5,7,3′,4′-Tetramethoxyflavone suppresses TGF-β1-induced activation of murine fibroblasts in vitro and ameliorates bleomycin-induced pulmonary fibrosis in mice

Abstract

Objective

This study aimed to investigate the use of 5,7,3′,4′-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. In vitro and in vivo models were used to examine the impact of TMF on PF.

Methods

NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factor‑β1 (TGF-β1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT–PCR), respectively. Downstream molecules affected by TGF-β1 were examined by western blotting. In vivo, mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques.

Results

The in vitro results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-β1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/β-catenin. The in vivo experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed.

Conclusion

These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-β1/Smad and non-Smad pathways.

Keywords:

• Idiopathic pulmonary fibrosis
• 5,7,3′,4′-tetramethoxyflavone
• TGF-β1
• bleomycin
• smad

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was financially supported in part by the Advanced Plant and Food Crop Biotechnology Center from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.