Sinomenine attenuates bleomycin-induced pulmonary fibrosis, inflammation, and oxidative stress by inhibiting TLR4/NLRP3/TGFß 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/TGFß 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/TGFß 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/TGFß signaling, indicating that sinomenine might become a therapeutic candidate to treat pulmonary fibrosis.