Human Mesenchymal Stem Cell-derived Exosomes Reduce Ischemia/Reperfusion Injury by the Inhibitions of Apoptosis and Autophagy

Background: Human mesenchymal stem cell-derived exosomes (hMSC-Exo) have been shown to reduce ischemia/reperfusion injury (I/R) in multiple models. I/R-induced apoptosis or autophagy play important roles in cell death. However, little or no reports demonstrate any roles of hMSC-Exo in this regards. Objective: To test the hypothesis that the inhibition of I/R-induced apoptosis and autophagy play a pivotal role in the cardioprotection of hMSC-Exo. Methods: Myoblast H9c2 cells and isolated rat hearts underwent hypoxia/re-oxygenate (H/R) or ischemia/ reperfusion (I/R) respectively. H9c2 were treated with 1.0 μg/ml Exo, in comparison with 3-MA or rapamycin (Rapa), a known anti- or pro-autophagic agent respectively. Hearts were treated with 0.5, 1.0 and 2.0 μg/ml Exo for 20 min in the beginning of reperfusion. Cell viability, WST assay, LDH release, Annexin-V staining apoptosis assay and GFP-LC3 labeled autophagosomes formation, cardiac function and Western blot were measured. Results: Exo significantly reduced H/R injury as indicated by increased cell viability and reduced LDH and apoptosis. 3-MA, while Rapa, showed increased or decreased protective effects. Rapa-induced injury was partially blocked by Exo. Exo decreased LC3-II/I ratio and increased p62, inhibited autophagosome formation, an indication of autophagy inhibition. In isolated heart, Exo increased cardiac functional recovery and reduced LDH release in I/R. Bcl-2 was significantly upregulated by Exo but not 3-MA. Exo downregulated Traf6 and upregulated mTORC1/p-4eBP1. Conclusion: Exo reduce I/R-induced apoptosis and autophagy. Up-regulation of Bcl-2 is the cross-talk between these two processes. The down-regulation of Traf6 and activation of mTORC1 are additional mechanisms in the inhibition of apoptosis and autophagy.