Active Induction Of Anti-NMDA Receptor Encephalitis In Mice And The Pharmacological Study Of Chinese Medicine In Treating Anti-NMDA Receptor Encephalitis

ObjectiveAnti-N-methyl-D-aspartate receptor(NMDA)receptor encephalitis is a central nervous system autoimmune disease that primarily affects children and young women.The clinical symptoms of anti-NMDA receptor encephalitis include progressive psychosis and symptoms,behavioral and cognitive impairment,memory deficits,speech disorders,seizures,dyskinesia,autonomic symptoms,and ataxia.The treatments for anti-NMDA receptor encephalitis is limited in clinical practices.The pathogenesis and immune mechanisms against NMDA receptor encephalitis remains unclear,mainly due to the lack of effective animal models.In this study,clinical samples and data of patients with anti-NMDA receptor encephalitis were collected,and relevant immune indicators were analyzed to explore the immune mechanism of anti-NMDA receptor encephalitis.We also aimed to establish an anti-NMDA receptor encephalitis mouse model by active induction.The study screens the compounds that inhibit the anti-NMDA receptor encephalitis antibody in Chinese medicine by simulation screening and on the cell model.It may provide research basis for studying the treatment of Chinese medicine against anti-NMDA receptor encephalitis.Methods(1)A total of 114 subjects including 46 patients with anti-NMDA receptor encephalitis were recruited.Blood,cerebrospinal fluid(CSF)samples were collected and relevant clinical data and follow-up information were collected.Luminex microarray and ELISA were used to detect the levels of cytokines,chemokines and other related inflammatory factors in serum and CSF,their correlation with disease severity were also investigated.(2)Designing peptides based on GluN1 protein,and performing screening in mice to test the antigenic properties.Cell transfection,immunofluorescence,site-directed mutagenesis,flow cytometry,Western blotting,patch clamp and other techniques were used to study the immune characteristics and functions of the active induced antibodies.The pathogenicity of active immune-inducing antibodies in mice was analyzed(3)Based on EphB2 protein,a compound simulating anti-NMDA receptor encephalitis antibody in Chinese medicine was screened and analyzed by immunocytochemistry on the primary cultured cells in vitro.Results(1)Expression of immune factors and their correlation with diseases severity in patients with anti-NMDA receptor encephalitisCompared with the control group,the levels of IL-6,IL-23,IL-1r??,IFN-?,April,BAFF,MIP-1?in serum and CSF samples of patients with anti-NMDA receptor encephalitis were significantly increased.The T cell-associated cell injury factor Fas/FasL is also elevated in patients with anti-NMDA receptor encephalitis and is positively correlated with disease severity.Adhesion molecules such as ICAM-1,VCAM-1,and L-selectin are closely related to anti-NMDA receptor encephalitis diseases and have potential diagnostic value.(2)Active induction of anti-NMDA receptor encephalitis in miceThrough design and screening,it was found that LE30 peptide can induce and maintain high expression of specific antibodies against GluN1 in mice.The site-directed mutagenesis test confirmed the property of mice CSF antibodies is like the antibody in the CSF patients.mice CSF antibodies mediated the internalization of NMDA receptor from the surface of neurons,thereby inhibiting NMDA receptor-mediated Ca2+ influx,immunity-induced antibodies have certain pathogenicity.(3)Screen the active ingredient of Chinese medicine for inhibiting the anti-NMDA receptor encephalitis antibody based on EphB2 proteinThe molecular library of 67 traditional Chinese medicine compounds was established.The molecule docking of traditional Chinese medicine compounds and target proteins was performed,and then physical and chemical analysis of selected drugs was conducted.A total of 12 compounds were screened,which have good bioavailability.The inhibitory effect of the compounds in HT-22 cells was examined.It was found that the compounds perlolyrine and asarinin could inhibite the binding of antibodies,suggesting that these two compounds may intervene of antibody-mediated neuronal damage.ConclusionLevels of Th1 and B cell-related factors were higher in the serum and CSF samples in patients with anti-NMDA receptor encephalitis.Levels of Fas/FasL and adhesion molecules such as ICAM-1,VCAM-1,and L-selectin are correlated to the severity of anti-NMDA receptor encephalitis and may be involved in the regulation of inflammation against NMDA receptor encephalitis.Through active immunization,a functional anti-GluN1 antibody can be induced in mice and has a certain pathogenic effect in mice.The compounds perlolyrine and asarinin have a certain inhibitory effect on the binding of antibodies,which may help to intervene in antibody-mediated neuronal damage.