The Role And Mechanism Of Microglia Activation In Neuronal Injury Induced By 2-chloroethanol

Objective: 1,2-Dichloroethane(1,2-DCE)is a halogenated hydrocarbon compound mainly used in the production of chlorinated hydrocarbons,and can also be used as a binder,degreaser and cleaning agent.Neurotoxicity is the most common toxic reaction after acute or subacute 1,2-DCE poisoning in humans,toxic encephalopathy is the main clinical manifestation,and cerebral edema is the main pathological process.2-chloroethanol(2-CE)is an intermediate metabolite of 1,2-DCE in the body and is more biotoxic,so 2-CE is likely to play a more important toxic role in the development of 1,2-DCE toxic cerebral edema.Microglia(MG)are the central nervous system’s first line of defense against damage from foreign chemicals.Activated MG has proven to be a double-edged sword,on the one hand,it protects neurons from damage through phagocytosis,and on the other hand,it induces an inflammatory response in the brain,causing damage to neurons.Our previous results showed that 2-CE exposure activates MG and increases the synthesis and release of its inflammatory mediators,but how MG-mediated inflammatory responses cause neuronal damage is unclear.Therefore,this study intends to explore the mechanism of neuronal damage induced MG activation induced by 2-CE through in vitro experimental studies,so as to provide an experimental reference for revealing the pathogenesis of subacute 1,2-DCE toxic cerebral edema.Methods: 1.Preparation of BV2 cell conditioned culture medium: take mouse microglia line BV2 cells as experimental objects,treat BV2 cells with 2-CE containing 0,40,80 and160 m M for 12 h,the old culture was discarded and a new culture was added and incubated for a further 12 h,collect the cell culture medium and put it in a sterile EP tube,centrifuge at 1200 rpm for 10 min,and immediately transfer the supernatant into a new sterile EP tube as a conditioned culture medium(BCM).Effects of 2-CE exposure on BV2 cell activation,NF-κB signaling pathway activation and pro-inflammatory cytokine secretion:BV2 cells and BCM were collected after 12 h exposure of BV2 cells to different concentrations of 2-CE,and the expression levels of p65 and p-p65 proteins in BV2 cells were detected by Western blotting method,and TNF-α and IL-1β m RNA expression levels in BV2 cells were detected by q RT-PCR.BCM was collected and the secretion levels of TNF-α and IL-1β were measured by ELISA.3.The damaging effect of 2-CE-induced BV2 cell activation on neurons: taking PC12 neurons as experimental objects,BCM was added to the culture medium of PC12 cells,and the cells were randomly divided into blank control group and 0 m M,40 m M,80 m M and 160 m M BCM co-culture groups,and PC12 cells were collected after 12 h of culture,and protein expression levels of Bax,Bcl-2,NR1,NR2 A,NR2B and Ca MKII were detected in PC12 cells by Western blotting method.Flow cytometry was used to detect apoptosis of PC12 cells.The fluorescence intensity of SYP in PC12 cells was detected by immunofluorescence method.4.The role of NF-κB signaling pathway in 2-CE activation of BV2 cells and its effect on neuronal damage: BV2 cells were pretreated by NF-κB signaling pathway inhibitor PDTC for 1 h,exposed to 160 m M 2-CE for 12 h,BCM was collected according to the above method,and co-cultured with PC12 neurons,and the cells were randomly divided into 0 m M BCM(control),PDTCBCM(inhibitor control),160 m M 2-CE-BCM(2-CE treatment)and 2-CE+PDTC-BCM(inhibitor intervention)co-culture group,after 12 h of co-culture,cells were collected for the above indexes.Results:1.Effects of 2-CE exposure on pro-inflammatory factor secretion in BV2 cells: Compared with the control group,the m RNA expression levels of TNF-α and IL-1β and the secretion levels in BCM in BV2 cells of the 2-CE exposure group were significantly increased(P<0.05).2.Effect of 2-CE exposure on NF-κB signaling pathway in BV2 cells: Compared with the control group,the protein expression level of p-p65 in BV2 cells in the 2-CE exposure group was significantly increased(P<0.05).3.Effects of 2-CE-induced BV2 cell activation on neuronal function and apoptosis:Western blotting results showed that compared with the simple neuronal culture group,there was no significant difference in Bax/Bcl-2 ratio and protein expression levels of NR1,NR2 A and NR2 B in PC12 cells in the co-cultured control group,and Ca MKII protein expression levels were significantly higher(P<0.05);Compared to the co-culture control group,the Bax/Bcl-2 ratio and the protein expression levels of NR1,NR2 A and NR2 B in PC12 cells in the 2-CE-treated co-culture group were significantly increased,but the expression level of Ca MKII protein decreased significantly(P<0.05).The results of flow cytometry showed that compared with the neuron culture alone,there was no significant difference in the apoptosis level of PC12 cells in the co-cultured control group.Compared with the co-cultured control group,the apoptosis level of PC12 cells in the 2-CE-treated co-culture group was significantly higher(P<0.05).In addition,the results of immunofluorescence staining also showed that there was no significant difference in SYP fluorescence intensity in PC12 cells in the co-cultured control group compared with the neuron culture group alone.Compared with the co-culture control group,SYP fluorescence intensity was weakened in PC12 cells in the 2-CE-treated co-cultured group.4.The role of NF-κB signaling pathway in 2-CE activation of BV2 cells and its effect on neuronal damage: Compared with the 160 m M 2-CE exposure group,the protein expression level of p-p65 in BV2 cells in the PDTC intervention group decreased significantly,and the expression level of m RNA and secretion level of TNF-α and IL-1βin BV2 cells and the secretion level in BCM decreased significantly(P <0.05).Compared with the 2-CE-treated co-culture group,the Bax/Bcl-2 ratio and the protein expression levels of NR1,NR2 A and NR2 B in PC12 cells in the PDTC intervention co-culture group decreased significantly,the expression level of Ca MKII protein and SYP fluorescence intensity increased significantly,and the apoptosis level of PC12 cells was significantly reduced(P<0.05).Conclusion:1.2-CE activates BV2 cells through the NF-κB signaling pathway and upregulates the synthesis and release of pro-inflammatory cytokines.2.Activated BV2 cells adversely affect the function of PC12 cells and promote the occurrence of neuronal apoptosis.3.The release of inflammatory factors caused by activation of NF-κB signaling pathway plays an important role in the damage of BV2 cells to neurons.