| Objective: This study constructs the SD rats myocardial ischemia/reperfusion(I/R)injury model to explore the protective effects of Tanshinone ⅡA on against I/R injury;and use the H9c2 cells to build anoxia/reoxygenation model to further investigate the cardioprotective mechanism of AMPKα2 protein.Methods:(1)The Male Sprague-Dawley rats(250-280 g,7-to-8-week-old)were given Tanshinone ⅡA 30 d by intragastric administration 15mg/kg/d and transfected AMPKα2-shRNA lentivirus by intramyocardial injection and occluded the left anterior descending coronary to construct I/R injury model.60 rats were randomly divided into 5 groups:(1)Sham group;(2)I/R group;(3)Tanshinone ⅡA+I/R(TanⅡA+I/R)group;(4)Tanshinone ⅡA+Negative+I/R group(Tan ⅡA+NC+I/R);(5)Tanshinone ⅡA+AMPKα2-siRNA+I/R group(Tan ⅡA+AMPKα2-siRNA+I/R).(2)Firstly,we use Powerlab system collect ECG to realize the situation of rats modeling;myocardial tissue frozen section was observed lentivirus transfection efficiency and the effects of Tanshinone ⅡA and AMPKα2-siRNA on the expression of AMPKα2 was assayed by western blotting analysis.(3)The myocardial infarct size of rats were measured by TTC staining;TUNEL was used to evaluate the apoptosis of cell,then we analyzed Malonic Dialdehyde(MDA),Superoxide Dismutase(SOD),Glutathione peroxidase(GSH-Px)and Lactate dehydrogenase(LDH)activityin in the serum by Spectrophotometry,to comparative study the relationship between AMPKα2 protein level and Tan ⅡA anti-I/R injury in the myocardium.(4)H9c2 cells were transfected with AD-scrRNAi adenovirus and subjected hypoxia /reoxygenationmodel.Then we use Co-IP and double immunofluorescence staining to explore the interaction of AMPKα2 and 14-3-3η and the mechanism of AMPKα2 mediated myocardial protection of Tanshinone ⅡA.Results:(1)The I/R model was successfully constructed by observing the changes of electrocardiogram in rats;After transfection of lentivirus into rat myocardium for 30 days,the expression of green fluorescent protein(GFP)inmyocardium was observed by fluorescence microscope shows transfection of AMPKα2-siRNA successfully.Western blotting showed that the interference effect of AMPKα2 in the AMPKα2-siRNA#2 group was significant.(2)Western blotting showed that the expression of AMPKα2 of Tan ⅡA+I/R group were significantly increased compared with I/R group;while the expression of AMPKα2 were interfered by AMPKα2-siRNA,the effect of Tan ⅡA on the expression of AMPKα2 was reversed.(3)Compared with I/R group,Tan ⅡA+I/R group caused great decrease of infarct size and apoptosis;mean while,the releases of the LDH and MDA were greatly decreased and SOD and GSH-Px were greatly increased(p<0.01).While interference AMPKα2 expression with AMPKα2-siRNA lentivirus,the role of Tanshinone ⅡA against myocardial I/R injury has been cancelled which showed that the effects of Tanshinone ⅡA were associated with the role of AMPKα2 protein.(4)Co-IP method showed the interaction between AMPKα2 and 14-3-3ηenhanced with H9c2 cells subjected by A/R,while interfere 14-3-3η expression with AD-scrRNAi,the interaction enhancement trend was reversed,suggesting 14-3-3ηplay a key role in the action of AMPKα2;Confocal showed the expression of AMPKα2 protein in nuclear of H9c2 cells with A/R were increased,and after 14-3-3ηAD-scr RNAi pretreatment,14-3-3η protein expression decreased,while also reducing AMPKα2 protein expression in the nuclear.The results showed AMPKα2may interact with 14-3-3η and co-localized in the nucleus and play a protective role in H9c2 cells with A/R.Conclusion: 1.Tanshinone ⅡA sodium preconditioning has a significant protective effect on rat heart against myocardial ischemia-reperfusion injury.2.The cardioprotection of Tanshinone ⅡA preconditioning may conducted by up-regulation the expression of AMPKα2/14-3-3η protein. |
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