| Objective: Epilepsy is a clinical syndrome of highly synchronous abnormal discharge of brain neurons caused by a variety of reasons,and the occurrence of drug-resistant epilepsy further increases the social and medical burden.This study intends to conduct proteomics research on serum exosomes of drug-resistant epilepsy,and explore the pathogenesis of key differential proteins involved in drug-resistant epilepsy through animal experiments while looking for potential biomarkers of drug-resistant epilepsy,so as to explore a new direction of diagnosis and treatment for drug-resistant epilepsy.Methods:1.10 patients with drug-resistant epilepsy,10 patients with seizure free and10 age-gender matched healthy controls were included in the study.Proteomics study was conducted on serum exosomes in each group,and bioinformatics analysis was performed on GO and KEGG.2.Through bioinformatics enrichment analysis,ITGAX was selected to study the possible mechanism of epilepsy in animal experiments,and western blotting was used to detect the expression of ITGAX in serum exosomes and hippocampus of normal group and epileptic group.3.The epilepsy model was treated with tail vein injection of ITGAX adeno-associated knockout virus.And it was divided into the normal control group(Control),the epilepsy group(Pilo),the pilcarpine + ITGAX Knock group(Pilo+ITGAX-),the pilcarpine + Vehicle group(Pilo+Vehicle).Western blotting was used to detect the expression of ITGAX in the hippocampus of each group.The latency of epileptic seizure was observed.The changes of blood-brain barrier(BBB)in each group were detected: albumin expression in hippocampus was detected by western blot and serum S100β level was detected by ELISA.4.The epileptic model was treated with intraperitoneal injection of exosome inhibitor,and divided into normal Control group(Control),pilcarpine group(Pilo),pilcarpine +GW4869 exosome inhibitor group(Pilo+GW4869),pilcarpine + solvent Control group(Pilo+DMSO).The changes of ITGAX in hippocampus were detected by western blotting,the latency of epileptic seizure was evaluated by behavioral science.The changes of BBB in each group were detected: albumin expression in hippocampus was detected by western blot and serum S100β level was detected by ELISA.Results: 1.Proteomic analysis showed that 66 proteins were significantly up-regulated and 63 proteins were significantly down-regulated in the drug-resistant epilepsy group compared with the healthy control group.In the drug-resistant epilepsy group,289 proteins were significantly up-regulated and 301 proteins were significantly down-regulated,compared with the group of seizure free.Venn diagram analysis revealed 43 significantly changed proteins in the drug-resistant epilepsy group compared with the normal group and the clinically effective epilepsy group.Bioinformatics showed that these 43 proteins were mainly enriched in immune response,cell adhesion molecule binding and amino acid metabolism,and immune response played a dominant role.The differential protein ITGAX was the most significant protein in immune response and cell adhesion.2.Expression of ITGAX in serum exosomes of normal and epileptic models:compared with the normal group,the protein level of ITGAX in serum exosomes of epileptic group was up-regulated,P<0.05.3.Expression of ITGAX in the hippocampus of normal and epileptic models:compared with the normal group,the protein level of ITGAX in the hippocampus of epileptic group was up-regulated,P<0.05.4.After tail vein injection of ITGAX adeno-associated knockout virus,in pilcarpine+ITGAX knockdown group(Pilo+ITGAX-),ITGAX expression in the hippocampus was down-regulated,the latency of seizures was significantly prolonged,albumin expression in the hippocampus was decreased,and serum S100β level was decreased,compared with the group of pilcarpine+Vehicle(Pilo+Vehicle),P<0.05.5.After intraperitoneal injection of exosome inhibitor,in pilcarpine +GW4869exosome inhibitor group(Pilo+GW4869),ITGAX expression in the hippocampus was down-regulated,the latency of seizures was significantly prolonged,albumin expression in the hippocampus was decreased,and serum S100β level was decreased,compared with the group of pilcarpine + solvent Control group(Pilo+DMSO),P<0.05.Conclusion: 1.Compared with the healthy control group and the group with seizure free,the differential proteins of serum exosomes in the drug-resistant epilepsy group were mainly enriched in immune response,cell adhesion molecule binding and amino acid metabolism.2.ITGAX was highly expressed in the hippocampus from peripheral blood to central nervous system mediated by exosomes,and its involvement in epileptic seizures may be related to changes in the blood-brain barrier permeability. |
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