本维莫德对HaCaT细胞增殖、炎症因子分泌及皮肤屏障因子产生的影响  

Effect of benvitimod on the proliferation of,inflammatory cytokine secretion by and skin barrier factor production by HaCaT cells

作　　者：胡宇晴 刘萍[1] 慕彰磊[1] 张建中[1] Hu Yuqing;Liu Ping;Mu Zhanglei;Zhang Jianzhong(Department of Dermatology,Peking University People′s Hospital,Beijing 100044,China)

机构地区：[1]北京大学人民医院皮肤科,100044

出　　处：《中华皮肤科杂志》2020年第12期984-991,共8页Chinese Journal of Dermatology

基　　金：北京大学人民医院研究与发展基金(RDY2019-24)。

摘　　要：目的研究本维莫德对人角质形成细胞增殖、炎症细胞因子分泌、皮肤屏障蛋白合成以及信号转导与转录激活蛋白1(STAT1)磷酸化的影响。方法体外培养HaCaT细胞,采用0.1~1000μmol/L本维莫德处理24 h,用CCK8法检测细胞增殖。部分HaCaT细胞分为6组,对照组仅加入DMEM培养基,刺激剂组加入10μg/L肿瘤坏死因子α(TNF-α)和干扰素γ(IFN-γ),本维莫德组加入10μg/L TNF-α和IFN-γ以及终浓度为1~10或1~100μmol/L本维莫德,AhR拮抗剂组加入10μg/L TNF-α和IFN-γ、10或100μmol/L本维莫德以及10 nmol/L StemRegenin1(SR1),处理24 h后,酶联免疫吸附实验检测细胞培养上清液中白细胞介素(IL)-4、IL-10、IL-22和胸腺活化调节趋化因子(TARC)的水平,RT-PCR检测HaCaT细胞芳香烃受体(AhR)、细胞色素P4501A(CYP1A1)、聚丝蛋白、内披蛋白、胸腺基质淋巴细胞生成素(TSLP)和TARC mRNA的表达水平,Western印迹法检测聚丝蛋白、内披蛋白、TSLP和STAT1及磷酸化STAT1(p-STAT1)的蛋白表达水平,免疫荧光检测本维莫德对HaCaT细胞中AhR核转位的影响。计量资料采用非配对Student t检验和单因素方差分析进行比较,Spearman检验分析各指标间的关系。结果0.1、1、10、100、1000μmol/L本维莫德干预HaCaT细胞24 h后,细胞存活率分别为(90.2±2.4)%、(85.4±11.9)%、(52.8±14.0)%、(39.4±7.9)%、(27.5±3.4)%,各组间差异有统计学意义(F=162.5,P<0.001),50%抑制浓度为48.54μmol/L。与刺激剂组相比,10和100μmol/L本维莫德组HaCaT细胞分泌的IL-10水平上升(F=16.110,P<0.001),但100μmol/L组IL-22(F=6.884,P<0.001)和10、100μmol/L组TARC水平(F=7.052,P<0.001)显著下降。与刺激剂组相比,1和10μmol/L本维莫德组CYP1A1 mRNA表达(P=0.004)和10μmol/L本维莫德组FLG mRNA表达(P=0.040)水平显著增高,而10μmol/L组TARC mRNA和10μmol/L组TSLP mRNA表达显著降低(均P<0.01),而刺激剂组和本维莫德组间AhR mRNA的表达差异无统计学意义(P=0.193)。与刺激剂组相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 0.1-1000μmol/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 10μg/L tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ),benvitimod groups treated with benvitimod at final concentrations of 1-10 or 1-100μmol/L followed by the treatment with 10μg/L TNF-αand IFN-γ,aryl hydrocarbon receptor(AhR)antagonist group treated with 10 or 100μmol/L benvitimod and 10 nmol/L StemRegenin1(SR1)followed by the treatment with 10μg/L TNF-αand 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 P4501A(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 1000μmol/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 concentratio

关 键 词：皮炎 特应性 角蛋白细胞 细胞增殖 细胞因子类 本维莫德 芳香烃受体 

分 类 号：R758.2[医药卫生—皮肤病学与性病学]