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Journal of Peking University (Health Sciences) ›› 2024, Vol. 56 ›› Issue (3): 505-511. doi: 10.19723/j.issn.1671-167X.2024.03.018

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Tofacitinib inhibits the transformation of lung fibroblasts into myofibroblasts through JAK/STAT3 pathway

Shan HE1,2,Xin CHEN1,2,Qi CHENG1,Lingjiang ZHU1,Peiyu ZHANG1,Shuting TONG1,Jing XUE1,Yan DU1,*()   

  1. 1. Department of Rheumatology, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China
    2. Department of Rheumatology, the Affiliated Jinhua Hospital of Zhejiang University School of Medicine, Jinhua, 321000, Zhejiang, China
  • Received:2022-08-31 Online:2024-06-18 Published:2024-06-12
  • Contact: Yan DU E-mail:duyan2014@zju.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(82271817);the Natural Science Foundation of Zhejiang Province(LY22H100004)

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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. 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

CLC Number: 

  • R563.13

Figure 1

Inhibitory effect of tofacitinib on HFL-1 cells' viability and migration ability * P<0.05, ** P<0.01, *** P<0.001, **** P<0.000 1. A, cell viability in cultured HFL-1 cells maintained in normal growth medium treated with vehicle (DMSO) and treated with tofacitinib at a concentration of 0.5-5.0 μmol/L in combination with stimulation with TGF-β1 for 24 h, 48 h, or 72 h; B and C, migration ability of HFL-1 cells maintained in normal growth medium treated with vehicle (DMSO) and treated with tofacitinib at a concentration of 0.5-5.0 μmol/L in combination with stimulation with TGF-β1 for 10 h or 24 h. TGF-β1, transforming growth factor-beta 1; HFL-1, human fetal lung fibroblast 1."

Figure 2

Effects of tofacitinib on the expression of α-SMA, FN1 and COL1A1 in HFL-1 cells induced by TGF-β1 * P<0.05, ** P<0.01. A, quantitative real-time PCR of α-SMA, FN1 and COL1A1 in cultured HFL-1 cells maintained in normal growth medium treated with vehicle (DMSO) and treated with tofacitinib at a concentration of 0.5-5.0 μmol/L in combination with stimulation with TGF-β1 for 48 h; the final results were obtained from 4 independent in vitro experiments (mean±SE). B and C, Western blotting and related densitometric analysis of α-SMA, FN1 and COL1A1 in cultured HFL-1 cells maintained in normal growth medium treated with vehicle (DMSO) and treated with tofacitinib at a concentration of 0.5-5.0 μmol/L in combination with stimulation with TGF-β1 for 48 h; the final results were obtained from 4-6 independent in vitro experiments (mean±SEM). α-SMA, α-smooth muscle actin; FN1, fibronectin 1; COL1A1, type Ⅰ collagen a1; TGF-β1, transforming growth factor-beta 1; HFL-1, human fetal lung fibroblast 1; RQ, relative quantity; SE, standard error; SEM, standard error of the mean."

Figure 3

Effects of tofacitinib on related pathways of HFL-1 cells in FMT * P<0.05, ** P<0.01, **** P<0.000 1. A, relative protein abundance of p-Smad2/3 and p-STAT3; B and C, relative RNA transcription level of IL-6 in HFL-1 cell (B) and sIL-6 protein level in the cell culture supernatant (C). The final results were obtained from 3-4 independent in vitro experiments (mean±SEM). Smad2/3, small mothers against decapentaplegic 2 and 3; p-Smad2/3, phosphorylation-Smad2/3; STAT3, signal transducer and activator of transcription 3; p-STAT3, phosphorylation-STAT3; TGF-β1, transforming growth factor-beta 1; HFL-1, human fetal lung fibroblast 1; IL-6, interleukin 6; sIL-6, soluble interleukin 6; FMT, fibroblast to myofibroblast transition; RQ, relative quantity; SEM, standard error of the mean."

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