Serum‑free‑medium‑type mesenchymal stem cell culture supernatant exerts a protective effect on A549 lung epithelial cells in acute lung injury induced by H2O2.

Abstract

The aim of the present study was to investigate the mechanisms and protective effect of serum‑free‑medium‑type fetal placental mesenchymal stem cell (fPMSC) culture supernatant on A549 lung epithelial cells following treatment with hydrogen peroxide(H2O2). A549 lung epithelial cells were stimulated with different concentrations of H2O2, and the survival rate of the cells was examined by Cell Counting Kit‑8 (CCK‑8) assay. It was concluded that the H2O2 concentration when the cell survival rate was at 50% was the optimum condition to create an oxidative damage model. Hoechst33258 staining and western blot analysis was used to validate the A549lung epithelial cell model. Serum‑free medium was used to culture fPMSCs, and A549 lung epithelial cells treated with H2O2 were cultured with passage 3MSC supernatant for 24h. This was termed the supernatant group. Simultaneously, a damage group that was stimulated with H2O2 only, and a vitaminC(VC) group that was treated with H2O2 followed by 100µmol/l VC in culture medium was also established. The apoptosis of the three groups was detected by flow cytometry, and western blotting was used to detect apoptosis‑associated and nuclear factor erythroid 2‑like2 (Nrf2)‑kelch‑like ECH‑associated protein 1 (Keap1)‑antioxidant response element/oxidative stress‑associated protein expression. Following the CCK‑8 test, 600µmol/l H2O2 was selected to stimulate the A549 lung epithelial cells for 24h, which resulted in a A549 cell survival rate of 56.41±3.31%. Hoechst33258 staining and western blotting also confirmed the reliability of the model. Flow cytometry demonstrated that the apoptotic rate of the cells in the VC and supernatant groups was reduced compared with that in the injury group. The difference between the supernatant group and the injury group was statistically significant. The detection of apoptosis‑associated proteins by western blotting revealed that the expression of apoptosis regulator BAX and Caspase‑3 in the VC and supernatant groups was decreased. Furthermore, the expression of B‑cell lymphoma‑2 was increased compared with that in the injury group, and the difference was statistically significant (P<0.05). Compared with that in the injury group, the expression of Nrf2 increased in the VC and supernatant groups, whereas the expression of Keap1 was decreased, and the difference was statistically significant (P<0.05). In conclusion, fPMSC supernatant exhibited an antioxidant capacity in A549 lung epithelial cells treated with H2O2 as a model of acute lung injury. The supernatant was found to reduce oxidative damage and inhibit apoptosis.