2022, Vol. 26 ›› Issue (1): 1-6

Hypoxic pretreatment enhances the protective effect of aged human bone marrow mesenchymal stem cells conditioned medium against H9C2 oxidative stress damage

Song Huifang1, 2, Tan Jiayin2, Kang Yi3, Li Bin2, Bi Zhifei2, Long Nü2, Xia Zhongnian2, Guo Rui4   

Abstract:

BACKGROUND: The therapeutic effect of autologous stem cell transplantation in aged patients suffered with ischemic heart injury is not good as young. It is important to explore effective methods to improve the repair and regenerate ability of aged stem cells.
OBJECTIVE: To investigate the protective effect of hypoxia preconditioning aged human bone marrow mesenchymal stem cells conditioned medium on myocardium and to provide experimental support for more effective autologous stem cell transplantation in aged patients.
METHODS: The young human bone marrow mesenchymal stem cells were placed in a normoxic incubator, and the aged human bone marrow mesenchymal stem cells were placed in a normoxic or hypoxic incubator respectively. The culture media were collected and divided into three groups: norCM-young, norCM-old and hypoCM-old. H9C2 cells were treated with three groups of conditioned media, and then treated with 300 μmol/L H2O2 for 30 minutes to establish an oxidative stress model. The cell survival rate of H9C2 was detected by the CCK-8 assay. Reactive oxygen species level detection by reactive oxygen species assay kit reflected the degree of oxidative stress damage. Bax and Bcl-2 expression levels by western blotting reflected the apoptosis level. p-AKT expression level detected by western blot assay reflected AKT activation level. In addition, H9C2 was cultured with Dulbecco’s Modified Eagle Medium containing 50 μmol/L LY294002 for 2 hours before hypoCM-old and H2O2 treatment to block the PI3K/AKT pathway and cell viability was detected by the CCK-8 assay.   
RESULTS AND CONCLUSION: (1) Compared with norCM-old group, hypoCM-old significantly improved the survival rate of H9C2 cells under oxidative stress damage (P < 0.01); significantly reduced the expression of apoptosis-related protein Bax (P < 0.05), increased the expression of apoptosis inhibitor protein Bcl-2 (P < 0.05); and reduced the ROS level in H9C2 cells (P < 0.05). (2) Compared with the norCM-old group, the p-AKT protein level in H9C2 cells treated with hypoCM-old was significantly increased (P < 0.05). (3) Compared with the hypoCM-old group, when the PI3K/AKT pathway of H9C2 was inhibited, the H9C2 cell survival was reduced (P < 0.05). (4) The results indicate that the conditioned media of aged human bone marrow mesenchymal stem cells pretreated with hypoxia could attenuate the oxidative stress damage of H9C2 cells, reduce apoptosis and enhance survival through the activation of AKT pathway.

Keywords:stem cells, human bone marrow mesenchymal stem cells, aged, conditioned medium, hypoxia, preconditioning, H9C2 cells, AKT, oxidative stress