TLR4/P38/JNK信号通路在海马神经元凋亡中的作用

doi:10.13459/j.cnki.cjap.2016.06.019

摘要/Abstract

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 cas-pase-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。

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

Abstract: 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 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. 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 significantly in TLR4 antibody +LPS group (P<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 (P<0.01). The apoptotic rate of hippocampal neurons was higher in LPS group than that in the control group (P<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 (P<0.05, P<0.01). The cell apoptosis rate had no significant differences between PD98059+LPS group and LPS group. Conclusion: 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.

Key words: hippocampal neurons, TLR4/P38/JNK pathway, Bcl-2/Bax, Active-caspase-3, cell apoptosis, lipopolysaccharides

中图分类号:

蒋伟, 张弦, 周爱玲. TLR₄/P38/JNK信号通路在海马神经元凋亡中的作用[J]. 中国应用生理学杂志, 2016, 32(6): 571-576.

JIANG Wei, ZHANG Xian, ZHOU Ai-Ling. The role of TLR₄/P38/JNK signaling pathway in apoptosis of hippocampal neurons[J]. CJAP, 2016, 32(6): 571-576.

参考文献

[1] Kayla M, Pate, Mc Call Rogers, et al. The Ability of Polyphenols to Reduce Aβ-Induced Apoptosis Associated with Alzheimer's Disease[J]. Biophysical Journal, 2015, 108(2):66a.
[2] McDonald JM, Cairns NJ, Taylor-Reinwald L, et al. The levels of water-soluble and triton-soluble Aβ are increased in Alzheimer's disease brain[J]. Brain Res, 2012, 1450:138-147.
[3] Watson K, Fan GH. Macrophage inflammatory protein 2 in-hibits beta-amyloidpeptide (1-42)-mediated hippocampal neuronal apoptosis through activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways[J]. Molpharmacol, 2005, 67(3):757-765.
[4] Tang SC, Arumugam TV, Xu X, et al. Pivotal role for neu-ronal Toll-like receptors in ischemic brain injury and func-tional deficits[J]. Proc Natl Acad Sci U S A, 2007, 104(34):13798-13803.
[5] Mao J, Hu Y, Zhou A, et al. Oxymatrine reduces neuroin-flammation in rat brain A signaling pathway[J]. Neural Re-gen Res, 2012, 7(30):2333-2339.
[6] Hu Y, Mao J, Zhang Y, et al. Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons[J]. Neu-ral Regen Res, 2013, 8(16):1465-1472.
[7] Walter S, Letiembre M, Liu Y, et al. Role of the Toll-Like Receptor 4 in Neuroinflammation in Alzheimer's Disease[J]. Cell Physiol Biochem, 2007, 20(6):947-956.
[8] Tang SC, Lathia JD, Selvaraj PK, et al. Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid β-peptide and the membrane lipid peroxidation product 4-hydroxynone-nal[J]. Exp Neurol, 2008, 213(1):114-121.
[9] Emerit J, Edeas M, Bricaire F. Neurodegenerative diseases and oxidative stress[J]. Biomed Phamracother, 2004, 58(1):39-46.
[10] 施晓容, 邵巧燕, 刘俊红, 等. 脂多糖联合海人藻酸诱导的热性惊厥导致幼鼠海马神经元凋亡[J]. 福建医科大学学报, 2012, 46(2):104-107.
[11] He Y, Zhou AL, Jiang W. Toll-like receptor 4-mediated sig-naling participates in apoptosis of hippocampal neurons[J]. Neural Regen Res, 2013, 8(29):2744-2753.
[12] Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases[J]. Biochim Biophys Acta, 2010, 1802(4):396-405.
[13] Di Benedetto B, Kallnik M, Weisenhorn DM, et al. Activa-tion of ERK/MAPK in the lateral amygdale of the mouse is required for acquisition of a fear-potentiated startle response[J]. Neuropsychopharmacol, 2009, 34(2):356-366.
[14] Ramiro-Cortés Y, Morán J. Role of oxidative stress and JNK pathway in apoptotic death induced by potassium deprivation and staurosporine in cerebellar granule neurons[J]. Neu-rochem Int, 2009, (5597):581-592.
[15] Reed JC. Double identity for proteins of the Bcl-2 family[J]. Nature, 1997, 38(6635):773-776.

