| Objective: This study constructs the SD rats myocardial ischemia/reperfusion(I/R)injury model to explore the protective effects of Tanshinone ?A on against I/R injury;and use the H9c2 cells to build anoxia/reoxygenation model to further investigate the cardioprotective mechanism of AMPK?2 protein.Methods:(1)The Male Sprague-Dawley rats(250-280 g,7-to-8-week-old)were given Tanshinone ?A 30 d by intragastric administration 15mg/kg/d and transfected AMPK?2-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 ?A+I/R(Tan?A+I/R)group;(4)Tanshinone ?A+Negative+I/R group(Tan ?A+NC+I/R);(5)Tanshinone ?A+AMPK?2-siRNA+I/R group(Tan ?A+AMPK?2-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 ?A and AMPK?2-siRNA on the expression of AMPK?2 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 AMPK?2 protein level and Tan ?A 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 AMPK?2 and 14-3-3? and the mechanism of AMPK?2 mediated myocardial protection of Tanshinone ?A.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 AMPK?2-siRNA successfully.Western blotting showed that the interference effect of AMPK?2 in the AMPK?2-siRNA#2 group was significant.(2)Western blotting showed that the expression of AMPK?2 of Tan ?A+I/R group were significantly increased compared with I/R group;while the expression of AMPK?2 were interfered by AMPK?2-siRNA,the effect of Tan ?A on the expression of AMPK?2 was reversed.(3)Compared with I/R group,Tan ?A+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 AMPK?2 expression with AMPK?2-siRNA lentivirus,the role of Tanshinone ?A against myocardial I/R injury has been cancelled which showed that the effects of Tanshinone ?A were associated with the role of AMPK?2 protein.(4)Co-IP method showed the interaction between AMPK?2 and 14-3-3?enhanced with H9c2 cells subjected by A/R,while interfere 14-3-3? expression with AD-scrRNAi,the interaction enhancement trend was reversed,suggesting 14-3-3?play a key role in the action of AMPK?2;Confocal showed the expression of AMPK?2 protein in nuclear of H9c2 cells with A/R were increased,and after 14-3-3?AD-scr RNAi pretreatment,14-3-3? protein expression decreased,while also reducing AMPK?2 protein expression in the nuclear.The results showed AMPK?2may interact with 14-3-3? and co-localized in the nucleus and play a protective role in H9c2 cells with A/R.Conclusion: 1.Tanshinone ?A sodium preconditioning has a significant protective effect on rat heart against myocardial ischemia-reperfusion injury.2.The cardioprotection of Tanshinone ?A preconditioning may conducted by up-regulation the expression of AMPK?2/14-3-3? protein. |
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