托法替布通过JAK/STAT3通路抑制肺成纤维细胞向肌成纤维细胞转化
Tofacitinib inhibits the transformation of lung fibroblasts into myofibroblasts through JAK/STAT3 pathway
何珊 1陈炘 1程琦 2朱灵江 2张培玉 2童淑婷 2薛静 2杜燕2
作者信息
- 1. 浙江大学医学院附属第二医院风湿免疫科,杭州 310009;浙江大学医学院附属金华医院风湿免疫科,浙江金华 321000
- 2. 浙江大学医学院附属第二医院风湿免疫科,杭州 310009
- 折叠
摘要
目的:研究泛Janus激酶(Janus kinase,JAK)抑制剂托法替布(tofacitinib)对转化生长因子-β1(transfor-ming growth factor-beta 1,TGF-β1)诱导的肺成纤维细胞向肌成纤维细胞转化的作用及机制,为临床治疗结缔组织病相关的间质性肺疾病提供理论依据.方法:(1)体外培养人胚胎肺成纤维细胞(human fetal lung fibroblast 1,HFL-1),设立6个组,分别为DMSO空白对照组、TGF-β1诱导组、TGF-β1联合不同浓度托法替布(0.5、1.0、2.0、5.0 μmol/L)药物干预实验组.采用CCK-8法检测细胞活力,划痕愈合实验检测细胞迁移能力.(2)采用实时荧光定量 PCR(quantitative real-time PCR,RT-PCR)、蛋白免疫印迹实验(Western blotting)检测 α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)、Ⅰ 型胶原蛋白(collagen Ⅰ,COL1)基因及蛋白的表达水平.应用RT-PCR和酶联免疫吸附试验检测各组白细胞介素-6(interleukin-6,IL-6)的基因和细胞培养上清液中的蛋白水平.(3)在不同组的细胞培养基中加入DMSO载体对照,以1.0μmol/L和5.0 μmol/L的托法替布预培养30 min,然后加入TGF-β1处理1 h、6 h和24 h.以Western blotting检测Smad2/3、信号传导和转录激活因子3(sig-nal transducer and activator of transcription 3,STAT3)的蛋白磷酸化水平.结果:(1)托法替布抑制TGF-β1诱导的HFL-1细胞活力及迁移能力.(2)与空白对照组相比,TGF-β1诱导组HFL-1的α-SMA、COL1A1和FN1基因表达显著上调(P＜0.05).5.0 μmol/L托法替布干预组的α-SMA基因表达与TGF-β1诱导组相比显著下调(P＜0.05)o 0.5～5.0 μmol/L托法替布各干预组与TGF-β1诱导组相比均可抑制FN1基因表达(P＜0.05).各干预组与TGF-β1诱导组相比,COL1A1基因的表达无明显变化.(3)Western blotting结果提示,TGF-(31诱导组细胞α-SMA、FN1蛋白水平较对照组显著增加(P＜0.05),COL1A1表达未见明显差异.托法替布不同浓度干预组与TGF-β1诱导组相比,α-SMA蛋白表达均有下降,其中2.0 μmol/L和5.0 μmol/L干预组与诱导组间的差异有统计学意义(P＜0.05).托法替布不同浓度干预组的FN1蛋白水平均有下降,但与TGF-β1诱导组相比差异无统计学意义.各干预组的COL1A1蛋白表达与TGF-β1诱导组相比差异无统计学意义.(4)TGF-β1诱导48 h,HFL-1细胞中IL-6基因及培养上清液中IL-6的表达水平较对照组显著升高,各浓度托法替布干预组与TGF-β1诱导组相比均有所降低.TGF-β1诱导1 h、6 h和24 h,STAT3蛋白磷酸化水平增加,托法替布预刺激抑制了 6 h时Smad2/3的磷酸化水平,在1 h、6 h和24 h时均抑制了 STAT3的磷酸化水平.结论:托法替布能抑制TGF-β1诱导的HFL-1细胞向肌成纤维细胞方向转化,其机制可能是通过抑制Smad2/3经典通路以及抑制TGF-β1所诱导的STAT3磷酸化,从而对肺纤维化疾病进展发挥保护作用.
