白细胞介素17A调控小鼠角质形成细胞表达白细胞介素1β和白细胞介素23机制研究

Study on mechanisms of interleukin-17A regulating the expressions of interleukin-1β and interleukin-23 in mouse keratinocytes

目的探讨白细胞介素17A(IL-17A)调控小鼠角质形成细胞(KC)表达IL-1β和IL-23的机制。方法从400只新生雌雄不限C57BL/6野生型小鼠皮肤中分离原代KC,用含体积分数10%胎牛血清的RPMI 1640培养基培养于24孔板中,用于以下实验。(1)取细胞,采用随机数字表法(分组方法下同)分为磷酸盐缓冲液(PBS)对照组、IL-17A刺激组,分别加入10μL的PBS、质量浓度为100 ng/mL的IL-17A培养6 h,采用实时荧光定量反转录PCR法检测细胞中IL-1β和IL-23 mRNA表达水平,每组3个样本。(2)取细胞,分为二甲基亚砜(DMSO)对照组、IL-17A+DMSO组、IL-17A+核因子κB抑制剂组、IL-17A+信号转导及转录激活因子3(STAT3)抑制剂组、IL-17A+胞外信号调节激酶1(ERK1)抑制剂组、IL-17A+ERK2抑制剂组、IL-17A+c-Jun氨基端激酶(JNK)抑制剂组,分别加入相应试剂,各试剂体积均为10μL,IL-17A质量浓度为100 ng/mL,核因子κB、STAT3、ERK1、ERK2、JNK信号通路抑制剂PDTC、S3I-201、SCH772984、SCH772984、SP600125物质的量浓度分别为5μmol/L、100μmol/L、4 nmol/L、1 nmol/L、10μmol/L,均培养6 h。采用实时荧光定量反转录PCR法检测细胞中IL-1β和IL-23 mRNA表达水平,每组3个样本。(3)取细胞,同实验(1)分组处理,采用蛋白质印迹法检测细胞中核因子κB磷酸化、STAT3磷酸化、ERK磷酸化、JNK磷酸化水平,每组3个样本。对数据行双尾Student t检验、单因素方差分析、t检验和Bonferroni校正。结果(1)培养6 h,与PBS对照组比较,IL-17A刺激组细胞中IL-1β和IL-23 mRNA表达水平均明显升高(t=13.46、6.72,P<0.01)。(2)培养6 h,DMSO对照组、IL-17A+DMSO组、IL-17A+核因子κB抑制剂组、IL-17A+STAT3抑制剂组、IL-17A+ERK1抑制剂组、IL-17A+ERK2抑制剂组、IL-17A+JNK抑制剂组细胞中IL-1β与IL-23 mRNA表达水平分别为1.00±0.11、4.01±0.32、0.32±0.06、1.76±0.43、3.62±0.24、3.80±0.43、4.26±0.74和1.03±0.29、4.08±0.34、4.76±0.38、4.70±0.21、1.06±0.42、0.92±0.21、0.39±0.05。与DMSO对照组比较,IL-17A+DMSO组细胞中IL-1β和IL-23 mRNA表达水平均明显升高(t=9.24、12.60,P<0.01)。与IL-17A+DMSO组比较,IL-17A+核因子κB抑制剂组与IL-17A+STAT3抑制剂组细胞中IL-1βmRNA表达水平均明显下降(t=11.34、6.91,P<0.01),IL-17A+ERK1抑制剂组、IL-17A+ERK2抑制剂组和IL-17A+JNK抑制剂组细胞中IL-23 mRNA表达水平均明显下降(t=12.44、13.03、15.21,P<0.01)。(3)培养6 h,与PBS对照组比较,IL-17A刺激组细胞中核因子κB磷酸化、STAT3磷酸化、ERK磷酸化、JNK磷酸化水平均明显升高。结论IL-17A分别通过促进核因子κB、STAT3信号通路磷酸化与ERK、JNK信号通路磷酸化促进小鼠KC转录表达IL-1β与IL-23。

