Asiatic Acid Attenuates Oxygen-glucose Deprivation/Reoxygenation-Induced Myocardial Injury Via Activating Akt/GSK-3?/HIF-1? Signaling Pathway In Rat H9c2 Cells

Background and aims:Myocardial ischemic/reperfusion injury results from severe impairment of coronary blood supply and leads to irreversible cell death,with limited therapeutic possibilities.Asiatic acid is a pentacyclic triterpenoid derived from the tropical medicinal plant Centella asiatica and serves a variety of bioactivities.In this study,we used rat H9c2 cardiomyocytes model of oxygen-glucose deprivation/reoxygenation(OGD/R)injury,which is established to mimic myocardial ischemic/reperfusion injury in vivo,to determine the effect of asiatic acid on myocardial ischemia/reperfusion injury and investigate the molecular mechanisms to provide new underlying medicine in vitro.Methods:1.Establishment of rat H9c2 cells model of oxygen-glucose deprivation/reoxygenation(OGD/R)injury: Rat H9c2 cells cultured in vitro were pretreated with asiatic acid of various concentrations(0.1,1,10,100?M)for 4 h.H9c2 cells were first suffered from 1h hypoxia and then 12 h reoxygenation.The protective effect of asiatic acid with varying concentrations in H9c2 cells was determined by MTT assay and LDH release.2.The effect of asiatic acid(10?M)on OGD / R-induced apoptosis was observed by Hoechst33258 staining.The activities of Caspase-3 and Caspase-9 were examined.The levels of Bax and Bcl-2 was determined by Western blot.3.Production of ROS was monitored Fluorimetrically,using the Fluorescent probe 2',7'-dichlorofluorescein(DCF).The intracellular calcium concentration was detected by Fluo-3 AM.The mitochondrial membrane potential was measured,using mitochondrial membrane potential assay kit with JC-1.These experiments were performed to evaluate the effect of asiatic acid on mitochondrial function in cardiomyocytes after OGD/R injury,perspectively.4.The effects of asiatic acid on Akt,phosphorylated Akt,GSK-3?,phosphorylated GSK-3? and HIF-1? were analyzed after OGD/R treatment.The Akt inhibitor MK2206 and HIF-1? inhibitor 2-ME were applied to elucidate possible molecular mechanisms of the protective role of asiatic acid in cardiomyocytes.Results:1.Asiatic acid at 0.1 to10?M had no effect on the viability of H9c2 cells under normaxia,however,it could effectively antagonized OGD/R-induced injury in a concentration-dependent manner.2.Asiatic acid at 10?M significantly prevented apoptosis in cardiomyocytes after OGD/R,decreased the activity of Caspase-3 and Caspase-9 and reversed the ratio of Bax/Bcl-2.3.Treatment with 10?M asiatic acid significantly decreased intracellular ROS production in cardiomyocytes following OGD/R injury and prevented loss of mitochondrial membrane potential reduction,inhibited calcium overload,thus improving mitochondrial function under hypoxia.4.Asiatic acid increased the levels of phosphorylated Akt and phosphorylated GSK-3?(Ser9),and promoted the expression of HIF-1? after OGD/R.Akt inhibitor MK2206 and HIF-1? inhibitor 2-ME attenuated the protective effects of asiatic acid.Conclusion:Pre-treatment with asiatic acid had protective effects on OGD/R-induced injury in rat H9c2 cells.These effects may be associated with the modulation of Akt/GSK-3?/HIF-1? signaling pathway,which in turn,prevented myocardial apoptosis and improved mitochondrial function.