Effects Of Aspirin-triggered Lipoxin A₄ On Lipopolysaccharide-induced Inflammatory Responses In Microglial Cells

Part one Effects of aspirin-triggered lipoxin A4 on the production of inflammatory mediators in lipopolysaccharide-activated microgliaObjective:To investigate whether aspirin-triggered lipoxin A4 (ATL) attenuates excessive production of nitric oxide (NO) and pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-a) in lipopolysaccharide (LPS)-stimulated mouse microglial cell.Methods:In all experiments, BV-2 microglial cells were treated with ATL (1nM, 10nM, 100nM) or vehicle (0.035% ethanol) for 30min before addition of 100ng/ml LPS under serum-free conditions.To investigate the involvement of ALX in the anti-inflammatory effects of ATL, the cells were treated with 100μM Boc-2 (a specific receptor antagonist) prior to the treatment with ATL for 30min. The effects of LPS and/or ATL on the expression of NO, inducible nitric oxide synthase (iNOS), IL-1βand TNF-a were analysed by nitrae reductase method, ELISA, Western blotting and real-rime PCR.Results:Both ALX1/FPR-rsl and ALX2/FPR2 were expressed in BV-2 microglial cells. ATL and vehicle did not affect BV-2 cells viability. Stimulation of BV-2 cells with LPS markedly increased NO, IL-1βand TNF-a production. Pretreatment with ATL significantly inhibited LPS-induced production of NO, IL-1βand TNF-a in a concentration dependent manner and these effects was inhibited by Boc-2.The mRNA expression of iNOS, IL-1βand TNF-a in response to LPS were also decreased by ATL, concentration dependently. Inhibitory effects of ATL on the LPS-induced iNOS mRNA up-regulation were accompanied by the attenuation of iNOS protein induction.Conclusion:These findings suggest that ATL exerts anti-inflammatory effects by inhibiting LPS-induced expression of iNOS, IL-1βand TNF-a in BV-2 microglial cells via ALX.Part two Effects of ATL on LPS/Toll-like receptor 4-mediated signalling pathways in microgliaObjective:To investigate the effects of ATL on the activation of LPS/Toll-like receptor 4 (TLR4) signalling pathways, including NF-κB and mitogen-activated protein kinases (MAPKs) in microglia.Methods:In all experiments, BV-2 cells were treated with 100nM ATL or vehicle (0.035% ethanol) for 30min before addition of 100ng/ml LPS under serum-free conditions. The effects of ATL on LPS-induced nuclear translocation of NF-κB p65, degradation of inhibitor of KB-a (IKB-a) and phosphorylation of MAPKs were analysed by immunofluorescence confocal microscopy and Western blotting. Moreover, we investigated the effects of ATL on LPS-induced DNA-binding activity of NF-κB and activator protein-1 (AP-1)by electrophoretic mobility shift assay (EMSA).Results:LPS stimulation caused obvious translocation of the NF-κB p65 from the cytoplasm into the nucleus in BV-2 cells at 60min after the activation, whereas the presence of 100nM ATL blocked it. Western blot analysis showed that LPS-induced degradation of IκB-αwas significantly reversed by 100nM ATL in BV-2 cells. ATL markedly inhibited extracellular signal-regulated kinase (ERK) 1/2 and p38 activation, while phosphorylation of c-jun N-terminal kinase (JNK) was not affected. Strikingly, ATL could induce JNK phosphorylation without effect on ERK1/2 and p38 activity. Moreover, pretreatment with ATL markedly reduced the LPS-induced DNA-binding activity of NF-κB and AP-1.Conclusion:This study indicates that ATL exerts anti-inflammatory effects at least in part via blocking NF-κB, ERK1/2,p38 MAPK and AP-1 signaling pathway in LPS-activated microglia.Part three Effects of ATL on LPS-induced oxidative stress in microgliaObjective:To investigate whether ATL attenuates excessive production of reactive oxygen species (ROS) in LPS-stimulated mouse microglial cell and to explore the potential molecular mechanism.Methods:In all experiments, BV-2 cells were treated with ATL or vehicle (0.035% ethanol) for 30min before addition of 100ng/ml LPS under serum-free conditions. The levels of intracellular ROS were detected by fluorescence microplate reader. The effects of ATL on the expression of heme oxygenase-1 (HO-1)were analysed by immunofluorescence confocal microscopy and Western blotting. Moreover, we investigated the effects of ATL on the expression and nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) by Western blotting.Results:The ROS level was higher in LPS-treated group than in control group at 6, 12 and 24h points and reached its peak at 12h after LPS stimulation; significant decrease of ROS was observed when the cells were pretreated with 100nM ATL. ATL induced HO-1 protein expression in a concentration- and time-dependent manner. The induction of HO-1 by ATL was evident as early as 3h, and augmentation lasted for at least 24h. This was also evident on the immunofluorescent staining of HO-1 in the BV-2 cells. Western blot analysis showed 100nM ATL increased Nrf2 protein expression in a time-dependent manner. Moreover, Nrf2 translocated to the nucleus was increased by 100nM ATL within 120min. In addition to this, the translocation to the nucleus of Nrf2 in LPS-stimulated cells was also increased when pretreated with 100nMATLConclusion:These findings suggest that ATL suppresses LPS induced ROS generation in microglia, likely via induction of microglial expression of HO-1 and Nrf2 and nuclear translocation of Nrf2.