Nox2 Impairs Cognition By Modulating Microglial Polarization In EAE Model

Background:Multiple sclerosis（MS）is an autoimmune nervous system disease with demyelination as an important clinical pathological change.Its pathogenesis involves complex processes,which mainly includes inflammatory cell infiltration,demyelination and axonal injury.Extensive injury of neurons in the brain of MS patients occurs in the early stage of the disease.Cognitive impairment is one of the common clinical symptoms,which seriously affects the work and life of patients.The neuronal axons in the brain of patients with MS have been extensive injured at the early stage.MS is often associated with excessive activation of microglia,and activated microglia can be polarized into M1 classical activation type and M2 alternative activation type.Different types of polarization play different roles in the regulation of immune inflammation in the pathogenesis of the disease,and whether microglial polarization is involved in MS induced cognitive dysfunction is still unclear.NADPH oxidase 2（Nox2）is highly expressed in activated microglia and Nox2 plays an important role in regulating the polarization of microglia.The present study was designed to explore the role of Nox2 in the regulation of microglial polarization in a MS model—Experimental autoimmune encephalomyelitis（EAE）.Objective:Defining the existence of cognitive impairment in the early EAE mice,and we initially explored the role and mechanism of Nox2 regulation of microglia polarization in cognitive impairment in the early EAE mice.Methods:（1）The EAE model was established by immunizing C57BL/6 mice with the MOG_35-55antigen peptide.The behavioral changes were observed and the clinical scores of the mice were recorded daily.The pathological changes of the spinal cord were observed by LFB staining at specific time points.In addition,Behavioral experiments were performed on mice from the 6th day after immunization,including Morris water maze test and conditional fear test.After the behavioral test was completed,at the 11th day（early disease course）,the mice were taken for corresponding detection.Postsynaptic density protein 95（PSD95）,Synapsin-1,Brain derived neurophic factor（BDNF）and cysteine were detected by Western blot.Expression and distribution of aspartic protease3（Caspase-3）.（2）The expression of microglia marker Iba-1 in hippocampus of early EAE mice was detected by immunohistochemistry.Western blot and q-PCR were used to detect the expression of M1 microglia markers CD16,i NOS and TNF and M2 microglia marker Arg-1.（3）The expression of Nox2 protein in hippocampus of early EAE mice was detected by Western blot.（4）Western blot was used to detect the protein expression of M1 microglia marker CD16 and M2 microglia marker Arg-1 in the EAE group and EAE+Nox2 inhibitor group.BV2 microglia were cultured,LPS and IFN-γwere used to induce the inflammatory response（LPS+IFN-γstimulation group）,and M1 type in vitro polarization model was established;or Nox2 inhibitor（GKT,GKT137831）was given1 h before stimulation（LPS+IFN-γ+Nox2 inhibitor group）,cell samples were collected 24 h later.The expression of M1 marker i NOS and CD16 was detected by immunofluorescence,Western blot and q-PCR.（5）The expression and distribution of PSD95 in Ctrl,EAE and EAE+Nox2 inhibitors were detected by Western blot.The supernatant of BV2 microglia stimulated by Ctrl group,LPS+IFN-γgroup,LPS+IFN-γ+Nox2 inhibitor（GKT）group was extracted,and the supernatant was incubated with primary cultured neurons for 24 hours.Immunofluorescence was used to detect the fluorescence signal intensity of neuronal PD95.Results:（1）Behavioral results showed that dysmotility began to appear on the 12th day after immunization of EAE mice,and demyelinating lesions on the 18th day（peak）of spinal cord LFB staining showed that EAE mouse model successfully constructed.On the 6th day after immunization,the behavioral experiments such as water maze and conditional fear were started.The results showed that the mice had memory impairment and decreased learning ability in the early stage of the disease.The results of Western blot showed that the expression of PSD95,Synapsin-1 and BDNF in hippocampus of EAE early mice was down-regulated,while the expression of cleaved-caspase3 was up-regulated,indicating that the expression of cognitive-related proteins was down-regulated in early EAE mice.The above results indicated that there is cognitive impairment in the early EAE mice.（2）The results of immunohistochemistry showed that the number of Iba-1 positive cells in the hippocampus of EAE early mice was up-regulated,indicating that the microglia in EAE early mice was significantly activated.The results of Western blot and q-PCR showed that the protein and m RNA levels of microglia M1 marker CD16 were up-regulated and the m RNA levels of i NOS and TNF were up-regulated,while the protein and m RNA levels of microglia M2 marker Arg-1 showed no significant changes in protein and m RNA levels.It was shown that microglia were polarized to M1 type in the early EAE mouse model.（3）Western blot analysis showed that Nox2 protein expression was up-regulated in hippocampus of early EAE mice.These results indicated that high expression of Nox2may be associated with M1 type polarization in microglia in early EAE mice.（4）The results of q-PCR and Western blot showed that Nox2 inhibitor could significantly decrease the expression of M1 microglia marker CD16 and up-regulate the expression of M2 microglia marker Arg-1 in early EAE mice.The M1 markers CD16 and i NOS of microglia in the combined stimulation group of LPS and IFN-γwere significantly up-regulated compared with the Ctrl group,suggesting that they successfully induced the polarization of BV2 microglia to M1.After pre-incubation of Nox2 inhibitor with BV2 microglia,the protein expression of CD16 was significantly down-regulated,and the number of i NOS-positive cells was significantly decreased.The above results indicate that the inhibition of Nox2 can significantly reduce the polarization of microglia to M1 type.（5）Western blot results showed that protein expression of PSD95 increased in early EAE mice after inhibition of Nox2.The results of cellular immunofluorescence showed that LPS+IFN-γ-stimulated BV2 microglial supernatant could significantly decrease the expression of PSD95 in neurons,while the inhibition of Nox2 could significantly restore PSD95 expression.The above results indicate that inhibition of Nox2 can reduce the polarization of microglia to M1 and improve neuronal synaptic loss.Conclusions:（1）There are cognitive impairment in early EAE mice.（2）The EAE early mouse microglia were polarized to M1 type.（3）Intervention of Nox2 inhibitor in EAE early mice significantly reduced the microglia to M1 type polarization,up-regulated the expression of the cognitive-related protein PSD95,and reduced neuronal synaptic loss.