MiR-199a-modified mesenchymal stem cell-derived exosomes promote mitochondrial repair in H9c2 cardiomyocytes of hypoxia/reoxygenation model through Akt/HIF-1α/DRP1 axis
Objective To investigate the mechanism of miR-199a-modified mesenchymal stem cell(MSC)-derived exosomes in repairing mitochondria of H9c2 cardiomyocytes in a model of hypoglycemia/hypoxia/reoxygenation with hypoglycemia.Method Transfect miR-199a mimics or miR-NC into MSCs and collect exosomes 48-72 h later.Fluorescence quantitative PCR(qRT-PCR)method was used to detect miR-199a levels in different exosomes.H9c2 cells were divided into control group,model group,Exosmimic group(final concentration of 50 μg·mL-1),ExosNC group(final concentration of 50 µg·mL-1),and Exosmimic+MK2206(10 μg·mL-1)group.Except for the control group,the model of hypoglycemia/hypoxia/reoxygenation with hypoglycemia was established.The survival rate of cells in each group was detected by CCK8 assay,and the levels of ATP,8-hydroxy-2-deoxyguanosine(8-OHdG),lactate dehydrogenase(LDH),superoxide dismutase(SOD),malondialdehyde(MDA)in each group were detected by enzyme-linked immunosorbent assay(ELISA).The changes of mitochondrial membrane potential(AΨm)and dynamics were detected by confocal microscopy.The expression changes of hypoxia-inducible factor 1α(HIF-1α)and mitochondrial dynamics-related protein 1(DRP1)were detected by Western blotting.Result Compared with control group,the expression level of miR-199a in Exosmimicc was significantly increased(P＜0.05),and the average diameter of extracted exosomes was 109.3 nm,with a concentration of 1×106 particles·mL-1.Compared with control group,the model group showed a significant decrease in cell survival rate and ATP,SOD,and AΨm levels,while LDH,MDA,8-OHdG levels,HIF-1α and DRP1 protein expression levels were significantly increased(P＜0.01,0.001),and mitochondrial division levels were increased.Compared with model group,the Exosmimic group showed a significant increase in cell survival rate,ATP,SOD,and AΨm levels,while LDH,MDA,8-OHdG levels,HIF-1α,and DRP1 protein expression levels were significantly decreased(P＜0.01,0.001),and mitochondrial division levels were reduced.MK2206 can significantly reverse the Exosmimic effect(P＜0.05,0.01).Conclusion miR-199a modified MSCs exosomes exert mitochondrial protective effects by AKT/HIF-1α/DRP1 axis.
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