ABSTRACT
BACKGROUND: Preliminary experimental studies have shown that the supernatant of human placental fetal mesenchymal stem cells (fPMSCs) has a certain ability to scavenge reactive oxygen species and itself has a certain antioxidant enzyme activity. OBJECTIVE: To investigate the protective role and mechanism of fPMSCs supernatant in serum-free culture on oxidative stress-injured lung epithelial cells. METHODS: Different concentrations of hydrogen peroxide produced oxygen stimulation to lung epithelial cell lines A549 for 6, 12, 24 hours, and the survival rate of lung epithelial cells was detected using cell counting kit-8 method. When the survival rate of lung epithelial cells was 50%, the concentration of hydrogen peroxide was most suitable to make an oxidative damage model. The validity of the model was verified using Hocheest33258 staining and western blot. fPMSCs were cultured in serum-free culture medium, and the supernatant of passage 3 cells was collected. Afterwards, the injured lung epithelial cells were cultured in the fPMSCs cell supernatant for 24 hours. Meanwhile, injury group (oxidative damage only) and vitamin C group (100 μmol/L vitamin C was added in the medium) were established. In the three groups, cell apoptosis was detected by flow cytometry; and western blot was used to detect apoptosis-related proteins and proteins related to the Nrf2-Keap1-ARE signaling pathway. RESULTS AND CONCLUSION: After oxygen stimulation by 600 μmol/L hydrogen peroxide for 24 hours, the survival rate of A549 cells was (56.41±3.31)% as ascertained by the cell counting kit-8 assay. Findings from Hocheest33258 staining and western blot further confirmed the reliability of this model. Flow cytometry results showed that the apoptosis rate in the vitamin C group and the supernatant group decreased to some extent compared with the injury group, and the difference between the supernatant group and the injury group was statistically significant (P < 0.05). In addition, the expression of Bax significantly decreased and the expression of Bcl-2 significantly increased in the vitamin C group and the supernatant group as detected by western blot assay, in comparison with the injury group (P < 0.05). Compared with the injury group, the expression of Nrf2 protein increased and the expression of Keap1 decreased in the vitamin C group and the supernatant group (P < 0.05). These findings suggest that fPMSCs supernatant has a certain antioxidant capacity, and may attenuat oxidative damage and inhibit apoptosis in A549 cells. The mechanism is probably related to the Nrf2-Keap1-ARE signaling pathway.