Magnolol Modulates the RIG-I/NF-κB Signaling Pathway to Alleviate JEV Infection-Induced ST Cell Damage

Japanese encephalitis virus (JEV) is a zoonotic pathogen. Infection in breeding boars triggers severe testicular inflammatory storms, resulting in testicular swelling, asthenospermia, oligozoospermia and even irreversible loss of reproductive capacity. Magnolol, a natural extract, exhibits remarkable anti-inflammatory and antioxidant activities. This study utilized swine testicular (ST) cells as an in vitro model to explore magnolol’s protective effects against JEV-mediated inflammation and its underlying mechanism. ST cells were infected with JEV at an MOI of 1 and treated with magnolol at 8, 12 and 16 μg/mL. We detected pro-inflammatory cytokine expression, performed 4D-FastDIA quantitative proteomics to screen differentially expressed proteins, verified key RIG-I and NF-κB cascade molecules at mRNA and protein levels, and tracked p65 nuclear translocation through immunofluorescence. The data showed that JEV significantly elevated TNF-α, IL-1β, IL-6, IL-8 and CCL5, while magnolol reduced these cytokines in a dose-dependent manner. JEV reshaped the proteome of innate immunity and inflammation, and excessively activated the RIG-I/NF-κB axis to increase IKKβ, TRAF2, TRAF6 transcription and p65 phosphorylation and nuclear import; 24 h treatment with 16 μg/mL magnolol greatly restrained this overactivation. In summary, magnolol alleviates JEV-triggered inflammatory injury by inhibiting the RIG-I/NF-κB pathway, offering experimental evidence for its use as a natural agent to mitigate JEV-induced cellular inflammatory response in boars.