The Role And Mechanism Of NLRP3 Inflammasome In EAN/EAE

BackgroundMultiple sclerosis(MS)is an inflammatory demyelinating autoimmune disease of the central nervous system.Experimental autoimmune encephalomyelitis is a classic animal model of MS,which has been widely used in basic research of MS for many years.Guillain-Barre syndrome(GBS)is an autoimmune disease of the peripheral nervous system,and experimental autoimmune neuritis is a common animal model of GBS.Both MS and GBS are common neurological diseases which lead to the disability of young adults.Inflammasome is a multiprotein complex expressed in myeloid cells and plays a key role in the innate immune response.There are various subtypes of inflammasome,and NLRP3 is the most clarified one in terms of structure and function.NLRP3 inflammasome consists of NLRP3,ASC(apoptosis-associated speck-like protein containing a caspase recruitment domain)and precursor caspase-1.NLRP3 inflammasome is activated when cells are stimulated or infected by external signals,and then cleavages pro-IL-1β and pro-IL-18 to IL-1β and IL-18.As important pro-inflammatory factors,IL-1β and IL-18 are capable of triggering immune responses.Since IL-1β and IL-18 are involved in the pathogenesis of EAE/EAN,NLRP3 inflammasome,as the upstream production factors of IL-1β and IL-18,might play a role in EAE/EAN.Besides,the mechanism of NLRP3 inflammasome activation has not been fully elucidated.Studies have shown that NF-κB signaling pathway is related to the activation of NLRP3 inflammasome,but whether NF-κB inhibits or promotes the activity of NLRP3 inflammasome remains controversial.Objective:To explore the role of NLRP3 inflammasome in the pathogenesis of experimental autoimmune encephalomyelitis/experimental autoimmune neuritis.The EAE/EAN model was established to observe(1)the role of NLRP3 inflammasome in the nervous system pathology and peripheral immune response of EAE/EAN(2)the relationship between NF-κB signaling pathway and NLRP3 inflammasome in EAE.(3)the prevention and treatment effect of NF-κB /NLRP3 inhibitor on EAE/EAN.Methods:(1)A mixture of MOG35-55 peptide,complete freund’s adjuvant(CFA)and mycobacterium tuberculosis,supplied with pertussis toxin(PTX),could efficiently induce EAE in C57BL/6 mice.(2)Mixture of P0180-199 peptide,complete Fresno adjuvant(CFA)and mycobacterium tuberculosis,supplied with pertussis toxin(PTX)could efficiently induce EAN in C57BL/6mice.(3)To assess the role of NLRP3 inflammasome and NF-κB in EAE: the EAE mice were sacrificed at onset and peak phage.(1)Spinal cord inflammation and myelin loss in EAE mice were observed by HE and myelin staining.(2)The mRNA and protein levels of NLRP3 inflammasome were detected by qPCR and Western blot in EAE mouse spinal cord,and P-NF-κB-p65 and NF-κB-p65 protein levels were detected by Western blot.(3)detecting mRNA and protein levels of NLRP3 inflammasome in mononuclear cells from EAE mice spleen by qPCR and Western blot.(4)assessing anti-inflammatory cytokines level in EAE at onset and peak phage.(4)Inhibiting NF-κB /NLRP3 could prevent and treat EAE: The EAE mice were treated with NLRP3 inflammasome inhibitor BAY11-7082 at dose of 5mg/kg/i.p.or 20mg/kg/i.p..(1)to assess symptom score and the pathology changes in spinal cord lesion.(2)to investigate the expression levels of NF-κB and NLRP3 in the spinal cord of EAE mice.(3)CBA method was used to detect the changes in cytokine levels of EAE mice.(5)To assess the role of NLRP3 inflammasome in EAN: EAN mice were(1)observing the pathology changes in sciatic nerve by HE staining.(2)isolating the sciatic nerve of EAN mice,evaluating the mRNA levels of NLRP3 inflammasome by qPCR.(3)isolating the mononuclear cells in spleen of EAN mice,evaluating the mRNA levels of NLRP3 inflammasome by qPCR.(6)Inhibiting NLRP3 could mitigate EAN:(1)observing the clinical score of EAN changes after treatment with BAY11-7082.(2)isolating the sciatic nerve of EAN mice,evaluating the mRNA levels of NLRP3 inflammasome by qPCR.(3)isolating the mononuclear cells in spleen of EAN mice,evaluating the mRNA levels of NLRP3 inflammasome by qPCR.(7)All the data was expressed as x ±SD.Checking whether the data conforms to the normal distribution with Shapiro-Wilk and Kolmogorov-Smirnov.T test was used for comparison between the two groups.Anova was used for comparison among groups.SPSS21.0 software was used for statistical analysis.P < 0.05 was statistically significant.Result:(1)The mixture of MOG35-55,CFA and tuberculin can effectively induce C57BL/6mice to the EAE model.The onset of the disease appears on the 10 th day after immunization,and the peak appears on the 20 th day.(2)The mixture of P0180-199,CFA and tuberculin can effectively induce C57BL/6 mice to become the EAN model.The onset of the disease appears on the 7th day after immunization,and the peak appears on the 15 th day.(3)The level of NLRP3 inflammasome in the spinal cord of EAE mice at onset and at peak stage is higher than that in the control group,and the level at the peak is higher than that at the early stage.The content of NLRP3 inflammasome in spleen mononuclear cells did not show any difference compared with the control group.(4)The P-NF-κB-p65/NF-κB-p65 ratio in the spinal cord of EAE mice at the early stage and at the peak is higher than that in the control group,and the peak is higher than that at onset stage.(5)Preventive and therapeutic application of BAY11-7082,which is capable of inhibiting the NF-κB/NLRP3 inflammasome pathway,contributed to the drop of EAE clinical symptom score and alleviation of pathological changes of spinal cord.(6)Both 5mg/kg/i.p.and 20mg/kg/i.p.BAY11-7082 showed preventive and therapeutic effect on EAE mice,while 20mg/kg/i.p.BAY11-7082 was more significantly effective than5mg/kg/i.p.BAY11-7082.(7)The mRNA level of NLRP3 inflammasome raised in sciatic nerve and spleen mononuclear cells of C57BL/6 EAN mice.(8)BAY11-7082 mitigates the symptom of EAN mice,and the mRNA level of NLRP3 inflammasome in the sciatic nerve decreased.The mRNA level of spleen mononuclear cells declined gently.Conclusion:(1)NLRP3 inflammasome plays a synergistic role with NF-κB in C57BL/6 mouse(2)BAY11-7082 could effectively inhibit the activity of NF-κB signaling pathway and NLRP3 inflammasome activity,and it can prevent and treat EAE.The effect of BAY11-7082 on prevention and treatment of EAE mice could be effected by dose and timingof treatment.(3)NLRP3 inflammasome is related with EAN model,and BAY11-7082 is capable of mitigating EAN.