Effect Of Metformin On NLRP3 Inflammasome And Mechanism In EAE Mouse Model

Objective To investigate the regulatory mechanism of metformin on NLRP3 inflammasome and reduce the inflammatory response of EAE based on the establishment of the EAE mouse model.Methods Female C57BL/6 mice were used to construct the EAE model,divided into EAE+MET group,EAE+AMPK inhibitor group,EAE group and control group;the EAE+MET group was intraperitoneally injected with metformin,the EAE+AMPK inhibitor group was intraperitoneally injected with AMPK inhibitor(Compound C),the same volume PBS was intraperitoneally injected to the control group and EAE group until the peak of clinical disease.To observe the mice neurological deficit scores before and after intervention,HE staining and Fast Blue(FB)staining observe inflammatory cell infiltrations and the demyelination of the spinal cord;Immunohistochemical staining(ICH)was performed to detect the proteins expression associated with the NLRP3 inflammasome at organizational level.Western blot was used to detect the proteins AMPK,NeK7 and associated with the NLRP3 inflammasome expression level at the molecular level.The levels of MOG antibody,proinflammatory cytokine interleukin 18(IL-18),cytokine interleukin 1β(IL-1β)and suppressive inflammatory cytokine interleukin 10(IL-10)were assayed by Enzyme-linked immunosorbent assay(ELISA).Results 1.The mouse model of EAE has been established : C57BL/6 mice were injected subcutaneously with the emulsion mixed with MOG35-55 peptide,complete Freund’s adjuvant(CFA)and the tuberculin H37 Ra,intraperitoneally injected with pertussis toxin(PTX)twice(0h and 24 h);EAE-associated symptoms began to appear in model mice on the 9th day after immunization and the clinical scores of EAE mice reached a maximal level around 25 th day.2.Metformin reduces the EAE mice neurological deficit scores and the level of MOG antibody: statisticing and analysising the mice neurological deficit scores in experiment procedure found,after the MET treatment,the EAE mice decreases average neurological deficit scores(P<0.05),and reduces the MOG antibody level(P<0.05).3.Metformin alleviates inflammatory cell infiltrations and the demyelination of the spinal cord in EAE mice: HE staining observes that inflammatory cell infiltrations in EAE+MET group less than the model group(P<0.05);LFB staining results was found that metformin therapy improved the demyelination of the spinal cord(P<0.05),the demyelination was aggravated after the AMPK inhibitor intervention(P<0.05).4.The therapy of metformin alleviates the proteins associated with the NLRP3 inflammasome express level: Immunohistochemical staining was performed to detect the proteins NLRP3,Caspase-1,ASC associated with the NLRP3 inflammasome express level less than EAE group(P<0.05).5.Metformin inhibits the expression of the NeK7 and regulates NLRP3 inflammasome activation procedure: Metformin as an activitor of AMPK was used in the course of the experiment,Western Blot verify the results,in the EAE+MET group,AMPK highly expresses,NeK7 expression was inhibited and reduced the expression of proteins associated with the NLRP3 inflammasome(P<0.05);in EAE+AMPK inhibitor group,the AMPK inhibitor decreased the expression of AMPK,but NeK7 expression was uncontrol(P<0.05),the expression of protein that makes up NLRP3 inflammasome was consistent with NeK7.6.Metformin impacts the interleukin 18(IL-18),cytokine interleukin 1β(IL-1β)and interleukin 10(IL-10)secretion: proinflammatory cytokine IL-1β,IL-18 expression in MET group significantly was inhibited(P<0.05),but suppressive inflammatory IL-10 enhances the express level(P<0.05),the inflammatory factors secretion in the AMPK inhibitor group was opposite to that in the EAE+ MET group.Conclusion(1)Metformin could reduce the neurological deficit scores of the EAE mice.(2)Metformin regulates the expression of NLRP3 inflammasome and alleviates the inflammatory response of EAE,the mechanism may be related to the inhibition of NeK7 expression and the activation of NLRP3 inflammasome.