TLR_4/P38/JNK信号通路在海马神经元凋亡中的作用  

The role of TLR_4/P38/JNK signaling pathway in apoptosis of hippocampal neurons

作　　者：蒋伟[1] 张弦[2] 周爱玲[3] 

机构地区：[1]南通大学科技与产业处 [2]南通大学附属医院感染性疾病科 [3]南通大学医学院病理生理学系,江苏南通226001

出　　处：《中国应用生理学杂志》2016年第6期571-576,共6页Chinese Journal of Applied Physiology

基　　金：南通市应用研究科技计划项目(BK2013007)资助

摘　　要：目的:探讨Toll样受体4(TLR4)/P38/JNK信号通路在海马神经元凋亡中的作用及其机制,为神经退行性疾病(ND)的发病机制与防治研究提供新的实验依据。方法:采用体外培养7 d的新生大鼠海马神经元,免疫荧光双标法鉴定海马神经元纯度。用TLR4配体脂多糖(LPS)或TLR4抗体预处理海马神经元,以激活或阻断TLR4的作用。实验1设正常对照组、LPS组及TLR4抗体+LPS组;免疫荧光法检测P-P38,P-JNK的表达。实验2分为6组:正常对照组,LPS组,TLR4抗体+LPS组,SB202190(抑制P38)+LPS组,SP600125(抑制JNK)+LPS组,PD98059(抑制ERK)+LPS组;分别用TLR4抗体、P38、JNK及ERK的抑制剂预处理海马神经元后再给以LPS刺激24 h,Western blot法检测Bcl-2,Bax,Active-caspase-3的表达变化;流式细胞术检测海马神经元凋亡率。结果:LPS组海马神经元P-P38、P-JNK的表达明显高于正常对照组(P<0.01),TLR4抗体+LPS组P-P38,P-JNK表达显著低于LPS组(P<0.01)。与正常对照组相比,LPS组海马神经元Bcl-2/Bax表达减少、Active-caspase-3表达增加,海马神经元凋亡率增加(P<0.01)。而TLR4抗体+LPS组、SB202190+LPS组、SP600125+LPS组Bcl-2/Bax显著高于LPS组、Active caspase-3显著低于LPS组(P<0.01),海马神经元凋亡率显著低于LPS组(P<0.05,P<0.01)。PD98059+LPS组与LPS组海马神经元凋亡率无明显差异。结论:1海马神经元中有TLR4介导的P38/JNK信号通路。2海马神经元TLR4激活后,P-P38、P-JNK表达增加,使Bcl-2/Bax的比例降低和Active-caspase-3表达增加,从而促进海马神经元的凋亡。海马神经元凋亡过程中有TLR4介导的P38/JNK信号通路的参与。Objective： To investigate the effects of the Toll like receptor 4 （TLR4） -P38-JNK signaling pathway in the apoptosis of hippocampal neurons in rats and its mechanisms. And to provide new experimental evidences for the pathogenesis research, prevention and treatment of neurodegenerative diseases （ND）. Methods： The hippocampal neurons derived from newborn rat were cultured for 7 d in vitro. The purity of hippocampal neurons was identified by immunofluorescence method. In order to activate or block the action of TLR4, the hippocampal neurons were pretreated with TLR4 ligand lipopolysaccharide （LPS） or TLR4 antibody. In the experiment 1, the hippocampal neurons were divided into normal control group, LPS group and TLR4 antibody ＋ LPS group. The expressions of P-P38 and P-JNK were deteced by immunoflu- orescence. In the experiment 2, the hippocmnpal neurons were divided into 6 groups： normal control group, LPS group, TLR4 antibody ＋ group, SB202190（inhibitor P38） ＋ LPS group, SP600125（inhibitor iNK） ＋ LPS group, PD98059（inhibitor ERK） ＋ LPS group. The cells in above mentioned groups were pretreated with TLR4 antibody, the inhibitors of P38, JNK or ERK for 2 h respectively. Then, all the six groups were stimulated by LPS for 24 h. The expressions of Bcl-2, Bax and Aetive-caspase-3 were detected by Western blot. The hippocampal neuronal apoptosis rate were tested with flow cytometry. Results： The expressions of P-P38 and P-JNK of hippocampal neurons in LPS group were higher than those in normal control group （ P 〈 0.01 ）. Compared with LPS group, the expressions of P-P38 and P-JNK were decreased signifi- cantly in TLR4 antibody ＋ LPS group （ P 〈 0.01）. Compared with the normal control group, the expressions of Bel-2/Bax were decreased, while the expression of Aetive-caspase-3 was increased in the hippocampal neurons after LPS stimulation （ P 〈 0.01 ）. The apoptotic rate of hippocampal neurons was higher in LPS group than that in the control group （ P 〈 0.

关 键 词：海马神经元 TLR4/P38/JNK信号通路 BCL-2/BAX 凋亡蛋白酶-3 细胞凋亡 脂多糖 

分 类 号：R3[医药卫生—基础医学]