Studies On The Role Of Glaucocalyxin A(GLA) In Protection Against Myocardial Ischemia-reperfusion Injury In Mice

Background:Coronary heart disease is becoming a major public burden of both developed and developing countries, with acute myocardial infarction(AMI) as the canonical cause for premature mortality. Myocardial reperfusion has been proved to be the most effective approach for myocardial infarction. Paradoxically, inevitable cardiac damages secondary to reperfusion substantially compromise the benefits from percutaneous coronary intervention therapy. To date, extensive studies on reperfusion injury have shed light on its underlying mechanism, although limited approaches have been determined to be effective in RI protection. Developing optimal therapeutic approaches for myocardial reperfusion injury remains the most challenging field in invasive cardiology.Glaucocalyxin A(GLA) is a novel diterpenoid isolated from a plant previously used as traditional medicine for treating gastrointestinal disorders, tumors, and inflammatory diseases in Asia. The pharmacological mechanism of GLA has been studied mostly in platelets and cancer cell lines, uncovering its role in regulating platelet activation, cell death, redox signaling and inflammation. Our recent studies show that GLA inhibits platelet activation and thrombosis via the GPVI dependent pathway. A recent report that GLA confers protection in cardiomyocytes against oxidative stress leads us to explore its putative therapeutic role in cardiac RI.Material and Methods:Myocardial ischemia-reperfusion in wild-type C57BL/6J mice was induced by transient ligation of the left anterior descending artery. Glaucocalyxin A was administrated intraperitoneally to the mice before reperfusion started. After 24 hours of myocardial reperfusion, ischemic size was revealed by histochemistry using Evans blue/TTC staining. Cardiac function was evaluated by echocardiography and microvascular thrombosis was assessed by immunofluorescence staining of affected heart tissue. We also measured the activation of the Reperfusion Injury Salvage Kinase(RISK) pathway, including Akt and ERK1/2, in the myocardium.Results:(1)To evaluate the effect of GLA on infarct size in myocardial RI, mice were given intraperitoneal injection of GLA before reperfusion after one hour of LAD occlusion. After 24 hours of reperfusion period, mice treated with GLA showed significantly decreased infarct sizes(IA/AAR) compare to those treated with vehicle(GLA 13.85±2.08% vs Control 18.95%±0.97%, p<0.05).(2)Following 24 hours of reperfusion after one hour of LAD occlusion, two-dimensional and M mode echocardiography demonstrated that intraperitoneal injection of GLA prior to myocardial reperfusion significantly improved LVEF(GLA 53.13±1.11% vs Control 49.99±1.25%, p<0.05) and LVFS(GLA 28.34±0.71% vs Control 25.11±0.74%, p<0.05) in myocardial I/R stressed mice.(3)According to our previous findings, GLA is able to inhibit the GPVI pathway of platelet activation. To further delineate how GLA confers protection against cardiac RI, we performed immunohistochemistry studies to quantify microvascular thrombosis in the heart. Our results displayed that GLA administration before reperfusion significantly decreased the degree of microvascular thrombosis.(4)To explore the molecular mechanism underlying the cardio-protective role of GLA, we measured the activation of the Reperfusion Injury Salvage Kinase(RISK) pathway. Interestingly, phosphorylation of the pro-survival kinase Akt, but not ERK1/2, was significantly increased by GLA in mice subjected to cardiac I/R.Conclusion:Our findings suggest that GLA protects the heart against RI in a mouse model of myocardial I/R. The cardio-protective roles of GLA may be mediated through the attenuation of microvascular thrombosis and the recruitment of antipoptotic pathways. Considering its low toxicity and minor bleeding risk, GLA may serve as a promising therapeutic option for ameliorating myocardial RI during cardiac revascularization. Further studies on the mechanism by which GLA protects heart against RI as well as the pharmacodynamics are required for the potential application of GLA for myocardial RI.