Abstract:

Objective To investigate DJ-1 expression and protective effect against H2O2-induced oxidative stress in primary human melanocytes. Methods The expression and location of DJ-1 in primary melanocytes were identified by immunofluorescence. After cultured melanocytes were exposed to H2O2 for 24 hours, modified MTT assay was used to assess the proliferation of cells and to choose the suitable concentration of H2O2 for the following experiment. Western blot was used to detect DJ-1 expression in melanocytes after being treated with 0.5 mmol/L H2O2 for 24 hours. Some melanocytes were divided into 3 groups to be reversely transfected with PBS （mock control group）, non-targeting siRNA （negative control group） and DJ-1 targeting siRNA （DJ-1 group）. Optical microscopy was utilized to observe the morphologic changes of transfected melanocytes. At 48 hours after the transfection, the melanocytes were stimulated with H2O2 for 24 hours. Subsequently, modified MTT assay, 2′,7′-dichlorofluorescein diacetate （DCFH-DA） and annexinⅤ-fluorescein isothiocyanate/propidium iodide were used to determine cell viability, intracellular reactive oxygen species （ROS） level and apoptosis rate respectively. Results DJ-1 was expressed in both melanocyte nucleus and cytoplasm, and predominantly in the nucleus. H2O2 inhibited the cell viability in a dose dependent manner. After treatment with H2O2 of 0.5 mmol/L for 24 hours, the cell viability began to decrease in melanocytes with the expression level of DJ-1 being 2.23 times that in the untreated melanocytes （both P < 0.05）. Compared with the mock control group, the dendrites of melanocytes in DJ-1 group were obviously shortened with cytoplasm vacuolization. After 24-hour treatment with H2O2 of 0.5 mmol/L, the cell viability in the DJ-1 group dropped to 35% of that in the mock control group （P < 0.05）, while the intracellular ROS fluorescence intensity （FI） and apoptosis rate were higher in the DJ-1 group than in the mock control group （902 ± 40 vs. 529 ± 32, 58% ± 6.1% vs. 30% ± 3.8%, both P < 0.05）. Conclusion DJ-1 can protect melanocytes against H2O2-induced oxidative stress likely by decreasing intracellular ROS production and inhibiting cell apoptosis.

Key words: Vitiligo