MIF Inhibitor Z-312 Inhibit Microglial Inflammatory Activation

Background:Microglial activation mediated neuroinflamamtion plays an important role in the progress of neurodegenerative diseases.Macrophage migration inhibitory factor（MIF）is a pleiotropic proinflammatory mediator that has been involved in the neuroinflammatory diseases.Previously,we demonstrated a small molecule compound3-[（biphenyl-4-ylcarbonyl）carbamothioyl]amino benzoic acid（Z-312）can inhibit MIF activity with docking-based virtual screening and experimental evaluation.Objective:To investigate the anti-inflammatory effects and molecular mechanisms of Z-312 on lipopolysaccharide（LPS）induced BV-2 microglia cells.Methods:The concentration of nitrite in BV-2 cell culture media was determined using Griess reagents.The amounts of tumor necrosis factor（TNF-α）,interleukin（IL）-6and prostaglandin（PG）E2 were tested using specific ELISA kits.The cell viability was evaluated by MTT assay.The mRNA expression of proinflammatory genes including iNOS,COX2,IL-6,TNF-αand IL-1?was determined by Quantitative real-time polymerase chain reaction（Q-RT-PCR）.The protein expression of proinflammatory genes,phosphorylation and degradation of IκB-αand activation of MAPKs were determined by Western blot.Subcellular location of p65 subunits of NF-κB was determined by immunofluorescence assay.Co-culture of BV-2 cell culture media with HT-22 neuroblastoma cells was used to determine the neuroprotective effect of Z-312.Results:Z-312 significantly inhibited production of NO,TNF-α,IL-6 and PGE₂ in LPS-stimulated BV-2 microglia cells.Z-312 markedly suppressed LPS induced expression of proinflammatory genes such as iNOS,COX-2,TNF-α,IL-6 and IL-1?in BV-2microglia cells in a dose dependent manner.Mechanistic study demonstrated that Z-312significantly inhibited LPS-induced IκB-αphosphorylation and subsequent degradation,nuclear translocation of p65 subunit of NF-κB and phosphorylation of MAPKs in BV-2microglia cells.Co-culture study found that Z-312 significantly reduced cell culture media of activated BV-2 microglia cell triggered cell death or growth inhibition of HT-22neuroblastoma cells.Conclusion:Z-312 process anti-inflammatory and neuroprotective activity in cultured microglia cells and neuroblastoma cells through inhibition of activation of NF-κB and MAPKs signal pathway.