In vitro effects of ascorbic acid on H2O2-induced oxidative injury in cultured melanocytes

Abstract

Objective To evaluate effects of ascorbic acid on proliferative activity of cultured melanocytes in vitro, as well as on H2O2-induced oxidative injury in melanocytes. Methods The optimal concentration of ascorbic acid solution and median lethal dose of H2O2 solution were determined by CCK-8 assay for the following experiment. Cultured melanocytes were classified into the control group, ascorbic acid group, H2O2 group and combination group. During the first 24 hours, the control group and H2O2 group were treated with M254 medium, while the ascorbic acid group and combination group with ascorbic acid solution. During an additional 24-hour period, the control group and ascorbic acid group were treated with M254 medium, while the H2O2 group and combination group with H2O2 solution at the median lethal dose. After 48-hour treatment, CCK-8 assay and flow cytometry were performed to determine the survival rate and apoptosis rate of melanocytes, respectively, in the 4 groups. Biochemical methods were used to evaluate the superoxide dismutase (SOD) activity and determine the malondialdehyde (MDA) concentration, and fluores-cent staining was conducted to detect the level of reactive oxygen species (ROS) in the control group, H2O2 group and combination group. Results The optimal concentration of ascorbic acid solution was 1 000 μmol/L, and the median lethal dose of H2O2 solution was 300 μmol/L. The cell survival rate, apoptosis rate, SOD activity, MDA concentration and ROS fluorescence intensity in the control group were 100% ± 4.99%, 6.90% ± 0.87%, 54.71 ± 4.75 U/mgprot, 263.39 ± 20.17 nmol/mgprot and 342.16 ± 27.36 respectively. Compared with the control group, H2O2 solution could significantly increase the cell apoptosis rate (16.47% ± 1.07%), SOD activity (103.62 ± 10.44 U/mgprot), MDA concentration (493.70 ± 31.36 nmol/mgprot) and intracellular ROS fluorescence intensity (782.48 ± 36.25), but decrease the survival rate of melanocytes (39.07% ± 2.94% ), while ascorbic acid solution markedly down-regulated the H2O2-induced apoptosis (11.83% ± 0.95%), SOD activity (76.73 ± 5.20 U/mgprot), MDA concentration (371.82 ± 23.05 nmol/mgprot) and ROS level (475.64 ± 52.18), but increased the cell survival rate (74.31% ± 5.53% ). Conclusion Ascorbic acid solution at the concentration of 1 000 μmol/L can not only promote proliferative activity of melanocytes, but also protect melanocytes from H2O2-induced oxidative injury. Key words: Melanocytes; Ascorbic acid; Hydrogen peroxide; Reactive oxygen species; Oxidative injury