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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.
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Objective To detect levels of aryl hydrocarbon receptor (AhR) and its downstream molecules in peripheral blood mononuclear cells (PBMCs) and sera from patients with atopic dermatitis (AD),and to analyze the correlation of their expression with serum cytokines and the severity of AD.Methods Real-time quantitative PCR (RT-PCR) was performed to analyze mRNA expression of AhR,cytochrome P4501A (CYP1A1),AhR repressor (AHRR),AhR nuclear translocator (ARNT) in PBMCs from 29 AD patients and 17 healthy controls,enzyme-linked immunosorbent assay (ELISA) to detect serum levels of interleukin (IL)-1β,IL-6,tumor necrosis factor (TNF)-α,IL-4,IL-22 and AhR in the AD patients,and immunohistochemical study to determine AhR expression in skin lesions of the AD patients and normal skin tissues of 21 patients with pigmented nevus.Measurement data were compared by using unpaired Student's t test,enumeration data were compared by using chi-square test,and correlations between indices were analyzed by using Pearson correlation analysis.Results The serum level of AhR was significantly higher in the AD group (41.26 ± 4.52 pmol/L) than in the healthy control group (33.73 ± 2.49 pmol/L,t =6.507,P ＜ 0.001).Compared with the healthy control group,the AD group showed significantly increased mRNA expression ofAhR (1.572 ± 0.392 vs.1.000 ± 0.173,t =6.819,P =0.007),AHRR (2.402 ±1.716 vs.1.000 ± 0.788,t =3.722,P =0.039),CYP1A1 (2.258 ± 1.598 vs.1.000 ± 0.796,t =3.400,P =0.002) and ARNT (1.383 ± 0.842 vs.1.000 ± 0.586,t =1.653,P =0.105) in PBMCs.The AhR expression in skin lesions in the AD group was significantly higher than that in normal skin tissues in the control group (0.191 ± 0.041 vs.0.087 ± 0.017,t =10.036,P ＜ 0.001).In the AD group,the mRNA expression of AhR in PBMCs was positively correlated with eczema area and severity index score (r =0.448,P =0.019) and the serum IL-6 level (r =0.377,P =0.046),and the AHRR mRNA expression was positively correlated with the serum IL-1β level (r =0.467,P =0.021).Conclusion AhR and its downstream molecules were highly expressed in the AD patients compared with healthy controls,and the AhR expression was positively correlated with the serum IL-6 level and AD severity in AD patients,suggesting that the AhR signaling pathway may play a certain role in pathogenesis of AD and AhR may serve as an efficient index for evaluating AD severitv.
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<p><b>BACKGROUND</b>Keratinocytes play a crucial role in the biological function of skin barrier. The relationship between sodium lauryl sulfate (SLS) and keratinocytes has been studied. However, the cytotoxicity and effects of sodium dodecyl benzene sulfonate (SDBS), a common detergent similar to SLS, on keratinocytes are still not known. This study aimed to investigate the effects of SDBS on cytotoxicity and expression of proinflammatory cytokines in cultured human keratinocytes.</p><p><b>METHODS</b>This study was carried out using the keratinocytes cell line, HaCaT cells. The cytotoxicity of SDBS on HaCaT cells was evaluated with cell counting kit-8 (CCK-8) and phase-contrast microscopy. After exposure to different concentrations of SDBS, the total RNA of the HaCaT cells was extracted for evaluating the relative mRNA expression of IL-1α, IL-6, IL-8, and TNF-α by qPCR. The supernatants of cells were collected for measuring the levels of IL-6 and IL-8 by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>SDBS at concentrations of 20 µg/ml and over showed direct cytotoxicity and induced morphological changes of the HaCaT cells. The mRNA expressions of IL-1α, IL-6, IL-8, and TNF-a in different concentrations of SDBS at different time were comparable with that of controls. SDBS at concentrations of 5, 10, and 15 µg/ml had no significant effects on IL-6 and IL-8 excretion from HaCaT cells after 24-hour exposure. Moreover, no significant effects on the IL-6 and IL-8 excretion were found after 10 and 15 µg/ml SDBS stimulations for 6, 12, and 24 hours, respectively.</p><p><b>CONCLUSION</b>SDBS at higher concentrations had cytotoxicity on HaCaT cells but had no effects on the mRNA expression of IL-1α, IL-6, IL-8, and TNF-a, that was different from SLS.</p>
Subject(s)
Humans , Benzenesulfonates , Pharmacology , Cell Line , Enzyme-Linked Immunosorbent Assay , Interleukin-1alpha , Metabolism , Interleukin-6 , Metabolism , Interleukin-8 , Metabolism , Keratinocytes , Metabolism , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
Objective To investigate the association of HLA-DRB31*03,*04 and *11 alleles with alopecia areata(AA)in Han Nationality in East China.Methods Polymerase chain reaction-sequence specific primer(PCR-SSP)method was conducted in 158 Chinese Han patients with AA as well as in 172 healthy human controls in East China.The relationships of HLA-DRB1 polymorphism to age of onset,episode frequency,clinical course,family history,and severity of AA were evaluated.Results No significant differences were observed for the frequency of HLA DRB1*03,*11 alleles between the patients and human controls,while increased frequency of HLA-DRB1*04 was observed in patients(OR=1.99,Pc=0.01).Multiple logistic regression analysis revealed that HLA-DRB1*04 was more prevalent in patients with an onset after 16 years of age(OR=1.94,Pc=0.02),those without family history(OR=1.97,Pc=0.02),those with recurrent AA(OR=2.49,Pc=0.02),those with a clinical course of more than 1 year(OR=2.94,Pc=0.01),those with severe AA(OR=3.53,Pc=0.00)and tbose with single episode of AA(OR=1.83,Pc=0.04)in comparison with the normal human controls.Conclusion This study demonstrates that HLA-DRB1*04 allele is associated with the occurrence and clinical types of AA in Han Nationality in East China.