| Aim:Acute myocardial infarction(AMI),a major deadly disease worldwide,is commonly caused by acute and persistent coronary artery occlusion.A common approach for treating patients with AMI is reconstituting the myocardial blood supply as quickly as possible through the implementation of myocardial reperfusion therapy,which includes percutaneous coronary intervention,coronary artery bypass grafting and thrombolytic therapy.However,during the implementation of myocardial reperfusion therapy,the rapid recovery of the blood supply to the ischemic myocardium does not ameliorate myocardial damage and causes extramyocardial insult,which is termed myocardial ischemia-reperfusion injury(MIRI).The occurrence of MIRI is difficult to predict in advance,and once it occurs,it greatly reduces the clinical benefit of myocardial reperfusion therapy.Even though MIRI was discovered more than half a century ago,the specific mechanism has yet to be fully elucidated.Oxidative stress,however,has long been regarded as a pivotal mechanism for MIRI.Brazilin,a major ingredient of Caesalpinia sappan L.,possesses multiple pharmaceutical activities,especially anti-oxidant activity,although whether or not brazilin exerts any protective effect on MIRI has not yet been reported.The present study determined the cardioprotective effects of brazilin,and elucidated the role of nuclear factor E2-associated factor 2(Nrf2)in this process.Methods:Following treatment with brazilin,H9c2 cells were subjected to 6 h of hypoxia/3 h of reoxygenation.CCK-8 assay and flow cytometry were employed to detect cell viability and apoptosis,respectively.The release of creatine kinase-MB(CK-MB)and lactate dehydrogenase(LDH)were measured by colorimetric method.Furthermore,after brazilin treatment,isolated rat hearts underwent 30 min of ischemia,followed by 90 min of reperfusion.Triphenyltetrazolium chloride(TTC)and terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL)staining were performed to measure myocardial infarct size and apoptosis,respectively.The changes of cardiac function,including heart rate,left ventricular developed pressure(LVDP),positive first order derivative of ventricular pressure(+dp/dt)and negative first-order derivative of ventricular pressure(-dp/dt)were recorded.Additionally,the antioxidant activity of brazilin were evaluated by measurement of reactive oxygen species(ROS)and malondialdehyde(MDA),the activities of superoxide dismutase(SOD)and glutathione peroxidase(GXH-Px).Nrf2 nuclear translocation was detected by immunofluorescence staining.The changes in the levels of proteins were detected by Western blotting.Results:Brazilin treatment dose-dependently led to a significant enhancement in cell viability,a reduction in myocardial infarct size,and a decrease in release of CK-MB and LDH.Moreover,brazilin also remarkably inhibited apoptosis and led to various improvements in cardiac function.Additionally,brazilin treatment caused a marked alleviation of oxidative stress,as evidenced by the fact that brazilin reduced the accumulation of ROS and MDA,while enhancing the activities of SOD and GXH-Px.Mechanistically,it was found that brazilin induced Nrf2 nuclear translocation,with a concomitant upregulation of both heme oxygenase 1(HO-1)and NAD(P)H quinone oxidoreductase(NQO1)expression.Furthermore,the phosphorylation level and transcriptional activity of Nfr2 were enhanced by brazilin,although these enhancements were abrogated by treatment with a protein kinase C(PKC)inhibitor.Finally,it was observed that the protective effects of brazilin could be negated through inhibition of Nrf2,which suggested that the cardioprotection afforded by brazilin was Nrf2-dependent.Conclusions:Taken together,our results have demonstrated that brazilin preconditioning may afford protection against MIRI through the activation of Nrf2 via the PKC signaling pathway.These results may lay the foundation for the further use of brazilin in the prevention of MIRI in clinical practice. |
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