铅致神经损伤AOP中NMDAR对BDNF的影响及在突触后信息传递中的效应  

Effect of NMDAR on BDNF and postsynaptic information transmission in lead⁃induced nerve injury AOP

作　　者：李雨露 杨海涛 王海华[1] 鱼涛[1] 陈宵[1] 刘黎[1] 张意[1] 段爱茹 乔梦芸 吴思雨 吕书俊 肖经纬[1] 李斌[1] LI Yu-lu;YANG Hai-tao;WANG Hai-hua;YU Tao;CHEN Xiao;LIU Li;ZHANG Yi;DUAN Ai-ru;QIAO Meng-yun;WU Si-yu;LV Shu-jun;XIAO Jing-wei;LI Bin(National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevetion,Key Laboratory of Chemical Pollution and Heath Safety,Chinese Center for Disease Control and Prevetion,Beijing 100050,China)

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,中国疾病预防控制中心化学污染与健康安全重点实验室,北京100050

出　　处：《毒理学杂志》2023年第4期323-331,共9页Journal of Toxicology

基　　金：职业健康风险评估与国家职业卫生标准制定项目(131031109000160004);国家自然科学基金面上项目(81773474)。

摘　　要：目的探讨铅暴露条件下,N-甲基-D-天冬氨酸受体(N⁃methyl⁃D⁃aspartate receptor,NMDAR)对脑源性神经营养因子(brain⁃derived neurotrophic factor,BDNF)的影响及其在突触后信息传递异常中的作用。方法以不同浓度梯度(0、12.5、50、100、200、300、400和500μmol/L)的铅溶液分别处理SH⁃SY5Y细胞,CCK⁃8法检测细胞的增殖活性并确定最终染毒浓度与时间。采用Real⁃time PCR法和Western blot法检测铅暴露48 h后BDNF、TrkB、p75NTR mRNA和蛋白相对表达情况。在不同浓度铅溶液中以D⁃AP5抑制NMDAR的表达,Western blot检测NMDAR亚基(NR1、NR2A和NR2B)、BDNF、TrkB、PI3K、ERK和PKC蛋白的相对表达情况。结果根据CCK⁃8结果选择铅染毒梯度为0、50、100和200μmol/L;染毒时间为48 h;随着铅染毒浓度的增加,与对照组相比,BDNF和TrkB mRNA逐渐减少,p75NTR mRNA增加(F值分别是7.656和12.402,P<0.05或P<0.01)。随着铅染毒浓度的增加,与对照组相比,BDNF表达呈现先逐渐减少后增加的变化过程,TrkB和p75NTR蛋白表达增加(F值分别是20.325和20.3425,P<0.05或P<0.01),且呈现剂量依赖性。给予D⁃AP5阻断后,与铅染毒组相比,Pb(Ac)2+D⁃AP5组的BDNF蛋白表达下调(t=3.760,P<0.01);TrkB的蛋白表达下调(t=3.231,P<0.05),p75NTR蛋白表达上调(t=4.849,P<0.01);NR2A和NR2B蛋白表达下调(t值分别是4.395和4.430,P<0.01);PI3 Kinase p110 beta蛋白表达下调(t值分别是4.518和10.068,P<0.01)。结论随着铅染毒剂量的增加,使得NMDAR亚基NR1、NR2A和NR2B表达上调,从而激活BDNF的转录和表达,激活PI3K⁃AKt信号通路。Objective To investigate the effect of NMDAR on BDNF and its role in abnormal postsynaptic information transmission under lead exposure.Methods The SH⁃SY5Y cells were treated with different concentrations of lead solution(0,12.5,50,100,200,300,400 and 500μmol/L),and the cell proliferation activity was detected by CCK⁃8 method to determine the final concentration and time of exposure.The mRNA and protein expressions of BDNF,TrkB and p75NTR were detected by Realtime PCR and Western blot after 48 hours of lead exposure.The relative expressions of NMDAR subunits(NR1,NR2A and NR2B),BDNF,TrkB,PI3K,ERK and PKC proteins were detected by Western blot.Results According to the CCK⁃8 result,the lead staining gradients were 0,50,100 and 200μmol/L,and the staining time was 48 h.As the lead staining concentration increased,BDNF and TrkB mRNA were gradually decreased and p75NTR mRNA was increased compared with the control group(F values:7.656,12.402,P<0.05 or P<0.01,respectively).As the concentration of lead staining increased,BDNF expression showed a gradual decrease followed by an increase,and TrkB and p75NTR protein expressions were increased compared with the control group(F values:20.325,20.3425;P<0.05 or P<0.01,respectively)in a dose⁃dependent manner.After administration of D⁃AP5 blockade,BDNF protein expression was down⁃regulated in the Pb(Ac)2+D⁃AP5 group compared with the lead⁃stained group(t value=3.760,P<0.01);TrkB protein expression was down⁃regulated(t value=3.231,P<0.05)and p75NTR protein expression was up⁃regulated(t value=4.849,P<0.01),NR2A and NR2B protein expression was down⁃regulated(t values:4.395,4.430,P<0.01,respectively).PI3 Kinase p110 beta protein expression was down⁃regulated(t values:4.518,10.068,P<0.01,respectively).Conslusion As the dose of lead exposure increased,it made the expression of NMDAR subunits NR1,NR2A and NR2B up⁃regulated,which activated the transcription and expression of BDNF and activated the PI3K⁃AKT signaling pathway.

关 键 词：铅 N-甲基-D-天冬氨酸受体 脑源性神经营养因子 神经损伤 突触后信息传递 

分 类 号：R114[医药卫生—卫生毒理学]