拉喹莫德改善神经炎症中突触可塑性的作用研究

Effect of Laquimod on Synaptic plasticity in Neuroinflammation

目的使用脂多糖(Lipopolysaccharide,LPS)诱导建立神经炎症模型研究拉喹莫德(Laquinimod,LAQ)对突触结构可塑性的改善作用。方法培养原代海马神经元、小胶质细胞株BV-2细胞,且分为对照[磷酸缓冲盐溶液(Phosphate buffer saline,PBS)]组(常规培养)、LPS组(低糖+5μg/mL LPS刺激4 h、复糖2 h)、LAQ组(预给药100 nM/mL LAQ 2 h、低糖+5μg/mL LPS刺激4 h、复糖+100 nM/mL LAQ 2 h);免疫荧光检测神经生长相关蛋白-43(Growth-associated protein,GAP-43)、微管相关蛋白-2(Microtubule associated protein 2,MAP-2)、突触素(Synaptophysin,SYN)、突触后密度蛋白-95(Postsynaptic density protein 95,PSD-95)、白细胞介素10(Interleukin-10,IL-10)、肿瘤坏死因子α(Tumor necrosis factor alpha,TNF-α)蛋白分布;Western blot检测蛋白激酶(Protein kinase B,Akt)、磷酸化蛋白激酶(Phosphorylated protein kinase B,p-Akt)、核因子κB(Nuclear factor kappa-B,NF-κB)、磷酸化核因子κB(Phosphorylated nuclear factor kappa-B,p-NF-κB)、核因子ΚB抑制因子Α(Recombinant nuclear factor kb inhibitor A,IκBα)、磷酸化核因子ΚB抑制因子Α(Phosphorylated recombinant nuclear factor KB inhibitor A,p-IκBα)、β-肌动蛋白(Actin beta,β-actin)蛋白表达水平。结果与PBS组比较,LPS组BV-2细胞TNF-α表达水平增高、IL-10表达水平降低,细胞胞体增大,轴突缩回;Akt,NF-κB,IκBα蛋白磷酸化水平升高;SYN与PSD-95蛋白表达减少、分布不连贯,树突棘数量减少,神经元间连接减少;LAQ组BV-2细胞TNF-α表达水平降低、IL-10表达水平增高,轴突延伸,胞体体积变回正常,且降低了Akt,NF-κB,IκBα的磷酸化水平;神经元SYN,PSD-95蛋白表达水平增高且分布于各神经元突触连接处,树突棘数量增多,神经元间连接增多。结论LAQ潜在机制是通过抑制Akt-NF-κB通路来减轻神经炎症、抑制突触蛋白的丢失,保护了树突棘使得突触连接增多,对突触结构可塑性起到积极作用。

Objective The neuroinflammation indu ced by Lipopolysaccharide(LPS)was used to investigate the role of laquinimod(LAQ)on the plasticity of synaptic structure.Methods The primary hippocampal neurons and Microglia cell line(BV-2 cells)were cultured,Divided into control(PBS)group(conventional culture),LPS group(low sugar+5μg/mL LPS stimulation for 4 hours,normal sugar for 2 hours),LAQ group(pre-administration 100 nM/mL LAQ for 2 hours,normal sugar+5μg/mL LPS stimulation for 4 hours,normal sugar+100 nM/mL LAQ for 2 hours).Immunofluorescence detection of GAP-43、MAP-2、Synaptophysin(SYN)、PSD-95、IL-10、TNF-αprotein distribution.Western blot detection of Akt、p-Akt、NF-κB、p-NF-κB、IκBα、p-IκBα、β-actin protein expression.Results Compared with the PBS group,the LPS group showed an increase of TNF-αand decrease of IL-10 in the BV-2 cell,cell body enlargement and axonal retraction;The level of p-Akt、p-NF-κB、p-IκBαprotein increased;Synaptophysin(SYN)and PSD-95 protein expression reduced and inconsistent distribution,neuron number of dendritic spines reduced,resulting in reduced intercellular connections.LAQ group showeddecrease d TNF-αand increased IL-10 in the BV-2 cell,axon extension,cell volume returned to normal,the level of p-Akt、p-NF-κB、p-IκBαprotein decreased;Synaptophysin(SYN)and PSD-95 protein increased and distributed at synaptic connections of various neurons,the number of dendritic spines also increased.Conclusion LAQ plays a positive role in synaptic structural plasticity by inhibiting the Akt-NF-κB pathway,which reduces neuroinflammation,inhibits the loss of synaptic proteins and protects synaptic connections.