Effect of benvitimod on the proliferation of, inflammatory cytokine secretion by and skin barrier factor production by HaCaT cells / 中华皮肤科杂志

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.