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Bruceine A inhibits TGF-β1/Smad pathway in pulmonary fibrosis by blocking gal3/TGF-β1 interaction
Chao Du1, Chong Ma1, Ruoyu Geng1
1Department of Pharmacognosy, School of Pharmacy, Xinjiang Medical University, Urumqi, China; Xinjiang Key Laboratory of Natural Medicines Active Components and Drug Release Technology, China.
Background:
Bruceine A(BA) has many pharmacological activities and significantly inhibits fibrosis in keloid fibroblasts. However, the underlying mechanisms have not yet been fully elucidated.
Objective:
This study aimed to investigate the effects of BA on pulmonary fibrosis(PF) and explore its underlying mechanisms.
Methods:
PF models were constructed by BLM-induced C57BL/6 J mice, TGF-β1- induced MRC-5 and HFL-1 cells. Cell proliferation, MMP, apoptosis, and ROS levels were analyzed in vitro. In vivo, experiments were performed to evaluate the therapeutic effect of BA on PF by detecting respiratory function, histopathology, and collagen level. Fibro-associated, ECM, and EMT key proteins were used to assess the degree of PF. To predict the target of BA by molecular docking technology, and verified by DARTS, CETSA, MST,and SPR. Then overexpression gal3-lentivirus, GB1107 gal3 inhibitor, and BA addition were used to verify the TGF-β1/Smad pathway key protein by western blot.
Results:
We found that BA inhibited PF both in vitro and in vivo. The predicted and validated results showed that gal3 was the target of BA, and the binding site was Arg144, His158, and Trp181. Mechanistically, BA disrupts the interaction between gal3 and TGF-β1. BA reduced Smad2/3 and p-Smad2/3 protein content and inhibited TGF-β1/Smad pathway in the overexpressing gal3 HFL-1 cells. After adding GB1107, the inhibitory effect of BA on TGF-β1/Smad pathway disappeared.
Conclusion:
This study is the first to demonstrate that BA can target gal3, interfere with the interaction between gal3 and TGF-β1 protein, inhibit the downstream TGF-β1/Smad pathway, and act as a "brake" to reverse the PF process. These findings provide a solid scientific basis for the clinical application of BA in the prevention and treatment of PF.
