Exosomes Derived from miR-214-Enriched Bone Marrow-Derived Mesenchymal Stem Cells Regulate Oxidative Damage in Cardiac Stem Cells by Targeting CaMKII

Figure 4

Exosomes released from hypoxia-pretreated BMSCs protect CSCs from oxidative stress injury. CSCs cultured with 100 μM H₂O₂ were pretreated with BMSC-exos (400 μg/ml) for 24 h and then subjected to analysis. (a) Representative dot plots of cell apoptosis after Annexin V/PI dual staining are shown. The left upper quadrant (% gated) shows necrotic cells (Annexin V−/PI+); the upper right quadrant (% gated) shows late apoptotic cells (Annexin V+/PI+); the left lower quadrant (% gated) shows live cells (Annexin V−/PI−); and the right lower quadrant (% gated) shows early apoptotic cells (Annexin V+/PI−). These cells were measured for comparison. (b) The percentage of apoptotic cells represents both early and late apoptotic cells. Compared with the H₂O₂-treated group, the BMSC-exo-treated group displayed a decreased percentage of apoptotic cells. In addition, Hypoxic-exos more markedly decreased apoptosis than did Nor-exos or Free-exos. (c) The intracellular ROS level was determined by FCM. The P2 percentage indicates the proportion of cells with increased ROS production, with signals above background 2,7-dichlorofluorescein (DCF) fluorescence levels. (d) Compared with the H₂O₂-treated group, the BMSC-exo-treated group had a significantly decreased intracellular ROS fluorescence intensity. In addition, Hypoxic-exos decreased ROS fluorescence to a greater degree than did Nor-exos or Free-exos. (e and f) The effects of BMSC-exos on cell apoptosis-related genes, such as procaspase-3, cleaved caspase-3, Bax, and Bcl-2 were detected by immunoblotting. Compared with the H₂O₂-treated cells, the BMSC-exo-treated cells had substantially decreased levels of cleaved caspase-3 and Bax and increased levels of Bcl-2. Additionally, Hypoxic-exos more markedly affected these protein levels than did Nor-exos. (g and h) Graph represents the SOD and MDA levels in CSCs; compared with H₂O₂ group, Hypoxic-exos inhibited MDA levels and increased SOD production. (i) Representative immunofluorescence staining for TUNEL (green), C-kit (red), DAPI (blue), and merged images. Photos were randomly captured using a fluorescence microscope. Scale bar = 20 μm. (j) The panel shows the percentage of TUNEL-positive cells. Compared with the H₂O₂-treated group, the BMSC-exo-treated group had significantly decreased percentage of TUNEL-positive cells. ; compared with the H₂O₂ group; compared with the Nor-exos group.

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