The Study Of Protective Effect And Mechanism Of CXCR2 Antagonist On Experimental Autoimmune Encephalomyelitis

Objective: multiple sclerosis?MS?is a common central nervous system disease.Presently,most studies suggest that it is an autoimmune disease with abnormal T lymphocyte function.Abnormal activated T lymphocytes infiltrate the central nervous system and destroy myelin,resulting in the loss of neurodegeneration.But anti-T lymphocyte therapy does not significantly improve the condition of MS,suggesting that there may be other immune cells involved in the occurrence and development of MS.experimental autoimmune encephalomyelitis?EAE?is an ideal animal disease model of MS.Studies have shown that neutrophils as non-specific immune cells may also play an important role in specific immunity.But the exact role and mechanism of neutrophils in MS and EAE are still unclear.CXCL2 is a major mediator in recruiting neutrophils.In this experiment,to establish an EAE model through the induction of MOG35-55,use CXCR2 small molecule antagonist to treat EAE,to explore the role of neutrophils in the development of EAE,and the protective effect of CXCR2 antagonist on EAE and its immunological mechanism.Method: Female C57BL/6 mice were randomly assigned to normal control group?n = 9?,the EAE model group?n = 9?and the CXCR2 treatment group?n = 9?,the MOG35-55 peptide was diluted with PBS and mixed with complete Freund's adjuvant?CFA?in the same volume,emulsion was repeatedly pushed and pulled into an oil-inwater emulsion with a syringe.Each mouse was injected with 200 ? L emulsion,in which the dose of MOG35-55 peptide was 300 ? g per mouse,100 ? L pertussis was injected intraperitoneally 0 h and 48 h after the first immunization.CXCR2 treatment group was injected intraperitoneally with 100 ? L every day after immunization.The neurological function was scored every day,and the mouse were killed and tissue samples were taken on the 7d and 14 d.Histomorphology was observed by HE staining,the number of neutrophils in central nervous system was observed by flow cytometry,esterase staining and immunofluorescence,and the permeability of blood-brain barrier was detected by Evans blue staining.Result: Compared with the EAE model group,the onset of CXCR2 antagonist group was delayed by 4 days,and the average clinical score of CXCR2 antagonist group?2.11 ± 0.75?was significantly lower than that of EAE mice?3.46 ± 0.62??P < 0.01?,HE staining was used to observe the infiltrating inflammatory cells and vascular cufflike formation in the spinal cord and brain tissue of mice in the EAE model group,while the inflammatory lymphocyte infiltration was significantly reduced and the inflammatory score decreased in the CXCR2 antagonist group?P < 0.01?.At the same time,pathogenic neutrophils(CD45^highCD11b⁺Ly6G⁺)infiltrated into the central nervous system in the early stage of EAE,and reached the peak in the meninges at the onset of the disease?compared with the brain and spinal cord,P < 0.05?,the neutrophil infiltration in the CXCR2 antagonist group was significantly decreased?P < 0.05?.The results of esterase staining showed that,no neutrophil positive cells were found in the brain parenchyma of blank group,but there were neutrophil positive cells in the brain of EAE group,the number of neutrophil positive cells in CXCR2 antagonist group was less than that in EAE group?P < 0.05?.Through the immunofluorescence confocal staining of CD31 and Ly6 G,it was found that most of the infiltrated neutrophils in the brain and spinal cord of EAE model group were located in or adhered to blood vessels,and the number of neutrophils decreased after treatment with CXCR2 antagonist?P < 0.05?.We found that NETs,was hardly found in the brain and spinal cord of normal mice,but NETs was significantly increased in EAE mice?P < 0.05?.Compared with EAE mice,CXCR2 antagonist significantly inhibited the release of NETs in brain and spinal cord,and the content of Evans blue in brain decreased significantly in CXCR2 antagonist group?P < 0.01?.Conclusion: In this experiment,an experimental autoimmune encephalomyelitis model?EAE?model,was used as the object to explore the protective effect of CXCR2 antagonist on the EAE model and its immune mechanism.It had been found that CXCR2 antagonists can effectively delay and reduce the clinical manifestations and inflammatory cell infiltration in the CNS;and alleviate the disease by preventing neutrophils from migrating across and inhibiting the formation of NETs in inflammatory sites,and reduce the destruction of BBB permeability,suggesting that CXCR2 inhibitors may by another potential inflammatory diseases.