The Role And Mechanism Of NF-?b Regulation In Macrophage Subtype In Experimental Autoimmune Neuritis

Guillain–Barré syndrome(GBS)is a common autoimmune disease characterized by peripheral nerve demyelination.Experimental autoimmune neuritis(EAN)is a classic animal model of GBS,which has been widely used in basic research of GBS.Current GBS treatments include intravenous immunoglobulin or plasma exchange,but some patients are not effective.Macrophages play an important role in the pathogenesis of guillain-barre syndrome and experimental autoimmune neuritis.Macrophages can be divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages.M1 macrophages secrete proinflammatory cytokines that damage the peripheral nervous system.M2 macrophages play an anti-inflammatory role and promote the tissue repair.The studys found that pro-inflammatory M1 macrophages were dominant in the actue of GBS.Neuropathological study found that the number of M1 macrophages increased in the sciatic nerve of EAN mice.The increase of M2 macrophages in the target organ was beneficial to the relief of EAN symptoms.Although some experiments have proved that the induction of M2 macrophages can improve the symptoms of EAN,the changes in the proportion of macrophages in different periods of EAN have not been completely clear.Therefore,in order to further explore the mechanism of macrophages in EAN,we conducted the following three experiments.Part 1 The changes of macrophage proportion and cytokines in different stages of EANObjective:To explore the changes in the proportions of spleen macrophages,M1 and M2 macrophages in different stages of EAN,and to clarify the role of macrophages in the pathogenesis of EAN.Methods:(1)Building EAN model;(2)Observe the changes of macrophage proportion in different stages of disease;(3)Detect the cytokines at different stages of disease;(4)Detect the cytokines in peripheral blood of GBS patients before and after immunoglobulin treatment.Results:(1)P0180-199 and complete freund's adjuvant(CFA)emulsion were used to construct EAN mouse model in C57BL/6 mice;(2)The proportion of macrophages in the spleen was significantly increased in the early stage of EAN disease,in which the proportion of M1 macrophages was significantly increased.With the development of the disease,the proportion of M1 macrophages was gradually reduced,while the proportion of M2 macrophages was gradually increased;(3)The expression of pro-inflammatory cytokines such as TNF,IL-1 and IL-17 A was significantly increased in the early stage of EAN onset,reaching the highest peak,while the concentrations of anti-inflammatory cytokines such as IL-4 and IL-10 gradually increased with the development of the disease;(4)The expressions of IL-1,TNF and IL-17 A were significantly increased in GBS patients,and the expression level of TNF was correlated with the severity of GBS.Immunoglobulin treatment can reduce the expression of proinflammatory cytokines,increase the expression of IL-4,and has little effect on IL-10.Conclusion: M1 macrophages play a pro-inflammatory role in the early stage of EAN onset by secreting pro-inflammatory cytokines,and M2 macrophages play an anti-inflammatory role in the peak period by inhibiting the expression of pro-inflammatory cytokines.Part 2 The therapeutic effect of M2 macrophage transfusion on EAN and its mechanismObjective: Investigate the therapeutic effect of M2 macrophage transfusion on EAN and its possible mechanism.Methods:(1)The induction of M2 macrophages in vitro;(2)Observe the therapeutic effect of M2 macrophage transfusion in EAN;(3)Detect the ratio of spleen macrophages in the peak period of EAN;(4)Detect the expression level of cytokines at the peak;(5)Detect the expression level of NF-?B protein.Results:(1)IL-4 and M-CSF could induce bone marrow stem cells into mature M2 macrophages;(2)M2 macrophage transfusion can alleviate the clinical symptoms of EAN;(3)M2 macrophage transfusion reduce the proportion of M1 cells in the peak period;(4)reduce the expression of pro-inflammatory cytokines;(5)inhibit the phosphorylation of NF-Bp65.Conclusion: M2 macrophages may relieve clinical symptoms of EAN by inhibiting the NF-B signaling pathway and reducing the ratio of M1/M2 and the expression of pro-inflammatory cytokines.Part 3 The effect of NF-? B inhibitor on EAN and its mechanismObjective: Explore the preventive and therapeutic effect of NF-?B inhibitor on EAN and its possible mechanismMethods:(1)Observe the effect of intraperitoneal injection of NF-?B inhibitor on EAN;(2)Detect the expression level of NF-?B protein;(3)Detect the proportion of spleen macrophages;(4)Observe effect of NF-?B inhibitor on the proliferation of M1/M2 macrophages;(5)Detect the expression of cytokines.Results:(1)The NF-?B inhibitor could alleviate the clinical symptoms of EAN and shorten the duration of symptoms.(2)NF-?B inhibitor can inhibit the phosphorylation of NF-?Bp65.(3)NF-?B inhibitor reduced the proportion of M1 macrophages in spleen.(4)NF-?B inhibitor can inhibit M1 macrophages in vitro.(5)NF-?B inhibitor inhibit the expression of pro-inflammatory cytokines.Conclusion: NF-?B inhibitor can relieve symptoms of EAN by inhibiting the proportion of M1 macrophages and the expression of pro-inflammatory cytokines.