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

doi:10.19723/j.issn.1671-167X.2024.03.018

摘要/Abstract

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(transforming 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1.0 μmol/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(signal 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)。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-β1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。

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

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 induction 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 significantly up-regulated compared with the blank control group (P < 0.05). Compared with the TGF-β1 induction 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 significant. There was no difference in COL1A1 protein expression between the intervention groups compared with the TGF-β1-induced group. (4) After TGF-β1 acted on HFL-1 cells for 48 h, the gene expression 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 myofibroblasts induced by TGF-β1, and the mechanism may be through inhibiting the classic Smad2/3 pathway as well as the phosphorylation of STAT3 induced by TGF-β1, thereby protecting the disease progression of pulmonary fibrosis.

Key words: Tofacitinib, Pulmonary fibrosis, Fibroblasts, Myofibroblasts, STAT3 transcription factor

中图分类号:

R563.13

何珊,陈炘,程琦,朱灵江,张培玉,童淑婷,薛静,杜燕. 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[J]. 北京大学学报（医学版）, 2024, 56(3): 505-511.

Shan HE,Xin CHEN,Qi CHENG,Lingjiang ZHU,Peiyu ZHANG,Shuting TONG,Jing XUE,Yan DU. Tofacitinib inhibits the transformation of lung fibroblasts into myofibroblasts through JAK/STAT3 pathway[J]. Journal of Peking University (Health Sciences), 2024, 56(3): 505-511.

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