Chikusetsusaponin IVa Protects against H9N2 Avian Influenza Virus Infection by Inhibiting Inflammation and TLR4/NF-κB Signaling Pathways in vitro and in vivo

Abstract

Background: The prevalence of Hemagglutinin 9 Neuraminidase 2 (H9N2) avian influenza virus (AIV) in poultry in Asia has raised serious concerns about its zoonotic transmission and the potential for a pandemic, underscoring the need for effective therapeutics. Chikusetsusaponin IVa (CHS), a bioactive triterpenoid saponin from Radix saposhnikoviae, has exhibited anti-inflammatory activity in preclinical studies. We hypothesized that CHS could mitigate immunopathology during tumor necrosis factor (TNF)-infection by suppressing excessive inflammation.Objectives: CHS is a natural saponin with anti-inflammatory activity. This research aimed to explore the in vitro and in vivo protective effects and mechanisms of CHS against H9N2 AIV infection.Materials and Methods: We employed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis for the prediction of the underlying mechanisms, followed by a network analysis approach. In animal experiments, the toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathways and associated proteins were assessed using immunohistochemistry and western blot analysis.Results: In A549 cells, CHS treatment reduced H9N2 AIV-induced apoptosis in a dose-dependent manner. CHS was found to have a suppressive effect on the mRNA expression of various inflammatory cytokines (including TNF-α, interleukin (IL)-6, IL-1β, and IL-8) as well as mediators such as inducible nitric oxide synthase (iNOS) and prostaglandin-endoperoxide synthase 2 (PTGS2). It can be seen that CHS does have anti-inflammatory effects. Bioinformatics analysis indicated that CHS might exhibit anti-inflammatory effects by modulating cytokine–cytokine receptor interaction and the NF-κB signaling pathways. Building upon this, it was found that CHS blocked the activation of TLR4 and NF-κB signaling pathways in A549 cells infected with H9N2 AIV. In vivo, CHS prolonged the survival time and increased the survival rate of H9N2 AIV-infected mice. CHS was found to have a protective effect on the lungs of infected mice, resulting in reduced damage and lower levels of viral load and inflammation. CHS inhibited the production of proinflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-8) and inhibited the activation of TLR4 and NF-κB signaling pathways in the pulmonary tissues of mice that were infected.Conclusion: CHS exerts protective effects against H9N2 AIV infection in vitro and in vivo. The anti-inflammatory mechanisms may involve the inhibition of cytokine production and the blockade of TLR4 and NF-κB signaling pathways.

Keywords: Chikusetsusaponin IVa; H9N2 avian influenza virus; anti-inflammation; TLR4; NF-κB Received 30 July 2023; accepted 27 October 2023