Study on the protective effect of folic acid against oxidative stress-induced damage to melanocytes in vitro
- VernacularTitle:叶酸对体外培养的黑色素细胞氧化应激损伤的保护作用研究
- Author:
Jiaxi CHEN
1
;
Xiuli YI
;
Chunying LI
;
Shuli LI
Author Information
- Keywords: Vitiligo; Melanocytes; Folic acid; Oxidative stress; Cell proliferation; Apoptosis; Reactive oxygen species; Membrane potential, mitochondrial; Melanin conten
- From: Chinese Journal of Dermatology 2024;57(6):547-552
- CountryChina
- Language:Chinese
- 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), H 2O 2 treatment group (normal culture for 24 hours followed by the treatment with 1 mmol/L H 2O 2 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 H 2O 2 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 H 2O 2 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 H 2O 2 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 H 2O 2 treatment group, characterized by a substantial number of vacuolated mitochondria, intimal swelling, and reduced ridges, compared with the control group; compared with the H 2O 2 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.
