Andrographolide Attenuates RSV-induced Inflammation by Suppressing Apoptosis and Promoting Pyroptosis after Respiratory Syncytial Virus Infection In Vitro.

Respiratory syncytial virus (RSV), which is the predominant viral pathogen responsible for causing acute lower respiratory tract infections in children, currently lacks specific therapeutic drugs. Despite andrographolide's demonstrated effectiveness against various viral infections, its effects on RSV infection remain unclear. In this study, RSV infection and andrographolide-intervened A549 cell lines were used. The virus load of RSV and the levels of IL-6 and IL-8 in the cell supernatant were quantified. The potential targets of andrographolide in the treatment of RSV-infected airway epithelial cells were analyzed using the Gene Expression Omnibus (GEO) database and the PharmMapper Database, and the changes in mRNA expression of these target genes were measured. To further illustrate the effect of andrographolide on the death pattern of RSV-infected airway epithelial cells, Annexin V-FITC/PI apoptosis assays and Western blotting were conducted. Andrographolide decreased the viral load and attenuated IL-6 and IL-8 levels in cell supernatant post-RSV infection. A total of 25 potential targets of andrographolide in the treatment of RSV-infected airway epithelial cells were discovered, and CASP1, CCL5, JAK2, and STAT1 were identified as significant players. Andrographolide noticeably suppressed the increased mRNA expressions of these genes post-RSV infection as well as IL-1β. The flow cytometry analysis demonstrated that andrographolide alleviated apoptosis in RSV-infected cells. Additionally, RSV infection decreased the protein levels of caspase-1, cleaved caspase-1, cleaved IL-1β, N-terminal of GSDMD, and Bcl-2. Conversely, andrographolide increased their levels. These results suggest that andrographolide may reduce RSV-induced inflammation by suppressing apoptosis and promoting pyroptosis in epithelial cells, leading to effective viral clearance.

Che S ，Xie X ，Lin J ，Liu Y ，Xie J ，Liu E ... -  《-》

Andrographolide exerts anti-respiratory syncytial virus activity by up-regulating heme oxygenase-1 independent of interferon responses in human airway epithelial cells.

Respiratory syncytial virus (RSV) is the leading cause of mortality and morbidity in children under the age of five. Despite this, there is still a lack of safe and effective vaccines and antiviral agents for clinical use. Andrographolide exerts antiviral functions against a variety of viruses, but whether (and how) it exerts antiviral effects on RSV remains unclear. In vitro RSV infection models using A549 and 16HBE cell lines were established, and the effects of andrographolide on RSV were analyzed via RSV N gene load and proinflammatory cytokine levels. The RNA transcriptome was sequenced, and data were analyzed by R software. Andrographolide-related target genes were extracted via network pharmacology using online databases. Lentiviral transfection was applied to knockdown the heme oxygenase-1 gene (Hmox1, HO-1). Results showed that andrographolide suppressed RSV replication and attenuated subsequent inflammation. Network pharmacology and RNA sequencing analysis indicated that the hub gene HO-1 may play a pivotal role in the anti-RSV effects of andrographolide. Furthermore, andrographolide exerted antiviral effects against RSV partially by inducing HO-1 but did not activate the antiviral interferon response. Our findings demonstrated that andrographolide exerted anti-RSV activity by up-regulating HO-1 expression in human airway epithelial cells, providing novel insights into potential therapeutic targets and drug repurposing in RSV infection.

Che S ，Zhou N ，Liu Y ，Xie J ，Liu E ... -  《-》

Late therapeutic intervention with a respiratory syncytial virus L-protein polymerase inhibitor, PC786, on respiratory syncytial virus infection in human airway epithelium.

Effective anti-respiratory syncytial virus (RSV) agents are still not available for clinical use. Current major targets are virus surface proteins, such as a fusion protein involved in viral entry, but agents effective after RSV infection is established are required. Here we have investigated the effects of late therapeutic intervention with a novel inhaled RSV polymerase inhibitor, PC786, on RSV infection in human airway epithelium. Air liquid interface-cultured bronchial or small airway epithelium was infected with RSVA2. PC786 was applied apically or basolaterally once daily following peak virus load on Day 3 post inoculation. Apical wash was collected daily for determination of viral burden by PCR and plaque assay (primary endpoints) and biomarker analyses. The effects were compared with those of ALS-8112, an anti-RSV nucleoside analogue, and GS-5806, a fusion-protein inhibitor, which were treated basolaterally. Late intervention with GS-5806 did not show significant anti-viral effects, but PC786 produced potent, concentration-dependent inhibition of viral replication with viral load falling below detectable limits 3 days after treatment commenced in airway epithelium. These effects were superior to those of ALS-8112. PC786 showed inhibitory activities against RSV-induced increases of CCL5, IL-6, double-strand DNA and mucin. The effects of PC786 were also confirmed in small airway epithelium. Late therapeutic intervention with the RSV polymerase inhibitor, PC786, reduced the viral burden quickly in human airway epithelium. Thus, PC786 demonstrates the potential to be an effective therapeutic agent to treat active RSV infection.

Brookes DW ，Coates M ，Allen H ，Daly L ，Constant S ，Huang S ，Hows M ，Davis A ，Cass L ，Ayrton J ，Knowles I ，Strong P ，Rapeport G ，Ito K ... -  《-》

[Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells].

Xie XH ，Liu EM ，Yang XQ ，Law HK ，Li X ，Wang LJ ，Liu W ，Xu WF ... -  《-》

Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infant and elderly populations worldwide. Currently, there is no efficacious vaccine or therapy available for RSV infection. The molecular mechanisms underlying RSV-induced acute airway disease and associated long-term consequences remain largely unknown; however, experimental evidence suggests that the lung inflammatory response plays a fundamental role in the outcome of RSV infection. High-mobility group box 1 (HMGB1) is a nuclear protein that triggers inflammation when released from activated immune or necrotic cells and drives the pathogenesis of various infectious agents. Although HMGB1 has been implicated in many inflammatory diseases, its role in RSV-induced airway inflammation has not been investigated. This study investigates the molecular mechanism of action of extracellularly released HMGB1 in airway epithelial cells (A549 and small airway epithelial cells) to establish its role in RSV infection. Immunofluorescence microscopy and Western blotting results showed that RSV infection of human airway epithelial cells induced a significant release of HMGB1 as a result of translocation of HMGB1 from the cell nuclei to the cytoplasm and subsequent release into the extracellular space. Treating RSV-infected A549 cells with antioxidants significantly inhibited RSV-induced HMGB1 extracellular release. Studies using recombinant HMGB1 triggered immune responses by activating primary human monocytes. Finally, HMGB1 released by airway epithelial cells due to RSV infection appears to function as a paracrine factor priming epithelial cells and monocytes to inflammatory stimuli in the airways. RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

Hosakote YM ，Brasier AR ，Casola A ，Garofalo RP ，Kurosky A ... -  《-》