Abstract: Yang Sailin, Xu Wen, Lin Fuquan, Zhou Miaoni, Xu Ai′e Department of Dermatology, Third Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310009, Zhejiang, China Corresponding author: Xu Ai′e, Email: xuaiehz@msn.com 【Abstract】 Objective To analyze effects of tacrolimus on the secretion of chemokines CXCL9 and CXCL10 by γ-interferon （IFN-γ）-simulated HaCaT cells, as well as phosphorylated Janus kinase 1 （p-JAK1） and phosphorylated signal transducer and activator of transcription 1 （p-STAT1）, and to explore the mechanism of tacrolimus in the treatment of vitiligo. Methods HaCaT cells were treated with 1, 10, 20, 40, 60, 80, 100, 120 mg/L tacrolimus solution separately for 4 hours, and methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the cellular proliferative activity. HaCaT cells were divided into 4 groups: blank control group receiving no treatment, IFN-γ group treated with 500 U/ml IFN-γ for 12 or 48 hours, tacrolimus group treated with 20 mg/L tacrolimus for 4 hours, and tacrolimus + IFN-γ group treated with 20 mg/L tacrolimus for 4 hours followed by the treatment with 500 U/ml IFN-γ for 12 or 48 hours. Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA of CXCL9 and CXCL10, Western blot analysis to determine the protein of CXCL9, CXCL10, p-JAK1, and p-STAT1, and enzyme-linked immunosorbent assay （ELISA） to detect the levels of CXCL9 and CXCL10 in the culture supernatants of HaCaT cells. Results Tacrolimus at the maximum concentration of 20 mg/L had no effect on the proliferation of HaCaT cells （P > 0.05）. After the pretreatment with 20 mg/L tacrolimus, the mRNA of CXCL9 and CXCL10 significantly decreased from 10 369.08 ± 7.99 and 290.02 ± 2.16 to 5 914.33 ± 4.59 and 114.96 ± 0.73, respectively, after the treatment with IFN-γ （both P < 0.01）, and the protein of CXCL9, CXCL10, p-JAK1, and p-STAT1 also significantly decreased from 8.47 ± 0.29, 7.87 ± 0.17, 4.20 ± 0.18 and 4.29 ± 0.11 to 7.36 ± 0.13, 7.36 ± 0.09, 2.60 ± 0.16 and 3.62 ± 0.19, respectively, after the treatment with IFN-γ （all P < 0.01）. Moreover, the levels of CXCL9 and CXCL10 in the culture supernatants of HaCaT cells significantly decreased in the IFN-γ group （1 213.36 ± 0.95, 1 722.41 ± 2.57, respectively） compared with the tacrolimus + IFN-γ group （426.45 ± 0.31, 554.12 ± 0.56, respectively, both P < 0.01）. Conclusion Tacrolimus can inhibit the secretion of CXCL9, CXCL10, p-JAK1 and p-STAT1 by HaCaT cells stimulated by IFN-γ.

Key words: Vitiligo, Interferon?gamma, Chemokine CXCL9, Chemokine CXCL10, Janus kinases, STAT1 transcription factor, HaCaT cells, Tacrolimus