Regulation And Mechanism Of Edaravone And Dexborneol On Polarization Of Lipopolysaccharide-induced BV-2 Cells

Background and ObjectiveMicroglia are intrinsic immune cells of the brain responsible for maintaining development and homeostasis of central nervous system(CNS).M1-phenotype microglia participate in the pro-inflammatory process of diseases,while M2-phenotype microglia play an anti-inflammatory role.There is growing evidence that modulating the polarization of microglia from a pro-inflammatory to an anti-inflammatory phenotype can restrain pathological processes such as neuroinflammation,oxidative stress,and neuronal apoptosis to ameliorate brain injury,which is a feasible approach for the treatment of CNS diseases.Edaravone and Dexborneol(ED)is a novel compound for the treatment of acute ischemic stroke.There are researches having shown that ED can suppress inflammation in some animal models of CNS diseases(such as cerebral trauma,autoimmune encephalomyelitis,and cerebral hemorrhage).However,whether ED affects inflammatory responses and phenotypic transformation related to microglia remains unclear.Therefore,this study was to investigate the regulation and mechanism of ED on polarization of BV-2 microglial cells induced by lipopolysaccharide(LPS).Methods1.The effects of LPS and ED alone or in combination on BV-2 cell viability(survival rate)were analyzed by CCK-8 assay to determine the treatment concentrations of LPS and ED in subsequent experiments.Grouping of follow-up experiments: control group,LPS group,LPS+ED(100 μM)group,LPS+ED(200 μM)group,and LPS+ED(400 μM)group.BV-2 cells were preincubated with different concentrations of ED for 2 h and then LPS was added to co-incubate with BV-2 cells for 12 h.2.Western blot analysis and Griess method were used to detect the expressions of LPS-induced inflammatory mediators(TNF-α,IL-1β,and NO)in BV-2 cells.3.Western blot and immunocytofluorescence methods were used to detect the expressions of microglial polarization markers(i NOS,Arg-1,and Ym-1)in BV-2 cells induced by LPS.4.Western blot analysis was used to detect the expressions of p65,STAT3,and STAT6 in the nucleus and cytoplasm as well as TLR4,My D88,p65,p-p65,IκBα,p-IκBα,IKKβ,p-IKKβ,NLRP3,pro-caspase-1,pro-IL-1β,cleaved-caspase-1,and cleaved-IL-1β in total proteins of BV-2 cells,respectively.And the nuclear translocation of p65 was also observed by immunofluorescence method.5.The DCFH-DA fluorescent probe was used to detect the expression of ROS in BV-2 cells.6.Colorimetric method was used to detect the expressions of antioxidant enzymes(SOD,GPx,and CAT)in BV-2 cells.Results1.ED could inhibit the promotion of LPS on BV-2 cell viability.2.Compared with the LPS group,ED reduced the production of inflammatory mediators(TNF-α,IL-1β,and NO)in BV-2 cells stimulated by LPS in a dose-dependent manner.3.Compared with the control group,LPS increased the expression of M1 microglial markers(i NOS)but had no effect on the expression levels of M2 microglial markers(Arg-1 and Ym-1).After ED pretreatment,the expression of i NOS could be significantly down-regulated and Arg-1 and Ym-1 could be up-regulated.4.LPS activated the transcription factors p65 and STAT3 in BV-2 cells,promoting them into the nucleus,but suppressed the nuclear translocation of STAT6.ED pretreatment only down-regulated the nuclear translocation of p65,the result of which was consistent with the result of immunofluorescence.In addition,ED pretreatment significantly reduced the expressions of key proteins TLR4,My D88,p-p65,p-IκBα,and p-IKKβ in the TLR4/Myd88/NK-κB pathway,but did not affect the activation of NLRP3 inflammasomes.5.LPS could induce BV-2 cells to produce excess ROS,while ED pre-intervention could remove ROS in cells.6.ED could enhance the enzymatic activity of the intracellular antioxidant enzyme GPx.Conclusions1.ED may exert an antagonistic effect on neuroinflammation by negatively regulating TLR4/My D88/NF-κB signal pathway mediating phenotypic transformation of LPS-induced BV-2 microglial cells from M1 to M2.2.ED displays the antioxidative property by depleting the intracellular excessive ROS caused by LPS through the enhancement of the enzymatic activity of GPx.