| Objective:Current therapeutic strategies for ischemic heart disease are being optimized and improved,but have a limited role in preventing further myocardial damage and the eventual development of heart failure.In recent years,cellular therapy has been considered as a promising strategy for repairing damaged myocardium.In this study,we prepared a rat cardiomyocyte injury model to systematically observe the regulatory role of exosomal miR-497 in the NF2/PAK1 signaling pathway at the cellular level in order to investigate the effect of miR-497 on myocardial injury and provide new research ideas for myocardial protection therapy after heart attack.Methods:1.Primary isolation of bone marrow mesenchymal stem cells(Bone mesenchymal stem cells,BMSCs)from rats was performed followed by culture and identification.2.Cells were transfected with negative reagent(NC)and mimetic agent(Mimic),and then exosomes were collected from the cultures and assayed.3.A rat cardiomyocyte injury model was constructed by hypoxia-reoxygenation treatment of rat H9C2 cells,and the extracted exosomes were used to treat hypoxia-reoxygenated cells according to the grouping.4.The study was divided into five groups:(1)Normal group,i.e.,normal culture of H9C2 rat cardiomyocytes.(2)Model group,in which H9C2 rat cardiomyocytes were given hypoxia-reoxygenation treatment alone without any additional substances.(3)Model+Blank exosome group,in which the BMSCs-exosome extracted in step 1 was added to H9C2 rat cardiomyocytes in co-culture and treated with hypoxia-reoxygenation.(4)Model+NC exosome group,adding the transfected NC group exosomes extracted in step 1.(5)Model+miR-497-5p exosome group,adding the transfected Mimic group exosomes extracted in step 1.5.The apoptosis of each group of H9C2 cells was measured by flow method,the expression of miR-497-5p in each group of H9C2 cells was detected by qPCR,and the expression of NF2,PAK1,Bax,Bcl-2 and VEGF in each group of H9C2 cells was detected by WB.Results:1.Myocardial modeling treatment exacerbated the cell damage,and correspondingly,the apoptosis rate of the remaining four groups of H9C2 cells increased significantly compared with the Normal group.Both the mimic-transfected exosome group and the NC-transfected exosome group showed an increase in apoptosis rate compared with the model group,and the former group showed a higher increase(p=0.008<0.05).2.miR-497-5p expression was significantly increased in the cells of transfected NC group and transfected mimic group compared with the model group,and the increase was greater in the transfected mimic group,with statistical differences(p<0.05).3.Compared with the model group,the expression of related proteins in H9C2 cells in all three groups showed a significant decrease(p<0.05),except for the control group.In addition,the expression of VEGF was significantly upregulated in the cells of the former group,while the expression of NF2,PAK1 and Bcl-2 proteins was significantly reduced in the Model+miR-497-5p exosome group compared with the Model+NC exosome group(p<0.05),while there was no significant difference in the expression of Bax protein.Conclusion:In this study,we mediated the downregulation of NF2 expression by miR-497-5p through BMSCs-exosome,which led to an increase in PAK1 autophosphorylation level degraded by ubiquitination and increased VEGF protein expression,as well as increased Bax expression and downregulated Bcl-2 expression,with an increase in the Bax/Bcl-2 ratio,showing overall that overexpression of miR-497-5p promoted apoptosis. |
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