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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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
目的：为了研究托法替尼的作用，泛Janus激酶（JAK）抑制剂，转化生长因子-β1(TGF-β1)诱导成纤维细胞向肌成纤维细胞转变(FMT)并探讨其机制。为结缔组织病相关间质性肺病(CTD-ILD)的临床治疗提供理论依据。
方法：(1)体外培养人胎肺成纤维细胞1(HFL-1),建立6组:DMSO空白对照组,TGF-β1诱导组,和TGF-β1用不同浓度的托法替尼（0.5、1.0、2.0、5.0μmol/L）药物干预实验组。CCK-8用于测量细胞活力，并进行伤口愈合试验以测量细胞迁移能力。联合治疗48小时后，采用实时定量PCR(RT-PCR)和免疫印迹法检测α-平滑肌肌动蛋白(α-SMA)基因和蛋白表达水平,纤连蛋白（FN），和Ⅰ型胶原(COL1)。(2)采用RT-PCR和酶联免疫吸附法(ELISA)检测白细胞介素-6(IL-6)基因和蛋白表达变化,分别。（3）DMSO载体对照，将1.0μmol/L和5.0μmol/L托法替尼加入不同组的细胞培养基中，预孵育30分钟。然后加入TGF-β1治疗1小时，6h和24h。通过蛋白质印迹法检测Smad2/3和信号转导和转录激活因子3（STAT3）蛋白的磷酸化水平。
结果：(1)Tofacitinib抑制TGF-β1诱导后HFL-1细胞的活力和迁移能力。(2)α-SMA的表达,与空白对照组相比,TGF-β1诱导组HFL-1的COL1A1和FN1基因表达明显上调(P<0.05)。与TGF-β1诱导组相比,5.0μmol/L托法替尼干预组α-SMA表达显著降低(P<0.05)。与TGF-β1诱导组相比,各干预组FN1基因在0.5~5.0μmol/L浓度下被显著抑制(P<0.05)。与TGF-β1诱导组相比,各干预组COL1A1基因表达无明显变化。(3)免疫印迹结果显示,TGF-β1诱导组α-SMA和FN1蛋白水平明显高于对照组(P<0.05),而COL1A1的表达无明显差别。与TGF-β1诱导组相比,不同浓度干预组的α-SMA蛋白水平下降。TGF-β1诱导组与2.0μmol/L、5.0μmol/L干预组比较差异均有统计学意义(P<0.05)。与TGF-β1诱导组相比,不同浓度干预组的FN1蛋白水平呈下降趋势,但差异无统计学意义。与TGF-β1诱导组相比，干预组之间的COL1A1蛋白表达无差异。(4)TGF-β1作用于HFL-1细胞48h后,IL-6的基因表达上调，培养上清液中IL-6增加，托法替尼干预部分抑制了TGF-β1诱导的IL-6基因表达和培养上清液中的IL-6.TGF-β1诱导HFL-1细胞Smad2/3蛋白磷酸化增加1h和6h,STAT3蛋白磷酸化在1h时增加，6h和24h，托法替尼预干预在6h时抑制TGF-β1诱导的Smad2/3磷酸化，并在1h时抑制TGF-β1诱导的STAT3磷酸化，
结论：托法替尼能抑制TGF-β1诱导的HFL-1细胞向肌成纤维细胞的转化，其机制可能是通过抑制经典的Smad2/3通路以及TGF-β1诱导的STAT3磷酸化，从而保护肺纤维化的进展。
方法：(1)体外培养人胎肺成纤维细胞1(HFL-1),建立6组:DMSO空白对照组,TGF-β1诱导组,和TGF-β1用不同浓度的托法替尼（0.5、1.0、2.0、5.0μmol/L）药物干预实验组。CCK-8用于测量细胞活力，并进行伤口愈合试验以测量细胞迁移能力。联合治疗48小时后，采用实时定量PCR(RT-PCR)和免疫印迹法检测α-平滑肌肌动蛋白(α-SMA)基因和蛋白表达水平,纤连蛋白（FN），和Ⅰ型胶原(COL1)。(2)采用RT-PCR和酶联免疫吸附法(ELISA)检测白细胞介素-6(IL-6)基因和蛋白表达变化,分别。（3）DMSO载体对照，将1.0μmol/L和5.0μmol/L托法替尼加入不同组的细胞培养基中，预孵育30分钟。然后加入TGF-β1治疗1小时，6h和24h。通过蛋白质印迹法检测Smad2/3和信号转导和转录激活因子3（STAT3）蛋白的磷酸化水平。
结果：(1)Tofacitinib抑制TGF-β1诱导后HFL-1细胞的活力和迁移能力。(2)α-SMA的表达,与空白对照组相比,TGF-β1诱导组HFL-1的COL1A1和FN1基因表达明显上调(P<0.05)。与TGF-β1诱导组相比,5.0μmol/L托法替尼干预组α-SMA表达显著降低(P<0.05)。与TGF-β1诱导组相比,各干预组FN1基因在0.5~5.0μmol/L浓度下被显著抑制(P<0.05)。与TGF-β1诱导组相比,各干预组COL1A1基因表达无明显变化。(3)免疫印迹结果显示,TGF-β1诱导组α-SMA和FN1蛋白水平明显高于对照组(P<0.05),而COL1A1的表达无明显差别。与TGF-β1诱导组相比,不同浓度干预组的α-SMA蛋白水平下降。TGF-β1诱导组与2.0μmol/L、5.0μmol/L干预组比较差异均有统计学意义(P<0.05)。与TGF-β1诱导组相比,不同浓度干预组的FN1蛋白水平呈下降趋势,但差异无统计学意义。与TGF-β1诱导组相比，干预组之间的COL1A1蛋白表达无差异。(4)TGF-β1作用于HFL-1细胞48h后,IL-6的基因表达上调，培养上清液中IL-6增加，托法替尼干预部分抑制了TGF-β1诱导的IL-6基因表达和培养上清液中的IL-6.TGF-β1诱导HFL-1细胞Smad2/3蛋白磷酸化增加1h和6h,STAT3蛋白磷酸化在1h时增加，6h和24h，托法替尼预干预在6h时抑制TGF-β1诱导的Smad2/3磷酸化，并在1h时抑制TGF-β1诱导的STAT3磷酸化，
结论：托法替尼能抑制TGF-β1诱导的HFL-1细胞向肌成纤维细胞的转化，其机制可能是通过抑制经典的Smad2/3通路以及TGF-β1诱导的STAT3磷酸化，从而保护肺纤维化的进展。
