Li Y S, Zhang X R, Yu M J, Hu X H, Yang J C, Huang Y, Luo G X, He W F
Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing Key Laboratory for Disease Proteomics, Chongqing 400038, China.
Zhonghua Shao Shang Za Zhi. 2020 Oct 20;36(10):923-929. doi: 10.3760/cma.j.cn501120-20200619-00315.
To investigate the mechanisms of interleukin-17A (IL-17A) regulating the expressions of IL-1β and IL-23 in mouse keratinocytes (KCs). 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 10 μL PBS or 10 μL 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 factor κB (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 10 μL, 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 5 μmol/L, 100 μmol/L, 4 nmol/L, 1 nmol/L, and 10 μmol/L, respectively. The expression levels of IL-1β mRNA 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 test, one-way analysis of variance, test, and Bonferroni correction. (1) After culture of 6 hours, compared with those in PBS control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A stimulation group were significantly increased (=13.46, 6.72, <0.01). (2) After culture of 6 hours, the expression levels of IL-1β and 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-1β and IL-23 mRNA in cells in IL-17A+ DMSO group were significantly increased (=9.24, 12.60, <0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-1β mRNA was significantly decreased in cells in IL-17A+ NF-κB inhibitor group and IL-17A+ STAT3 inhibitor group (=11.34, 6.91, <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 (=12.44, 13.03, 15.21, <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. IL-17A promotes the transcription of IL-1β in mouse KCs through the phosphorylation of NF-κB and STAT3 pathways and IL-23 through the phosphorylation of ERK and JNK pathways.
探讨白细胞介素-17A（IL-17A）调控小鼠角质形成细胞（KC）中IL-1β和IL-23表达的机制。从400只新生野生型C57BL/6雌雄小鼠的皮肤中分离原代KC，接种于含10%体积分数胎牛血清的Roswell Park Memorial Institute 1640培养基的24孔板中，进行以下实验。（1）按随机数字表将细胞分为磷酸盐缓冲液（PBS）对照组和IL-17A刺激组（以下分组方法相同），分别用10 μL PBS或质量浓度为100 ng/mL的10 μL IL-17A培养6小时。采用实时荧光定量逆转录聚合酶链反应（RT-PCR）检测细胞中IL-1β和IL-23 mRNA的表达水平，每组3个样本。（2）将细胞分为二甲基亚砜（DMSO）对照组、IL-17A+DMSO组、IL-17A+核因子κB（NF-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+细胞外信号调节激酶1（ERK1）抑制剂组、IL-17A+ERK2抑制剂组和IL-17A+c-Jun氨基末端激酶（JNK）抑制剂组。分别向相应组细胞中加入试剂培养6小时。各试剂体积均为10 μL，IL-17A质量浓度为100 ng/mL，NF-κB、STAT3、ERK1及ERK2、JNK信号通路抑制剂PDTC、S3I-201、SCH772984、SCH772984、SP600125的摩尔浓度分别为5 μmol/L、100 μmol/L、4 nmol/L、1 nmol/L、10 μmol/L。采用实时荧光定量RT-PCR检测细胞中IL-1β mRNA和IL-23 mRNA的表达水平，每组3个样本。（3）细胞分组及处理同实验（1）。采用蛋白质免疫印迹法检测NF-κB磷酸化、STAT3磷酸化、ERK磷酸化及JNK磷酸化水平，每组3个样本。数据采用双侧Student检验、单因素方差分析、检验及Bonferroni校正进行统计学分析。（1）培养6小时后，与PBS对照组相比，IL-17A刺激组细胞中IL-1β和IL-23 mRNA的表达水平显著升高（=13.46，6.72，<0.01）。（2）培养6小时后，DMSO对照组、IL-17A+DMSO组、IL-17A+NF-κB抑制剂组、IL-17A+STAT3抑制剂组、IL-17A+ERK1抑制剂组、IL-17A+ERK2抑制剂组和IL-17A+JNK抑制剂组细胞中IL-1β mRNA和IL-23 mRNA的表达水平分别为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对照组相比，IL-17A+DMSO组细胞中IL-1β和IL-23 mRNA的表达水平显著升高（=9.24，12.60，<0.01）。与IL-17A+DMSO组相比，IL-17A+NF-κB抑制剂组和IL-17A+STAT3抑制剂组细胞中IL-1β mRNA的表达水平显著降低（=11.34，6.91，<0.01）。与IL-17A+DMSO组相比，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的表达水平显著降低（=12.44，13.03，15.21，<0.01）。（3）培养6小时后，与PBS对照组相比，IL-17A刺激组细胞中NF-κB磷酸化、STAT3磷酸化、ERK磷酸化及JNK磷酸化水平显著升高。IL-17A通过NF-κB和STAT3途径的磷酸化促进小鼠KC中IL-1β的转录，并通过ERK和JNK途径促进IL-23的转录。
