Sinomenine attenuates bleomycin-induced pulmonary fibrosis, inflammation, and oxidative stress by inhibiting TLR4/NLRP3/TGFβ signaling

Abstract

Objective

The present work concentrated on validating whether sinomenine alleviates bleomycin (BLM)-induced pulmonary fibrosis, inflammation, and oxidative stress.

Methods

A rat model of pulmonary fibrosis was constructed through intratracheal injection with 5 mg/kg BLM, and the effects of 30 mg/kg sinomenine on pulmonary inflammation, fibrosis, apoptosis, and 4-hydroxynonenal density were evaluated by hematoxylin and eosin staining, Masson’s trichrome staining, TUNEL staining, and immunohistochemistry. Hydroxyproline content and concentrations of inflammatory cytokines and oxidative stress markers were detected using corresponding kits. MRC-5 cells were treated with 10 ng/ml PDGF, and the effects of 1 mM sinomenine on cell proliferation were assessed by EdU assays. The mRNA expression of inflammatory cytokines and the protein levels of collagens, fibrosis markers, and key markers involved in the TLR4/NLRP3/TGFβ signaling were tested with RT-qPCR and immunoblotting analysis.

Results

Sinomenine attenuated pulmonary fibrosis and inflammation while reducing hydroxyproline content and the protein expression of collagens and fibrosis markers in BLM-induced pulmonary fibrosis rats. Sinomenine reduced apoptosis in lung samples of BLM-challenged rats by increasing Bcl-2 and reducing Bax and cleaved caspase-3 protein expression. In addition, sinomenine alleviated inflammatory response and oxidative stress in rats with pulmonary fibrosis induced by BLM. Moreover, sinomenine inhibited the TLR4/NLRP3/TGFβ signaling pathway in lung tissues of BLM-stimulated rats. Furthermore, TLR4 inhibitor, TAK-242, attenuated PDGF-induced fibroblast proliferation and collagen synthesis in MRC-5 cells.

Conclusion

Sinomenine attenuates BLM-caused pulmonary fibrosis, inflammation, and oxidative stress by inhibiting the TLR4/NLRP3/TGFβ signaling, indicating that sinomenine might become a therapeutic candidate to treat pulmonary fibrosis.

Keywords:

• Pulmonary fibrosis
• sinomenine
• bleomycin
• inflammation
• oxidative stress

Acknowledgement

None.

Ethical approval

The procedures involving experimental animals were authorized by the Medical Ethics Committee of The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology.

Disclosure statement

None.

Authors’ contributions

Yijue Liu, Hong Chen, and Yan Wu were the main designers of this study. Yijue Liu, Hong Chen, Yan Wu, Fen Ai, Wei Li, Bo Yu, and Zhen Chen performed the experiments and analyzed the data. Yijue Liu, Hong Chen, Yan Wu, Bo Yu, and Zhen Chen drafted the manuscript. All authors read and approved the final manuscript.

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

1. Natural Science Fund of Hubei Science and Technology Department, General Project (2022CFC002). 2. Medical Research Fund of Wuhan Municipal Health Commission, Youth Project (WX20Q21).