Abstract: 【Abstract】 Objective To investigate the protective effect of folic acid on melanocytes under oxidative stress. Methods The normal human melanocyte cell line （PIG1） was cultured in vitro and divided into 5 groups to receive corresponding treatments: control group （normal culture for 48 hours without other treatment）, H2O2 treatment group （normal culture for 24 hours followed by the treatment with 1 mmol/L H2O2 for another 24 hours）, and 3 folic acid pretreatment groups （pretreatment with folic acid at concentrations of 50, 125, and 250 μmol/L for 24 hours followed by the treatment with 1 mmol/L H2O2 for another 24 hours）. The cell viability was assessed using the cell counting kit 8 （CCK8） assay, the intracellular melanin content was measured by the sodium hydroxide solubilization method, cell apoptosis rates were detected by annexin V-fluorescein isothiocyanate/propidium iodide double staining, levels of intracellular reactive oxygen species （ROS） were detected using the fluorescent probe CM-H2DCFDA, the mitochondrial membrane potential was determined using the fluorescent probe JC-1, and the mitochondrial ultrastructure was observed by transmission electron microscopy. Comparisons among multiple groups were performed using one-way analysis of variance, and multiple comparisons were performed using Tukey test. Results Compared with the control group, the H2O2 treatment group showed decreased cell viability （83.62% ± 3.77% vs. 99.99% ± 5.06%, P = 0.031）, intracellular melanin content （68.48% ± 4.17% vs. 100.11% ± 2.30%, P < 0.001）and mitochondrial membrane potential （2.96 ± 0.26 vs. 5.86 ± 0.56, P = 0.002）, but increased cell apoptosis rate （16.35% ± 1.20% vs. 6.45% ± 1.34%, P = 0.001） and intracellular ROS level （138.98% ± 2.74% vs. 100.00% ± 0.64%, P = 0.004）. Compared with the H2O2 treatment group, the 125-μmol/L and 250-μmol/L folic acid pretreatment groups showed increased cell viability （106.21% ± 6.34%, 101.64% ± 6.77%, respectively; both P < 0.05） and intracellular melanin content （77.24% ± 3.85%, 88.34% ± 2.65%, respectively; both P < 0.05）; the 50-μmol/L, 125-μmol/L and 250-μmol/L folic acid pretreatment groups all showed decreased cell apoptosis rates （9.40% ± 0.99%, 9.00% ± 1.13%, 6.50% ± 0.28%, P = 0.007, 0.005, 0.001, respectively）; the 125-μmol/L and 250-μmol/L folic acid pretreatment groups showed decreased intracellular ROS levels （112.99% ± 4.21%, 101.36% ± 10.60%, P = 0.023, 0.005, respectively）, but increased mitochondrial membrane potential （4.93 ± 0.25, 5.67 ± 0.35, P = 0.012, 0.003, respectively）. Transmission electron microscopy showed damaged mitochondrial ultrastructure in melanocytes in the H2O2 treatment group, characterized by a substantial number of vacuolated mitochondria, intimal swelling, and reduced ridges, compared with the control group; compared with the H2O2 treatment group, the 250-μmol/L folic acid pretreatment group exhibited decreased degree of mitochondrial damage, manifesting as reduced mitochondrial vacuolization, clearer mitochondrial ultrastructure, and slight swelling of mitochondrial ridges. Conclusion Folic acid could reduce the oxidative stress level in melanocytes, thus protecting melanocytes from oxidative stress.

Key words: Vitiligo, Melanocytes, Folic acid, Oxidative stress, Cell proliferation, Apoptosis, Reactive oxygen species, Membrane potential, mitochondrial, Melanin content