Abstract: 【Abstract】 Objective To evaluate the effect of benvitimod on the proliferation of, inflammatory cytokine secretion by, skin barrier protein synthesis by, and phosphorylation of signal transducer and activator of transcription 1 （STAT1） in human keratinocytes. Methods In vitro cultured HaCaT cells were treated with 0.1 - 1 000 μmol/L benvitimod for 24 hours, and cell counting kit-8 （CCK8） assay was performed to evaluate cell proliferative ability. Some HaCaT cells were divided into 6 groups: control group treated with DMEM medium alone, stimulant group treated with 10 μg/L tumor necrosis factor-α （TNF-α） and interferon-γ （IFN-γ）, benvitimod groups treated with benvitimod at final concentrations of 1 - 10 or 1 - 100 μmol/L followed by the treatment with 10 μg/L TNF-α and IFN-γ, aryl hydrocarbon receptor （AhR） antagonist group treated with 10 or 100 μmol/L benvitimod and 10 nmol/L StemRegenin1 （SR1） followed by the treatment with 10 μg/L TNF-α and IFN-γ. After 24-hour treatment, enzyme-linked immunosorbent assay （ELISA） was performed to detect levels of interleukin （IL）-4, IL-10, IL-22 and thymus- and activation-regulated chemokine （TARC） in the cell culture supernatant, reverse transcription （RT）-PCR to determine the mRNA expression of AhR, cytochrome P450 1A （CYP1A1）, filaggrin, involucrin, thymic stromal lymphopoietin （TSLP） and TARC in HaCaT cells, Western blot analysis to determine the protein expression of filaggrin, involucrin, TSLP, STAT1 and phosphorylated STAT1 （p-STAT1）, and immunofluorescence study to assess the effect of benvitimod on AhR nuclear translocation in HaCaT cells. Measurement data were compared by using unpaired Student′s t test and one-way analysis of variance, and relationship between the indicators was analyzed by using Spearman test. Results After 24-hour treatment with benvitimod at concentrations of 0.1, 1, 10, 100 and 1 000 μmol/L, the survival rate of HaCaT cells significantly differed among the different benvitimod groups （90.2% ± 2.4%, 85.4% ± 11.9%, 52.8% ± 14.0%, 39.4% ± 7.9%, 27.5% ± 3.4%, respectively, F = 162.5, P < 0.001）, and the 50% inhibitory concentration was 48.54 μmol/L. Compared with the stimulant group, the level of IL-10 secreted by HaCaT cells significantly increased in the 10- and 100-μmol/L benvitimod groups （F = 16.110, P < 0.001）, while the IL-22 level significantly decreased in the 100-μmol/L benvitimod group （F = 6.884, P < 0.001）, and the TARC level significantly decreased in the 10- and 100-μmol/L benvitimod groups （F = 7.052, P < 0.001）. Compared with the stimulant group, RT-PCR showed significantly increased CYP1A1 mRNA expression in the 1- and 10-μmol/L benvitimod groups （P = 0.004）, significantly increased FLG mRNA expression in the 10-μmol/L benvitimod group （P = 0.040）, but significantly decreased TARC and TSLP mRNA expression in the 10-μmol/L benvitimod group （both P < 0.01）, and there was no significant difference in the AhR mRNA expression between the stimulant group and benvitimod group （P = 0.193）. Compared with the stimulant group, Western blot analysis showed significantly increased filaggrin expression but significantly decreased TSLP expression in the 10-μmol/L benvitimod group （P = 0.02, < 0.001, respectively）, and significantly increased involucrin expression but significantly decreased p-STAT1 expression in the 1-, 10-μmol/L benvitimod groups （all P < 0.001）. Compared with the 100-μmol/L benvitimod group, the AhR antagonist group showed significantly decreased supernatant levels of IL-10 （t = 4.794, P = 0.003）, but significantly increased mRNA expression of TSLP （t = 3.769, P = 0.005）; compared with the 10-μmol/L benvitimod group, the AhR antagonist group showed significantly decreased protein expression of involucrin （t = 5.117, P = 0.002）, but significantly increased protein expression of TSLP （t = 3.117, P = 0.043）, and there was no significant change in protein expression of p-STAT1 （t = 1.400, P = 0.719）. Immunofluorescence staining showed green fluorescence of AhR in the cytoplasm of HaCaT cells in the control group and 1-μmol/L benvitimod group, but almost no fluorescence in the nuclei; both the 10- and 20-μmol/L benvitimod groups showed high-density green fluorescence in the cytoplasm and nuclei of HaCaT cells. Conclusion Benvitimod can inhibit the proliferation of HaCaT cells, regulate the secretion of inflammatory cytokines, upregulate production of skin barrier-related factors and inhibit STAT1 phosphorylation by activating the AhR signaling pathway.

Key words: Dermatitis, atopic, Keratinocytes, Cell proliferation, Cytokines, Benvitimod, Aryl hydrocarbon receptor