistochemical staining. mRNA expression levels were detected by reverse transcription-quantitative PCR; protein expression levels were detected by immunoblotting. When treated with metformin, the number of TUNEL-positive cells was significantly decreased. Reduced 4HNE immunoreactivity was observed in metformin-treated rats as determined via immunohistochemistry. Furthermore, NADPH oxidase 4 (NOX4) was downregulated by metformin at both the mRNA and protein levels, and adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation was increased by metformin. In a primary myocardial hypoxia-reoxygenation cell model, metformin increased the viability of cardiomyocytes and reduced the content of malondialdehyde. It was also found that metformin upregulated the phosphorylation of AMPK and decreased the expression of NOX4. Furthermore, pre-treatment with AMPK inhibitor compound-C could block the effect of metformin, indicated by increased NOX4 compared with metformin treatment alone. These results suggested that metformin was capable of reducing the oxidative stress injury induced by MIRI. In conclusion, the present study indicated that metformin activated AMPK to inhibit the expression of NOX4, leading to a decrease in myocardial oxidative damage and apoptosis, thus alleviating reperfusion injury.