Jiang Wei, Zhang Xian, Zhou Ai-Ling
Science Technology and Industry Department, Nantong University, Nantong 226001, China.
Department of Infectious Disease, Affiliated Hospital of Nantong University, Nantong 226001, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2016 Jun 8;32(6):571-576. doi: 10.13459/j.cnki.cjap.2016.06.019.
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).
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 immunofluorescence. In the experiment 2, the hippocampal neurons were divided into 6 groups:normal control group, LPS group, TLR4 antibody +LPS group, SB202190(inhibitor P38)+LPS group, SP600125(inhibitor JNK)+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 Active-caspase-3 were detected by Western blot. The hippocampal neuronal apoptosis rate were tested with flow cytometry.
The expressions of P-P38 and P-JNK of hippocampal neurons in LPS group were higher than those in normal control group (<0.01). Compared with LPS group, the expressions of P-P38 and P-JNK were decreased significantly in TLR4 antibody +LPS group (<0.01). Compared with the normal control group, the expressions of Bcl-2/Bax were decreased, while the expression of Active-caspase-3 was increased in the hippocampal neurons after LPS stimulation (<0.01). The apoptotic rate of hippocampal neurons was higher in LPS group than that in the control group (<0.01). Compared with LPS group, the expressions of Bcl-2/Bax were increasd and the expression of Active-caspase-3 was decreased in TLR4 antibody+LPS group, SB202190+LPS group, and SP600125+LPS group. The apoptotic rate of hippocampal neurons was significantly lower than that in the LPS group (<0.05, <0.01). The cell apoptosis rate had no significant differences between PD98059+LPS group and LPS group.
①TLR4-mediated P38/JNK signaling pathway exists in hippocampal neurons. ②After TLR4 activation in hippocampal neurons, the expressions of P-P38 and P-JNK are up-regulated, the expressions of Bcl-2/Bax are decreased and the expression of Active-caspase-3 is increased, which promote apoptosis of hippocampal neurons. TLR4/P38/JNK signaling pathway is involved in apoptosis of hippocampal neurons.
探讨Toll样受体4（TLR4）-P38-JNK信号通路在大鼠海马神经元凋亡中的作用及其机制，为神经退行性疾病（ND）的发病机制研究、防治提供新的实验依据。
取新生大鼠海马神经元进行体外培养7 d，采用免疫荧光法鉴定海马神经元纯度。为激活或阻断TLR4的作用，用TLR4配体脂多糖（LPS）或TLR4抗体预处理海马神经元。实验1：将海马神经元分为正常对照组、LPS组和TLR4抗体+LPS组，采用免疫荧光法检测P-P38和P-JNK的表达。实验2：将海马神经元分为6组：正常对照组、LPS组、TLR4抗体+LPS组、SB202190（P38抑制剂）+LPS组、SP600125（JNK抑制剂）+LPS组、PD98059（ERK抑制剂）+LPS组。上述各组细胞分别用TLR4抗体、P38、JNK或ERK抑制剂预处理2 h，然后6组均用LPS刺激24 h。采用蛋白质免疫印迹法检测Bcl-2、Bax和活化型半胱天冬酶-3的表达，用流式细胞术检测海马神经元凋亡率。
LPS组海马神经元P-P38和P-JNK的表达高于正常对照组（<0.01）。与LPS组比较，TLR4抗体+LPS组P-P38和P-JNK的表达明显降低（<0.01）。LPS刺激后，海马神经元Bcl-2/Bax的表达较正常对照组降低，活化型半胱天冬酶-3的表达升高（<0.01）。LPS组海马神经元凋亡率高于对照组（<0.01）。与LPS组比较，TLR4抗体+LPS组、SB202190+LPS组和SP600125+LPS组Bcl-2/Bax的表达升高，活化型半胱天冬酶-3的表达降低，海马神经元凋亡率明显低于LPS组（<0.05，<0.01）。PD98059+LPS组与LPS组细胞凋亡率差异无统计学意义。
①海马神经元中存在TLR4介导的P38/JNK信号通路。②海马神经元TLR4激活后，P-P38和P-JNK表达上调，Bcl-2/Bax表达降低，活化型半胱天冬酶-3表达增加，促进海马神经元凋亡，TLR4/P38/JNK信号通路参与海马神经元凋亡。
