Abstract: 【Abstract】 Objective To investigate the effect of the antioxidant Tempol on the skin depigmentation of a vitiligo-like mouse model induced by the combination of the endogenous reactive oxygen species （ROS） producer AAPH and tyrosinase-related protein 2 （TRP2）-180 nonapeptide. Methods A vitiligo-like skin depigmentation model was established by immunizing mice with injections of a TRP2-180 nonapeptide mixture into the foot pads twice and into the tails twice, with the injection interval being 1 week. After the first injection, 12 immune-induced mouse models of vitiligo were randomly divided into 4 groups （3 mice per group）: a model group, an AAPH group, a Tempol group, and a combined treatment group; additionally, 3 untreated mice injected with an ovalbumin （OVA） 257-264 peptide served as a sham control group. Mice in the AAPH group, the Tempol group, and the combined treatment group were subcutaneously injected with AAPH into the tails, intraperitoneally injected with Tempol, and received the above both treatments, respectively, while mice in the model group and the sham control group received phosphate-buffered saline injections into the tail and/or abdomen. Drug interventions were carried out 3 times per week for 3 consecutive weeks. Six weeks after the last immunization, mice were sacrificed. The area of depigmented macules on the tail was measured using a point-counting method, X-gal staining and double immunofluorescence staining were performed to assess the distribution and number of melanocytes, mast cells, and CD8+ T cells in depigmented macules on the tail. HaCaT cells were in vitro co-cultured with AAPH and/or Tempol, and a conventional culture group served as the control. Cellular ROS levels were measured by dichlorofluorescin diacetate labeling and flow cytometry; Western blot analysis was performed to determine the expression of matrix metalloproteinase 9 （MMP9） and stem cell factor （SCF） in cell lysates, and to detect soluble SCF levels in the culture supernatant. Comparisons among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were performed using least significant difference-t test. Results Depigmented macules were observed on the tails of mice in all groups except the sham control group. The area of depigmented macules was significantly larger in the AAPH group （7.27 ± 0.31 cm2） than in the model group and combined treatment group （3.53 ± 0.21 cm2, 4.07 ± 0.40 cm2; t = 13.48, 11.56, respectively, both P < 0.001）, while there was no significant difference between the Tempol group （3.30 ± 0.40 cm2） and the model group （P = 0.424）. X-gal staining and double immunofluorescence staining showed that the number of melanocytes in the normal skin around the depigmented macules was significantly lower in the AAPH group and the combined treatment group than in the model group （t = 6.31, 5.16, respectively, both P < 0.001）, and no significant difference was observed between the AAPH group and the combined treatment group （P = 0.516）. The numbers of CD8+ T cells and mast cells were significantly higher in the AAPH group than in the model group and the combined treatment group （all P < 0.001）. The numbers of the 3 types of cells mentioned above in the Tempol group did not differ from those in the model group （all P > 0.05）. The ROS levels in HaCaT cells in the AAPH group were the highest, and significantly higher than those in the control group and the combined treatment group （both P < 0.001）. Western blot analysis showed that the MMP9 level in the cell lysates and soluble SCF level in the culture supernatant were significantly higher in the AAPH group than in the control group and the combined treatment group （all P < 0.05）; no significant difference was observed in the membrane-bound SCF level in cell lysates among the groups （F = 0.06, P = 0.977）. Conclusion The antioxidant Tempol could inhibit the formation of skin depigmented macules in vitiligo-like mouse models under AAPH-induced oxidative stress.

Key words: Vitiligo, Oxidative stress, Antioxidants, Models, animal, Melanocytes, Mast cells