Effects Of Resolyin D1on Neuroinflammation And Microglia Polarization In Mice

Part one Effects of resolvin D1on lipopolysaccharide-induced neuroinflammation in miceObjective:To investigate the effects of resolvin D1(RvD1) on lipopolysaccharide (LPS)-induced excessive production of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), microglia activation and activation of nuclear factor kappa B (NF-κB).Methods:Adult male C57BL/6(2-3mouth) mice received an intraperitoneal injection of LPS (250μg/kg) followed by an intracerebroventricular injection of RvD1(1ng,10ng) or vehicle or combination of RvD1(10ng) and formyl peptide receptor2(FPR2) antagonist Boc-2(1μg).4h after administration of LPS, serum, cortex and hippocampus were collected. The effects of LPS and/or RvD1on production of TNF-α and IL-1β, microglia activation, expression of NF-κB p65and inhibitor of κB-α (IκB-α), DNA binding activity of NF-κB were analysed by ELISA, immunohistochemistry, Western blot and EMSA.Results:Stimulation with LPS markedly increased TNF-α and IL-1β in serum, cortex and hippocampus. Treatment with10ng RvD1inhibited LPS-induced production of TNF-α and IL-1β and microglia activation.10ng RvD1also inhibited LPS-induced activation of NF-κB pathway in cortex and hippocampus. The effects of RvD1were reversed by treatment with Boc-2.Conclusion:These findings suggest that RvD1inhibites LPS-induced neuroinflammation in mice via FPR2and NF-κB. Part two Effects of resolvin D1on LPS-induced microglia M1polarizationObjective:To investigate the effects and mechanisms of RvDl in LPS-induced microglia Ml polarization.Methods:BV-2cells were treated with RvD1(0.1nM,1nM,10nM,100nM) or vehicle for30min before stimulated with100ng/ml LPS. To investigate the involvement of FPR2in the effects of RvD1, BV-2cells were treated with10μM Boc-2prior to the treatment with RvD1. The expression of TNF-a, IL-1β,nitric oxide (NO), inducible nitric oxide synthase (iNOS), NF-κB p65, inhibitor of KB-a (IκB-a) and phosphorylation of mitogen-activated protein kinases (MAPKs) were measured by ELISA, nitrae reductase method and Western blot. The DNA-binding activity of NF-κB and activator protein-1(AP-1) were analysed by electrophoretic mobility shift assay (EMS A).Results:Pretreatment with RvD1mitigated microglia classical activation by reducing LPS-induced expression of TNF-a, IL-1β, NO and iNOS in BV-2cells. RvD1inhibited LPS-induced nuclear translocation of NF-κB p65, degeneration of IκB-a and phosphorylation of MAPK. RvD1also reduced DNA binding activity of NF-κB and AP-1. These effects of RvD1were reversed by Boc-2.Conclusion:RvD1mitigated microglia Ml polarization, these effects of RvDl may be involving in inhibition of NF-κB and MAPK/AP-1pathways. Part three Effects of resolvin D1on IL-4-induced microglia M2polarization Objective:To investigate the effects and mechanisms of RvD1in IL-4-induced microglia M2polarization.Methods:BV-2cells were treated with RvDl (100nM) or vehicle for30min before stimulated with10ng/mlIL-4. To investigate the effects of FPR2, signal transducer and activator of transcription-6(STAT6) and peroxisome proliferator-activated receptor gamma (PPARy), BV-2cells were treated with10μM Boc-2, or100μM leflunimide, or1μM GW9662prior to the treatment with RvD1. Western blot and immunofluorescence were performed to detect protein levels of alternative activation markers Arginase1(Argl), Chi-tinase3-like3(Yml). Moreover, the phosphorylation of STAT6, expression of PPARy and DNA binding activity of STAT6and PPARy were analysed by Western blod or EMSA.Results:RvD1enhanced IL4-induced microglia alternative activation by increasing the expression of Argl and Yml. RvDl also enhanced phosphorylation of STAT6, nuclear translation of PPARy, the DNA binding activity of STAT6and PPARy. These effects were abolishment by Boc-2. Further, the effects on Argl and Yml were blocked by the application of leflunomide or GW9662.Conclusion:RvDl promotes IL-4-induced microglia M2polarization via STAT6and PPARy signaling pathways.