Venenum bufonis and its active constituents alleviate RSV-induced pneumonia in mice by suppressing macrophage infiltration and NLRP3 inflammasome activation

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and the elderly, with limited treatment options. Venenum bufonis (Vb), a traditional Chinese medicine, exhibits broad pharmacological activities including antiviral and anti-inflammatory effects, but its potential against RSV-induced pneumonia remains unclear. This study evaluates Vb's therapeutic effects and mechanisms in RSV-infected pneumonia and identifies its key active constituents. RSV pneumonia was induced in C57BL/6 mice via intranasal inoculation. Mice were treated with Vb or ribavirin intraperitoneally for five days. Lung pathology, viral gene, inflammatory cytokine gene expression, NLRP3 pathway activation, and immune cell infiltration were assessed using H&E staining, electron microscopy, RT-qPCR, immunohistochemistry, RNA sequencing and flow cytometry. The results demonstrated that Vb treatment significantly reduced lung tissue damage, mRNA levels of RSV-N protein, proinflammatory cytokines (TNF-α, IL-6) and type II interferon (IFN-γ), and enhanced the mRNA levels of type I interferons (IFN-α/β). Furthermore, Vb markedly decreased macrophage infiltration and suppressed mRNA expression of NLRP3, Caspase-1, and IL-1β in lung tissue, suggesting it may alleviate RSV pneumonia by inhibiting macrophage-driven inflammation and NLRP3 activation. Additionally, bufalin, cinobufagin, and resibufogenin were identified as likely bioactive constituents mediating Vb's therapeutic effects. This study provides a scientific basis for the potential application of Vb in the treatment of RSV-induced pneumonia.