Objective To investigate the mechanisms of interleukin-17A(IL-17A)regulating the expressions of IL-1βand IL-23 in mouse keratinocytes(KCs).Methods Primary KCs were isolated from the skin of 400 newborn male and female wild type C57BL/6 mice and cultured in 24-well plates with Roswell Park Memorial Institute 1640 medium containing fetal bovine serum in the volume fraction of 10%for the following experiments.(1)The cells were divided into phosphate buffer solution(PBS)control group and IL-17A stimulation group according to the random number table(the same grouping method below),which were cultured with 10μL PBS or 10μL IL-17A in the mass concentration of 100 ng/mL for 6 hours,respectively.The expression levels of IL-1β and IL-23 mRNA in cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR),with 3 samples in each group.(2)The cells were divided into dimethyl sulfoxide(DMSO)control group,IL-17A+DMSO group,IL-17A+nuclear factorκB(NF-κB)inhibitor group,IL-17A+signal transduction and activator of transcription 3(STAT3)inhibitor group,IL-17A+extracellular signal-regulated kinase 1(ERK1)inhibitor group,IL-17A+ERK2 inhibitor group,and IL-17A+c-Jun N-terminal kinase(JNK)inhibitor group.The reagents were added to cells in corresponding groups respectively and cultured for 6 hours.The volume of each reagent was 10μL,the mass concentration of IL-17A was 100 ng/mL,and the molarity concentrations of NF-κB,STAT3,ERK1,ERK2,JNK signal pathway inhibitors PDTC,S3I-201,SCH772984,SCH772984,SP600125 were 5μmol/L,100μmol/L,4 nmol/L,1 nmol/L,and 10μmol/L,respectively.The expression levels of IL-1βmRNA and IL-23 mRNA in cells were detected by real-time fluorescence quantitative RT-PCR,with 3 samples in each group.(3)The cells were grouped and treated the same as those in experiment(1).The levels of NF-κB phosphorylation,STAT3 phosphorylation,ERK phosphorylation,and JNK phosphorylation were detected by Western blotting,with 3 samples in each group.Data were statistically analyzed with two-tailed Student t test,one-way analysis of variance,t test,and Bonferroni correction.Results(1)After culture of 6 hours,compared with those in PBS control group,the expression levels of IL-1βand IL-23 mRNA in cells in IL-17A stimulation group were significantly increased(t=13.46,6.72,P<0.01).(2)After culture of 6 hours,the expression levels of IL-1βand IL-23 mRNA in cells in DMSO control group,IL-17A+DMSO group,IL-17A+NF-κB inhibitor group,IL-17A+STAT3 inhibitor group,IL-17A+ERK1 inhibitor group,IL-17A+ERK2 inhibitor group,and IL-17A+JNK inhibitor group were 1.00±0.11,4.01±0.32,0.32±0.06,1.76±0.43,3.62±0.24,3.80±0.43,4.26±0.74 and 1.03±0.29,4.08±0.34,4.76±0.38,4.70±0.21,1.06±0.42,0.92±0.21,0.39±0.05,respectively.Compared with those in DMSO control group,the expression levels of IL-1βand IL-23 mRNA in cells in IL-17A+DMSO group were significantly increased(t=9.24,12.60,P<0.01).Compared with that in IL-17A+DMSO group,the expression level of IL-1βmRNA was significantly decreased in cells in IL-17A+NF-κB inhibitor group and IL-17A+STAT3 inhibitor group(t=11.34,6.91,P<0.01).Compared with that in IL-17A+DMSO group,the expression level of IL-23 mRNA was significantly decreased in cells in IL-17A+ERK1 inhibitor group,IL-17A+ERK2 inhibitor group,and IL-17A+JNK inhibitor group(t=12.44,13.03,15.21,P<0.01).(3)After culture of 6 hours,compared with those in PBS control group,the levels of NF-κB phosphorylation,STAT3 phosphorylation,ERK phosphorylation,and JNK phosphorylation in cells in IL-17A stimulation group were significantly increased.Conclusions IL-17A promotes the transcription of IL-1βin mouse KCs through the phosphorylation of NF-κB and STAT3 pathways and IL-23 through the phosphorylation of ERK and JNK pathways.