2023, Vol. 27 ›› Issue (6): 853-859

Effects of bone marrow mesenchymal stem cells-derived exosomes on hypoxia-treated myoblasts

Li Qicheng1, 2, Deng Jin2, Fu Xiaoyang1, 2, Han Na1, 2   

Abstract: BACKGROUND: The cellular functions will be changed in hypoxic conditions, and the exosomes secreted by mesenchymal stem cells can alleviate the pathological damage caused by hypoxia.  
OBJECTIVE: To investigate the effects of different concentrations of CoCl2 on the functional changes of myoblasts, and to further study the effects of exosomes derived from bone marrow mesenchymal stem cells on apoptosis and reactive oxygen species production of hypoxia-treated myoblasts.
METHODS: Bone marrow mesenchymal stem cells from SD rats were isolated in vitro. Exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot assay. Myoblasts were treated with 0, 50, 100, 200 μmol/L CoCl2 for 1, 3, 5, 7 days, and the proliferation activity of myoblasts was detected by CCK-8 assay. After 5 days of treatment, the expression levels of myoblasts differentiation genes MyHC, MyoD and MyoG were detected by qRT-PCR, and the fusion of myoblasts was observed by modified Giemsa staining. Flow cytometry was used to detect the apoptosis rate of myoblasts after treatment for 3 and 5 days. DCFH-DA staining was used to observe the changes of reactive oxygen species of myoblasts after 3 days of treatment. Myoblasts were divided into four groups: control group (0 μmol/L CoCl2), CoCl2 group (200 μmol/L CoCl2), exosomes group (200 μmol/L CoCl2+50 μg/mL exosomes), and NAC group (200 μmol/L CoCl2+2 mmol/L NAC). The apoptosis rate and reactive oxygen species level were detected after 3 days of treatment.  
RESULTS AND CONCLUSION: (1) The exosomes showed cup-like structure. The particle size distribution of exosomes was between 30-150 nm, and the expression of CD9 and TSG101 was positive. (2) CoCl2 had a significant inhibitory effect on the proliferation and differentiation of myoblasts (P < 0.05) and promoted the apoptosis of myoblasts (P < 0.05), and the high concentration of CoCl2 had a more obvious inhibitory effect on the functions of myoblasts. (3) Exosomes reduced the apoptosis rate of myoblasts and reactive oxygen species level induced by 200 μmol/L CoCl2 (P < 0.05), and it was similar to the anti-reactive oxygen species reagent NAC in reducing the level of reactive oxygen species (P < 0.05). (4) These findings suggest that CoCl2 had different inhibitory effects on the physiological functions of myoblasts in a concentration-time dependent manner, while the exosomes derived from bone marrow mesenchymal stem cells and NAC had similar effects of anti-hypoxia apoptosis and reducing the reactive oxygen species. The mechanism may be that the exosomes alleviated the hypoxia-induced apoptosis of myoblasts by reducing the production of reactive oxygen species.
Key words: stem cell, mesenchymal stem cell, bone marrow mesenchymal stem cell, exosome, myoblast, hypoxia, apoptosis, reactive oxygen species, CoCl2