Anti GM1 IgG Antibody Inhibits Axon-like Growth Of AMAN Cell Model By Activating AMPK

Objective: Guillain-Barré syndrome(GBS)is a group of autoimmune-mediated peripheral neuropathy,characterized by acute symmetrical paralysis of the limbs and effective immunotherapy,with electrophysiological manifestations are demyelination and/or axonal injury of multiple peripheral nerves and nerve root.Acute motor axonal neuropathy(AMAN)is one of the common subtypes of GBS,and the main pathological changes were extensive peripheral axonal lesions.Current studies suggest that pathogens induce the production of human autoimmune antibodies through molecular simulation mechanism,leading to the pathogenesis of AMAN.However,its exact pathogenesis is still unclear.Energy metabolism plays an important role in the process of cells repair.Recent studies have shown that energy metabolism plays a key role in the process of axonal growth or regeneration,which provides new ideas for the treatment of AMAN.Adenosine monophosphate-activated protein kinase(AMPK)is a cell energy monitor that regulates cell energy balance through phosphorylation.In this study,the AMAN cell model was established and compared the expression of phospho-adenosine monophosphate-activated protein kinase(p-AMPK)/AMPK with the cells in control group,investigating the effect of anti-GM1 Ig G antibody on the expression of p-AMPK/AMPK,and exploring the role of energy metabolism in the pathogenesis of AMAN by analyzing the changes of energy metabolites in AMAN cell model with metabonomics,so as to provide theoretical basis for the development of new therapeutic schemes of AMAN.Methods: Undifferentiated PC-12 cells were selected as the cell source of the model,and there were found to be induced by nerve growth factor(NGF)to differentiate into neuron-like cells;Cell morphology and GM1 expression on cell membrane at the different time points were analyzed by immunofluorescence,and the optimal time point of cell differentiation was selected(48h);Purification of anti GM1 Ig G antibodies in serum of AMAN patients by Protein G chromatography column;Basing on the optimal differentiation time point of PC-12 cells,the anti-GM1 Ig G antibody was added into the differentiated neuron-like cells at the different time points,and the AMAN cell model was established;Normal group and control Ig G treatment group was established at the same time.The expression of p-AMPK/AMPK at the different time points in the normal group,AMAN model group and control Ig G treatment group were compared by western blot,selecting the different time point(24h).Cell immunofluorescence was used to compare the pseudopod length of three groups of cells at 24 h,and the expression of metabolites was compared among the normal group,AMAN model group,control Ig G treatment group and AMPK activation inhibition group by targeted energy metabolite analysis.The length of pseudopod and the gray value of western blot were measured by Image J software.The expression of p-AMPK/AMPK and pseudopod length of the normal group,AMAN model group and control Ig G treatment group were compared by t test;The targeted qualitative analysis of energy metabolism was based on the self-established data base of target standard,and quantitative analysis was multi-reaction monitoring by triple quadrupole mass spectrometry;Analysis was performed with Graph Pad Prism 6.0 software,P value < 0.05 was considered to be significant.Results: In this study,PC-12 cells had neuronal morphogenetic response to NGF,and the increasing fucosyl-GM1 expression in PC-12 cells induced by NGF for 48 hours,and the content was about twice that of undifferentiated cells(P < 0.001).This study found that the expression of p-AMPK/AMPK in 24 h AMAN model was significantly increased than the normal group and control Ig G treatment group(P < 0.001).In addition,the pseudopod length of 24 h AMAN cell model was significantly shorter than that of the normal group and control Ig G treatment group(P < 0.001).Compared with the normal group,there were 13 kinds of metabolites changed in 24 h AMAN model group,but only one kind of metabolite changed in 24 h control Ig G treatment group and there were 24 kinds of metabolites changed in 24 h AMPK activation inhibition group.Compared with the control Ig G treatment group,9 kinds of metabolites were changed in AMAN model group.Conclusion: In AMAN cell model,the anti-GM1 Ig G antibody could promote p-AMPK/AMPK expression and inhibit ATP consumption or nutrient synthesis,so as to inhibit axon-like growth of AMAN cell model.The results of this study need to be further verified by animal experiments.