Protection Of Vitamin C On Human A375 Melanoma Cells Under H₂O₂-induced Oxidative Damage

Objective: To observe the anti-oxidation effect of Vitamin C on H2O2 damaged human A375 melanoma cells, and provide experimental evidence for Vitamin C as an antioxidant used to treat vitiligo.Methods:1. We selected the optimal dose and time by MTT of H2O2 from different concentrations(100μmol/L,250μmol/L,500μmol/L) and time(1h,2h,4h) on human A375 melanoma cells. Then we used them to complete H2O2 damaged model.2. We used different dose of Vitamin C(0.1μmol/ml,0.5μmol/ml,1.0μmol /ml,1.5μmol/m,2.0μmol/ml,2.5μmol/ml,3.0μmol/ml,4.0μmol/ml,5.0μmol/ml) on H2O2 damaged model. 12 hours later we tested A490 value to pick out optimal concentration by MTT. The optimal time was chosen from different time(2h,6h,12 h,24h,36 h,48h) by the same way.3. The final concentrations(0.1μmol/ml,1.0μmol/ml,2.0μmol/ml,4.0μmol /ml) and time(6h,12 h,24h,36h) of Vitamin C were applied to H2O2 damaged model. MTT colorimetry was used to observe proliferation, Annexin V/PI staining method was used to observe apoptosis, and NaOH assay was used to observe melanogenesis on each group.Results:1. H2O2 could take injury into cell morphology and suppress proliferation on human A375 melanoma cells by oxidative stress damage in concentration dependent manners.2. MTT showed: After adding four concentrations of Vitamin C(0.1μmol/ml,1.0μmol/ml,2.0μmol/ml,4.0μmol/ml), the proliferation level was higher than H2O2 damaged group, and the difference was remarkable(P<0.05). 4μmol/ml Vitamin C could get the maximal A490 value(0.945±0.044) after 12 hours cultivation.3. Flow cytometry showed: The cell viability rate of each Vitamin C group increased in concentration dependent manners. The early and late apoptotic rate declined gradually, and all of them were lower than H2O2 damaged group.4. NaOH assay showed: The four concentrations of Vitamin C(0.1μmol/ ml,1.0μmol/ml,2.0μmol/ml,4.0μmol/ml) could regain melanogenesis of H2O2 damaged human A375 melanoma cells. The melanin level was higher than H2O2 damaged group, and the difference was remarkable(P<0.05). 4μmol/ml Vitamin C could get the maximal A490 value(0.360±0.031) after 12 hours cultivation.Conclusion:1. H2O2 could lead oxidative damage on human A375 melanoma cells by inhibiting proliferation and contributing to apoptosis.2.A certain concentration of Vitamin C could not only promote cell proliferation and melanogenesis, but inhibit apoptosis rate on H2O2-damaged human A375 melanoma cells.