Exosomes Secreted From Mesenchymal Stem Cells Overexpressing MIF Inhibit Apoptosis Of Cardiomyocytes Via AMPK Signaling Pathway

Objective:Mesenchymal stem cells?MSCs?transplantation can significantly reduce the size of myocardial infarction,promote vascular regeneration in and around the infarction area,and reduce the apoptosis of cardiomyocytes.The paracrine of transplanted human MSCs?hMSCs?has been demonstrated in the mechanism of cardioprotection.Macrophage migration inhibitory factor?MIF?is a pro-inflammatory cytokine,which is expressed by a variety of cells,including MSCs.It has been reported that MIF is related to inhibit apoptosis.Exosomes are 30-150 nm vesicles,which involved in intercellular communication.This study aimed at the role of exosomes derived from human MSCs?hMSCs?overexpressed MIF for cardioprotection.Methods:hMSCs and neonatal rat cardiomyocytes?NRCM?were isolated and cultured.And then hMSCs was transfected by lentivirus of MIF or control.Exosomes from hMSCs(Exo^hMSCs)?hMSCs transduced with lentiviral MIF(Exo^MIF)or a null vector(Exo^Null)were isolated and characterized by size distribution and flow cytometry.The effect of exosomes on NRCM during hypoxia was evaluated by TUNEL staining and western blotting.The protein expression level of molecular marker of AMPK signal passway such as AMPK,p-AMPK,p53,and SIRT1 was analyzed by Western blotting.Results:Compared with NRCM-hypo group,NRCM apoptosis in NRCM-hypo+Exo^hMSCsMSCs group,NRCM-hypo+Exo^Nullull group,and NRCM-hypo+Exo^MIFgroup significantly reduced under hypoxia environment,p<0.01,respectively.And compared with NRCM-hypo+Exo^hMSCsMSCs group and NRCM-hypo+Exo^Nullull group,NRCM apoptosis in NRCM-hypo+Exo^MIFIF group also significantly reduced under hypoxia environment,p<0.01,respectively.Similarly,Compared with NRCM group,NRCM apoptosis in NRCM+Exo^hMSCsMSCs group,NRCM+Exo^Nullull group,and NRCM+Exo^MIFgroup significantly reduced under normal oxygen environment,p<0.01,respectively.And compared with NRCM+Exo^Nullull group,NRCM apoptosis in NRCM+Exo^MIFgroup,and NRCM+Exo^hMSCsMSCs group also significantly reduced,p<0.01,respectively.Protein expression of Bcl-2 significantly upregulated in NRCM-hypo+Exo^MIFIF group compared with NRCM-hypo group and NRCM-hypo+Exo^hMSCsMSCs group,p<0.01,respectively.Reversely,protein expression of Bax significantly downregulated in NRCM-hypo+Exo^MIFIF group than NRCM-hypo group and NRCM-hypo+Exo^hMSCsMSCs group,p<0.01,respectively.NRCM cocultured with exosomes derived from hMSCs with overexpression MIF.Protein expression of p-AMPK and SIRT1 significantly increased in NRCM-hypo+Exo^MIFIF group than NRCM-hypo group,P<0.001.And p53 significantly decreased in NRCM-hypo+Exo^MIFIF group than NRCM-hypo group,P<0.001.The agonist of AMPK can enlarge the effect of MIF and upregulated the protein expression of p-AMPK and SIRT1,P<0.01,respectively.Reversely,the inhibitorof AMPK?Dorsomorphin,Dor?can inhibit the effect of MIF and downregulated the protein expression of p-AMPK and SIRT1,P<0.05,respectively.Conclusion:Exosomes derived from hMSCs with overexpression MIF deceased apoptosis of cardiomyocytes by AMPK signaling pathway.