Tetrandrine ameliorates bleomycin-induced pulmonary fibrosis in systemic sclerosis-associated interstitial lung disease by inhibiting the Ras/Raf/MEK/ERK signaling pathway

Background: Systemic sclerosis (SSc)-associated interstitial lung disease (SSc-ILD) represents a severe complication of SSc for which effective therapeutic options remain limited. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, has demonstrated antifibrotic and anti-inflammatory activities in diverse models of organ fibrosis. However, its therapeutic efficacy and the underlying mechanisms in the context of SSc-ILD remain incompletely understood. Objective: This study was designed to evaluate the protective effects of Tet against bleomycin (BLM)-induced SSc-ILD in a murine model and to elucidate the potential molecular mechanisms involved. Methods: Male ICR mice were employed to establish a mouse model of SSc-ILD via subcutaneous injection of BLM (1 mg/kg/d) over a duration of 4 weeks. The animals were randomly allocated into 6 groups ( n = 8 per group): control, BLM, pirfenidone (100 mg/kg), and Tet at doses of 20, 40, or 80 mg/kg. Tet was administered intragastrically beginning in the second week. Cutaneous and pulmonary fibrosis were assessed by histopathological examination (hematoxylin and eosin staining and Masson trichrome staining), femtosecond label-free imaging, and hydroxyproline (Hyp) content assay. Serum cytokine levels were quantified via enzyme-linked immunosorbent assay. Network pharmacology analysis was performed to predict Tet-associated targets and signaling pathways. In addition, Western blot and immunofluorescence staining were applied to evaluate the rat sarcoma viral oncogene homolog (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade and the expression of transforming growth factor-β (TGF-β). Results: Administration of Tet significantly ameliorated BLM-induced cutaneous fibrosis across all tested dosages, as demonstrated by reductions in dermal thickness, diminished collagen deposition, and decreased Hyp content, with the most marked therapeutic effect observed at the 80 mg/kg dose. In pulmonary tissues, Tet treatment attenuated alveolar wall damage, mitigated inflammatory cell infiltration, and reduced fibrotic lesion formation, accompanied by significant declines in both alveolitis and fibrosis histopathological scores. Quantification via femtosecond label-free imaging and Hyp assay further corroborated the diminished collagen accumulation within lung parenchyma. Concurrently, Tet exerted a pronounced suppressive effect on systemic inflammation, as evidenced by markedly reduced serum levels of interleukin-1β, interleukin-6, TGF-β, and tumor necrosis factor-α. Network pharmacology analysis unveiled 73 intersecting targets common to Tet and SSc-ILD, with significant enrichment identified within the Ras signaling pathway. Subsequent mechanistic investigation demonstrated that Tet substantially downregulated the protein expression levels of Ras, the phosphorylated-to-total Raf ratio, the phosphorylated-to-total MEK ratio, and the phosphorylated-to-total ERK ratio, while concurrently suppressing TGF-β expression in lung tissues. Conclusion: In summary, Tet demonstrates broad-spectrum comprehensive therapeutic activity in the BLM-induced mouse model of SSc-ILD, characterized by the amelioration of both cutaneous and pulmonary fibrosis, the mitigation of systemic inflammatory cascades, and the blockage of the TGF-β-driven Ras/Raf/MEK/ERK signaling cascade. Taken together, these findings highlight the prospective utility of Tet as a viable pharmacological agent for the treatment of SSc-ILD.