TLR₄/P38/JNK信号通路在海马神经元凋亡中的作用

The role of TLR₄/P38/JNK signaling pathway in apoptosis of hippocampal neurons

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘凯丽, 都新新, 张文琴, 吴亚俐, 王茜, 王春芳, 崔香丽. 过表达miR-31对结肠炎模型小鼠TLR4/NF-κB信号通路及凋亡蛋白的调控[J]. 中国应用生理学杂志, 2020, 36(3): 211-215.
[2]	陈宇峰, 董凡赫, 楼云玮, 寿今豪, 张慧婷, 周一超, 严明, 毛红娇, 张云. 补骨脂素对TCP磨损颗粒所致大鼠成骨细胞损伤的干预作用及其机制[J]. 中国应用生理学杂志, 2020, 36(3): 255-260.
[3]	程鋆, 鲁萌萌, 张阳, 高凌云, 陈超, 蒋慧心, 刘平, 黄秀雯, 刘云路. 二甲双胍对D-半乳糖诱导雄性中年小鼠衰老的干预作用^*[J]. 中国应用生理学杂志, 2019, 35(5): 433-437.
[4]	杲修杰, 王尚, 刘伟丽, 王坤, 陈照立, 王新兴. Sestrin2对热暴露肺上皮细胞凋亡的干预作用及其机制^*[J]. 中国应用生理学杂志, 2019, 35(4): 289-292.
[5]	洪凯, 蒋盼若, 柯瑞君, 应佳豪, 张肖艳, 陈佳玉. 2-12烷基-6-甲氧基环己基-2,5-二烯-1,4-二酮(DMDD)抗弥漫大B淋巴瘤的作用及机制^*[J]. 中国应用生理学杂志, 2019, 35(4): 312-316.
[6]	袁辉, 扬国宏, 李姝, 李丽, 宋高臣, 徐长庆, 孙健. 心肌梗死大鼠钙敏感受体表达与心肌细胞凋亡的变化[J]. 中国应用生理学杂志, 2019, 35(3): 268-272.
[7]	林中民, 陈国荣, 张泉波, 王芳, 项兰婷, 曹琼洁. 髓样分化蛋白2基因沉默对高糖诱导的大鼠心肌细胞增殖抑制、凋亡及炎症反应的影响^*[J]. 中国应用生理学杂志, 2019, 35(3): 273-278.
[8]	王瑜, 柯瑞君, 蒋盼若, 应佳豪, 楼恩哲, 陈佳玉. 杨芽黄素对前列腺癌细胞凋亡的影响及其机制^*[J]. 中国应用生理学杂志, 2019, 35(3): 283-288.
[9]	柳卓, 刘检, 凌佳, 杨琴, 杨蕙, 孟盼, 杜青, 赵洪庆, 王宇红. ELK-1/JNK/c-Fos对左归降糖解郁方(ZGJTJYF)体外培养的模拟糖尿病并发抑郁症(DD)大鼠海马神经元凋亡中的作用[J]. 中国应用生理学杂志, 2019, 35(1): 50-54.
[10]	赵嘉涵, 贾雨涵, 汤雅婷, 林艺鑫, 王艳蕾. 参麦注射液对肠缺血/再灌注肺损伤大鼠肺组织p38MAPK及凋亡相关基因表达的影响[J]. 中国应用生理学杂志, 2019, 35(1): 65-68.
[11]	刘煜鑫, 闫东. 右美托咪定对大鼠海马神经元生长发育的影响及其机制[J]. 中国应用生理学杂志, 2019, 35(1): 69-73.
[12]	黄生辉, 巩婷, 李妍怡. 中风膏对氧糖剥夺/再复氧损伤大鼠海马神经干细胞增殖的影响[J]. 中国应用生理学杂志, 2018, 34(6): 554-557.
[13]	方小霞, 周易, 孙高林, 邱一华, 彭聿平. TGF-β1对Aβ_1-42诱导海马神经元-小胶质细胞共培养体系中细胞因子表达和分泌的影响[J]. 中国应用生理学杂志, 2018, 34(5): 385-388.
[14]	孙伟铭, 张源洲, 温馨, 席雨鑫, 袁迪, 王跃虹, 魏璨, 徐长庆, 李鸿珠. 外源性H₂S恢复缺氧后适应对衰老H9C2细胞的保护作用及机制[J]. 中国应用生理学杂志, 2018, 34(4): 289-293.
[15]	李朝晖, 张颖, 路艳, 陆庆明, 谢晓华. 大鼠双下肢骨折合并失血对心肌细胞损伤的作用及其机制[J]. 中国应用生理学杂志, 2018, 34(2): 115-119.