The Effects Of AMPK?2 Mediating Vanillic Acid Against Cardiomyocytes Anoxia/reoxygenation Injury

Objective:Vanillic acid(VA)is a phenolic derivative of edible plants,experimental studies have provided evidence of effectiveness on cardiovascular diseases for vanillic acid.In this study,the molecular mechanisms related to the protective effects of vanillic acid in H9c2 cells from anoxia/reoxygenation(A/R)injury were further investigated.Methods:The cultured H9c2 cells were pretreated with different concentrations of vanillic acid before A/R treatment.The cell viability was assayed by MTS.The expression levels of AMPK?2 protein were detected by western blot analysis.We accessed reactive oxygen species(ROS)production and the mitochondrial membrane potential(??_m)by flow cytometry.Finally,lactate dehydrogenase(LDH),creatine phosphokinase(CPK),mitochondrial permeability transition pore(mPTP)open level and the activity ofcysteine protease(caspase-3)were measured using the spectrophotometric method.The apoptotic H9c2 cells were analyzed by flow cytometry and TdT-mediated dUTP Nick-End Labeling(TUNEL).Results:Pretreated with different concentrations of VA could increase the viability of H9c2 cells significantly in a dose-dependent manner compared to A/R group.The viability of H9c2 cells reached the peak with a final concentration of 1.00mM VA(p<0.05,vs.A/R group);Pretreatment with VA significantly decreased the percentage of apoptotic cells,as well as LDH and CPK activity,which were accompanied by a reduction ROS and caspase-3 activity.VA pretreatment also restored mitochondrial membrane potential.Moreover,pre-incubation with VA significantly attenuated mPTP activity.Additionally,VA upregulated AMPK?2protein expression(p<0.05,vs.A/R group).However,pretreatment with the lentivirus AMPK?2-siRNA,effectively attenuated the cardioprotective effects of VA against A/R injury(p<0.05,vs.VA+A/R group).Conclusion:The protective effect of vanillic acid against anoxia/reoxygenation injury in H9c2 cells may be related to AMPK?2 signaling pathway,which depend on(at least partly depend on)upregulating the expression of AMPK?2 protein.