NLRP3对自身免疫性脑炎的影响及其作用机制  

Effect of NLRP3 on autoimmune encephalitis and its mechanism

作　　者：王莉[1] 于雪源 闾佳佳 陈飞[1] WANG Li;YU Xueyuan;L Jiajia;CHEN Fei(Department of Clinical Laboratory,Affiliated Nanjing Brain Hospital,Nanjing Medical University,Nanjing 210029,Jiangsu,China;Hospital-Acquired Infection Control Department,Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China;Department of Pediatrics,Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)

机构地区：[1]南京医科大学附属脑科医院检验科,南京210029 [2]上海交通大学医学院附属新华医院感染管理科,上海200092 [3]上海交通大学医学院附属瑞金医院儿科,上海200025

出　　处：《医学研究与战创伤救治》2023年第2期171-177,共7页Journal of Medical Research & Combat Trauma Care

基　　金：南京医科大学科技发展基金(NMUB2019103)。

摘　　要：目的NLRP3在自身免疫性脑炎中的作用尚不明确。文中旨在探讨炎症小体NLRP3对自身免疫性脑炎的影响及其机制研究。方法选取在2019年6月至2020年12月南京医科大学附属脑科医院确诊为抗NMDAR脑炎的患者(n=5)作为抗NMDAR组,选择非炎症性神经系统疾病的6例患者作为对照组。ELISA法检测对照组和抗NMDAR组脑炎患者脑脊液中NLRP3和IL-1β变化以及趋化因子CCL2的变化。利用MOG35-55皮下给药诱导C57BL/6J雌性小鼠发生实验性自身免疫性脑炎(EAE),建立EAE模型。实验动物分为3组:MOG组予MOG35-5550μg(溶于100μL完全弗氏佐剂),MOG+MCC950组予MOG35-5550μg(溶于100μL完全弗氏佐剂)及MCC95050 mg/kg(溶于100μL玉米油),CON组给予玉米油(100μL)。小鼠脑组织进行CD4及NLRP3免疫组化,Western blot检测脑组织髓磷脂碱性蛋白(MBP)表达,ELISA检测IL-1β和IL-17A水平,流式细胞术检测小鼠外周血Th17比例。结果与对照组比较,抗NMDAR组脑脊液中IL-1β含量显著增高[(5.93±1.26)pg/mL vs(16.31±6.28)pg/mL,P<0.01],NLRP3含量显著上升[(0.16±0.06)ng/mL vs(0.43±0.24)ng/mL,P<0.05]。MOG组小鼠脑组织中NLRP3+细胞较CON组小鼠明显增多[(21.00±4.85)/HP vs(6.80±1.92)/HP,P<0.001];同时CD4+T细胞亦显著增多[(73.40±11.70)/HP vs(15.60±6.02)/HP,P<0.001]。与CON组比较,MOG组小鼠脑组织中IL-1β显著上升(P<0.001),MBP表达显著降低(P<0.01),而利用MCC950抑制NLRP3后,MOG+MCC950组MBP表达显著增加(P<0.05),IL-1β显著降低(P<0.05)。与CON组比较,MOG组小鼠脑组织中IL-17A含量显著增加(P<0.001),而MOG+MCC950组小鼠脑组织中IL-17A含量显著降低(P<0.001)。与CON组比较,MOG组小鼠外周血Th17细胞比例显著升高(P<0.01),MOG+MCC950组外周血Th17细胞比例较MOG组显著降低(P<0.01)。结论自身免疫性脑炎时,脑组织NLRP3表达增加,激活Th17免疫反应,介导神经系统脱髓鞘和自身免疫性脑炎的发生发展,而抑制NLRP3可抑制Th17免疫反应,抑制脱髓鞘Objective The role of NLRP3 in autoimmune encephalitis(AE)is unclear.This study aims to explore the effect of NLRP3 activation in AE and associated mechanisms.Methods Patients diagnosed with anti-NMDAR encephalitis in the Affiliated Nanjing Brain Hospital,Nanjing Medical University,from June 2019 to December 2020(n=5)were selected as the anti-NMDAR group,and 6 patients with non-inflammatory nervous system diseases were selected as the control group.ELISA was used to determine NLRP3,IL-1βand CCL2 in cerebro spinal fluid(CSF)of control subjects and patients with anti-NMDAR encephalitis.MOG35-55 was subcutaneously injected to induce experimental autoimmune encephalitis(EAE)in female C57BL/6J mice to establish EAE model.Mice were divided into 3 groups:MOG with 50μg MOG35-55(dissolved in 100μL complete Freund's adjuvant,CFA),MOG+MCC950 with 50μg MOG35-55(dissolved in 100μL CFA)and 50 mg/kg MCC950(dissolved in 100μL corn oil),and control group(CON)with 100μL corn oil.CD4 and NLRP3 immunohistochemistry were performed in mouse brain tissues.The expression of myelin basic protein(MBP)in brain tissue was detected by Western blot;the levels of IL-1βand IL-17A by ELISA;the proportion of Th17 in peripheral blood by flow cytometry.Results Compared with the control group,the content of IL-1βin CSF of anti-NmdAR group was significantly increased[(5.93±1.26)pg/mL vs(16.31±6.28)pg/mL,P<0.01];the content of NLRP3 was significantly increased[(0.16±0.06)ng/mL vs(0.43±0.24)ng/mL,P<0.05].There were more NLRP3+cells in MOG group than those in CON group[(21.00±4.85)/HP vs(6.80±1.92)/HP,P<0.001];CD4+T cells also increased significantly[(73.40±11.70)/HP vs(15.60±6.02)/HP,P<0.001].Compared with CON group,IL-1βwas significantly increased(P<0.001)and MBP expression was significantly decreased(P<0.01)in MOG group,while MBP expression was significantly increased(P<0.05)and IL-1βwas significantly decreased(P<0.05)in MOG+MCC950 group after MCC950 was used to inhibit NLRP3.Compared with CON group,the content of IL-17A in the bra

关 键 词：实验性自身免疫性脑炎 NLRP3 MCC950 CD4+T细胞 IL-1Β IL-17A CCL2 

分 类 号：R742[医药卫生—神经病学与精神病学]