Abstract
Objective:To investigate the effect of tofacitinib,a pan-Janus kinase(JAK)inhibitor,on transforming growth factor-beta 1(TGF-β1)-induced fibroblast to myofibroblast transition(FMT)and to explore its mechanism.To provide a theoretical basis for the clinical treatment of connective tissue disease-related interstitial lung disease(CTD-ILD).Methods:(1)Human fetal lung fibroblast 1(HFL-1)were cultured in vitro,and 6 groups were established:DMSO blank control group,TGF-β1 in-duction group,and TGF-β1 with different concentrations of tofacitinib(0.5,1.0,2.0,5.0 μmol/L)drug intervention experimental groups.CCK-8 was used to measure the cell viability,and wound-healing assay was performed to measure cell migration ability.After 48 h of combined treatment,quantitative real-time PCR(RT-PCR)and Western blotting were used to detect the gene and protein expression levels of α-smooth muscle actin(α-SMA),fibronectin(FN),and collagen type Ⅰ(COL1).(2)RT-PCR and enzyme-linked immunosorbnent assay(ELISA)were used to detect the interleukin-6(IL-6)gene and protein expression changes,respectively.(3)DMSO carrier controls,1.0 μmol/L and 5.0 μmol/L tofacitinib were added to the cell culture media of different groups for pre-incubation for 30 min,and then TGF-β1 was added to treat for 1 h,6 h and 24 h.The phosphorylation levels of Smad2/3 and signal transducer and activator of transcription 3(STAT3)protein were detected by Western blotting.Results:(1)Tofacitinib inhibited the viability and migration ability of HFL-1 cells after TGF-β1 induction.(2)The expression of α-SMA,COL1A1 and FN1 genes of HFL-1 in the TGF-β1-induced groups was signifi-cantly up-regulated compared with the blank control group(P＜0.05).Compared with the TGF-β1 in-duction group,α-SMA expression in the 5.0 μmol/L tofacitinib intervention group was significantly inhi-bited(P＜0.05).Compared with the TGF-β1-induced group,FN1 gene was significantly inhibited in each intervention group at a concentration of 0.5-5.0 μmol/L(P＜0.05).Compared with the TGF-β1-induced group,the COL1A1 gene expression in each intervention group did not change significantly.(3)Western blotting results showed that the protein levels of α-SMA and FN1 in the TGF-β1-induced group were significantly higher than those in the control group(P＜0.05),and there was no significant difference in the expression of COL1A1.Compared with the TGF-β1-induced group,the α-SMA protein level in the intervention groups with different concentrations decreased.And the differences between the TGF-β1-induced group and 2.0 μmol/L or 5.0 μmol/L intervention groups were statistically significant(P＜0.05).Compared with the TGF-β1-induced group,the FN1 protein levels in the intervention groups with different concentrations showed a downward trend,but the difference was not statistically sig-nificant.There was no difference in COL1A1 protein expression between the intervention groups com-pared with the TGF-β1-induced group.(4)After TGF-β1 acted on HFL-1 cells for 48 h,the gene ex-pression of the IL-6 was up-regulated and IL-6 in culture supernatant was increased,the intervention with tofacitinib partly inhibited the TGF-β1-induced IL-6 gene expression and IL-6 in culture supernatant.TGF-β1 induced the increase of Smad2/3 protein phosphorylation in HFL-1 cells for 1 h and 6 h,STAT3 protein phosphorylation increased at 1 h,6 h and 24 h,the pre-intervention with tofacitinib inhibited the TGF-β1-induced Smad2/3 phosphorylation at 6 h and inhibited TGF-β1-induced STAT3 phosphorylation at 1 h,6 h and 24 h.Conclusion:Tofacitinib can inhibit the transformation of HFL-1 cells into myofi-broblasts induced by TGF-β1,and the mechanism may be through inhibiting the classic Smad2/3 path-way as well as the phosphorylation of STAT3 induced by TGF-β1,thereby protecting the disease progres-sion of pulmonary fibrosis.
关键词
托法替布/肺纤维化/成纤维细胞/肌成纤维细胞/STAT3转录因子Key words
Tofacitinib/Pulmonary fibrosis/Fibroblasts/Myofibroblasts/STAT3 transcription factor引用本文复制引用
基金项目
国家自然科学基金(82271817)
浙江省自然科学基金(LY22H100004)
出版年
2024
国家科技期刊平台
NSTL
万方数据