抗NMDAR脑炎小鼠血脑屏障破坏的实验性研究

Study on the destruction of blood-brain barrier in mice with anti-NMDAR encephalitis

目的:探讨抗N-甲基-D-天冬氨酸受体(NMDAR)NR1亚基氨基末端结构域主动免疫诱导NMDAR脑炎对小鼠紧密连接(TJ)跨膜蛋白Occludin、带状闭合蛋白1(ZO-1)和血脑屏障(BBB)完整性的破坏作用。方法:将C57BL/6小鼠随机分为对照组和模型组,每组10只。模型组用NMDAR NR1受体区域的N356/G385氨基酸肽段主动免疫C57BL/6小鼠,建立抗NMDAR脑炎小鼠模型,对照组注射不含肽段的等体积乳剂混合物,主动免疫6周。采用酶联免疫吸附试验(ELISA)检测小鼠血清抗NMDAR IgG抗体,水迷宫和旷场实验评估小鼠记忆和焦虑行为,荧光素钠法测定BBB通透性,实时荧光定量PCR(RTqPCR)法检测大脑皮质NMDAR NR1、Occludin、ZO-1 mRNA表达,western blotting法检测大脑皮质NMDAR NR1、基质金属蛋白酶9(MMP9)、Occludin、ZO-1和神经元特异性核蛋白(NeuN)蛋白表达。结果:与对照组比较,模型组小鼠血清中抗NMDAR IgG抗体为阳性,并出现明显的记忆障碍和焦虑行为(均P<0.05)。模型组小鼠脑荧光素钠渗漏量明显增加(P<0.001),大脑皮质NMDAR NR1、Occludin和ZO-1 mRNA及蛋白表达量降低,MMP9蛋白表达量升高,NeuN蛋白表达量降低(均P<0.05)。结论:利用NMDAR NR1356-385肽段主动免疫成功建立了抗NMDAR脑炎小鼠模型,产生抗NMDAR抗体,表达增加的MMP9可能破坏TJ蛋白从而使BBB功能障碍,导致大脑神经元损伤,并出现精神行为学改变。

Objective:To investigate the destruction of tight junction(TJ)transmembrane protein(Occludin),Zona occludens-1(ZO-1)and blood-brain barrier(BBB)integrity in mice with Anti-N-methyl-D-aspartate receptor(NMDAR)encephalitis immunized actively with the amino-terminal domain of NMDAR NR1 subunit.Methods:C57BL/6 mice were randomly divided into control group and model group,with 10 mice in each group.In the model group,C57BL/6 mice were actively immunized with N356/G385 amino-terminal domain peptide from NMDAR NR1 receptor region to establish a mouse model of anti-NMDAR encephalitis,while mice in the control group were injected with an equal volume of emulsion mixture without peptide and actively immunized for 6 weeks.Enzyme-linked immunosorbent assay(ELISA)was used to detect anti-NMDAR IgG antibody in mice serum.Morris water maze and open field test were used to evaluate the memory and anxiety behavior of the mice.Fluorescein sodium method was used to determine BBB permeability.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to analyze the mRNA expressions of NMDAR NR1,Occludin and ZO-1 in cerebral cortex.Western blotting was used to detecte the protein expressions of NMDAR NR1,matrix metalloproteinase 9(MMP9),Occludin,ZO-1 and neuron-specific nucleoprotein(NeuN)in cerebral cortex.Results:Compared with the control group,the serum anti-NMDAR IgG antibody in the model group was positive,and there were obvious memory impairment and anxiety behavior(all P<0.05).In model group,the brain sodium fluorescein leakage significantly increased(P<0.001),the mRNA and protein expressions of NMDAR NR1,Occludin and ZO-1 in the cerebral cortex decreased,MMP9 protein expression increased,and NeuN protein expression decreased(all P<0.05).Conclusion:The mouse model of anti-NMDAR encephalitis is established successfully by active immunization with the peptide of NMDAR NR1356-385 and anti-NMDAR antibody is generated.The increased expression of MMP9 may destroy TJ protein,leading to BBB dysfunction,which will cause brain neuron damage and psychobehavioral changes.