Anti-inflammatory Effects And Mechanism Of Agents In Lipopolysaccharide-induced BV2Microglia Cells

Microglia is the macrophage like cells resident in CNS, which play a central rolein the immune response of the central nervous system. Upon the injury or infectionsuch as lipopolysaccharide (LPS) and other incentives, microglia are activated quickly.The activation of microglia induces protease activation, promoting inflammatoryfactors and glutamate release, and generates a large number of reactive oxygen andnitrogen species, leading to other cells damage and loss. Microglia activation isinvolved in many neurodegenerative diseases and development. Therefore, manystudies have tried to through inhibiting activation of microglia cells to treatneurological diseases. Naloxone is an opioid receptor antagonist, anddextromethorphan is an opioid receptor agonist. They have been confirmed to haveneuroprotective effects, but the mechanism is not clear. Our study aimed to clarifywhether naloxone and dextromethorphan achieve neuroprotective effects throughinhibition of LPS (lipopolysaccharide) activated BV2microglia cells. The resultsshowed that naloxone and dextromethorphan significantly inhibited heat shockprotein60(HSP60) expression and release in BV2microglial cells. We hypothesizedthat microglia release HSP60extracellularly and extracelluar HSP60combines withToll-like receptor-4(TLR-4) and activates NF-?B(nuclear factor-kappa B) andCASPASE-3, inducing the release of different cytokines and inflammatory reaction.Then we examined effects of both drugs on TLR-4, NF-? B and caspase-3activity inLPS induced BV2microglia cells. It is found that they significantly inhibited theexpression of NF-?B, TLR-4and caspase-3. Naloxone and dextromethorphan also reduced inflammatory factors NO, iNOS, IL-6, IL-1?, TNF-? and HSP60release. These results suggest that naloxone and dextromethorphan significantly inhibited theactivation of microglia and may exert anti-inflammatory effects throughHSP60-TLR-4-NF-?B signal pathway.Objective:We aimed to investigate the neuroprotective effects of naloxone anddextromethorphan as well as the underlying mechanisms by focusing on the inhibitionof LPS induced BV2microglia cells activation.Methods:BV2microglia cells were treated with indicated concentration of of agents. Theeffect of agents on viability of LPS-stimulated BV2microglia was measured byCCK8assay. Using the western blot and immunofluorescence, we studied the effectsof naloxone and dextromethorphan on HSP60?HSF1, iNOS, caspase-3,NF-?B andTLR-4protein expression in LPS-stimulated BV2microglia. Finally, we investigatedthe effects of naloxone and dextromethorphan on the release of IL-6?IL-1??TNF-?and HSP60in LPS-stimulated BV2microglia. NO production and iNOS activity weremeasured by Griess Reagent by detection of cell culture supernatant.Results:1? CCK8assay demonstrated that treatment of BV2microglia with24h of exposureto LPS decreased the viability of LPS-stimulated BV2microglia (P?0.05).However, treatment of microglia with naloxone and dextromethorphan for up to24h could significantly increase the viability of LPS-stimulate BV2microglia incompared with LPS only ?P?0.05?. These results suggested that naloxone anddextromethorphan may have positive effects on viability of LPS-stimulated BV2microglia.2? Western blot and immunofluorescence assay suggested that treatment ofmicroglia with1?g/ml LPS for up to24h increased expression of HSP60, HSF1, iNOS, caspase-3, NF-?B and TLR-4?P?0.05?. However, naloxone anddextromethorphan could significantly increase the expression of HSP60, HSF1,iNOS, caspase-3, NF-?B and TLR-4compared with LPS only?P?0.05?. Ourresults indicated the anti-inflammatory effects of naloxone and dextromethorphanmay be associated with inhibition of HSP60-TLR-4-NF-?B signaling pathway.3? ELISA (Enzyme-linked immunosorbent assay) results showed that LPS alone(1?g/ml) markedly induced the release of IL-6, IL-1?, TNF-? and HSP60compared to control?P?0.05?. However, naloxone and dextromethorphansignificantly reduced IL-6, IL-1?, TNF-? and HSP60production induced by LPS?P?0.05). In summary, these results suggested naloxone anddextromethorphan could inhibit the release of Inflammatory factor inLPS-stimulated BV2microglia.4? By Griess Reagent assay, we found that, compared with the control group, in thesupernatant of LPS treated BV2microglia cells, NO production and iNOS activityincreased, while naloxone and dextromethorphan group effectively inhibited theinflammatory changes in LPS-induced BV2microglia cells.Conclusion:1?Naloxone and dextromethorphan may have a positive effect on viability ofLPS-stimulated BV2microglia.2?HSP60is involved in the protective effects of naloxone and dextromethorphan onLPS-stimulated BV2microglia and the mechanism is may be throughHSP60-TLR-4-NF-?B signal pathway.