Mechanism of anti-porcine circovirus type 2-induced inflammation of porcine alveolar macrophages by red clover isoflavones

【Objective】To elucidate the mechanism of red clover isoflavones in alleviating porcine circovirus type 2 （PCV2）-induced inflammation in porcine alveolar macrophages（3D4/2）and provide theoretical basis for developing red clover isoflavones into a veterinary anti-inflammatory drug.【Method】A 3D4/2 cell inflammation model was established via PCV2 infection. 3D4/2 cells were treated with red clover isoflavones solutions at different concentrations（0.25， 0.50 and 1.00 mg/mL）or dexamethasone（DXMS）. The secretion and gene expression levels of inflammatory cytokines， interleukin-1β（IL-1β），interleukin-6（IL-6），tumor necrosis factor-α（TNF-α）and interleukin-10（IL-10），were measured using ELISA and real-time fluorescence quantitative PCR（qRT-PCR）. Western blotting was employed to assess the expression and phosphorylation of NF-κB and MAPK signaling pathway related proteins（p65，IκB-α and p38）.【Result】 PCV2 induction greatly inhibited 3D4/2 cell growth and extremely significantly increased the contents of inflammatory cytokines IL-1β，IL-6，TNF-α，and IL-10（P<0.01，the same below），and their gene relative expression also extremely significantly increased. IκB-α protein degradation level，p38 protein expression，p65 protein phosphorylation and expression were extremely significantly elevated. The growth of PCV2-induced 3D4/2 cells treated with 0.25，0.50 and 1.00 mg/mL of red clover isoflavones showed no obvious difference compared with blank control group，compared with PCV2-induced 3D4/2 cells，the contents of pro-inflammatory factors IL-1β，IL-6 and TNF-α and their relative gene expression were obviously decreased，while the contents of anti-inflammatory factor IL-10 and its relative gene expression were obviously increased in a dose-dependent manner，and the effects of 1.00 mg/mL red clover isoflavones. Additionally，red clover isoflavones of different concentrations extremely significantly inhibited the phosphorylation of p65 protein and the degradation of IκB- α protein，and extremely significantly decreased the expression of p65 and p38 proteins，and the change trend was basically dose-dependent，that was，it exerted anti-inflammatory effects by interfering with the activation of NF- κB signaling pathway and MAPK signaling pathway. 【Conclusion】Red clover isoflavones exerts antiinflammatory effects on PCV2-induced 3D4/2 cells by suppressing inflammatory factor release，promoting IL-10 secretion of anti-inflammatory cytokines，and inhibiting NF-κB signaling pathway and MAPK signaling pathway.