Tnf α

The PK15 cell line was obtained from the A.T.C.C. fetal bovine serum (FBS), dulbecco's modified eagle medium (DMEM) and Opti-MEM medium were obtained from Gibco). The vectors pcDNA™6.2-GW/EmGFP-miR, pMD-18T, pcDNA3.1(+), pDONR221 and pLenti6.3/V5-DEST, and the vector construction kits BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP, Packaging Mix, T4 DNA ligase, high purity plasmid extraction kit, Lipofectamine 2000 and TRIzol were purchased from Invitrogen. Synergy Brands, Inc (SYBR) premix ExTaq was purchased from Takara. Total protein extraction kit and BCA protein detection kit were purchased from Nanjing Keygen Technology. Primary antibodies–MyD88 (1:800), cluster of differentiation antigen 14 (CD14) (1:400), IFN-α (1:600), IL-1β (1:600), TLR4 (1:1000), TNF-α (1:1000) and β-actin (1:4000)–were purchased from Abcam. Second antibody (IgG-HRP, 1:3000) was purchased from Jackson. LPS was purchased from Sigma–Aldrich. Porcine IL-1β, TNF-α, IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1α and MIP-1β ELISA kits were purchased from AssayPro.

At the end of the study, pro-inflammatory cytokines like TNF-α, TGF-β, IL-6, IL-10, and IL-1β (Assaypro, USA) were evaluated by following the manufacturer’s procedures.
Endogenous antioxidant parameters like catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH), and malondialdehyde (MDA) levels were measured as previously described⁵⁹ .