丁苯酞对缺血性脑卒中大鼠海马神经元凋亡的抑制作用及其对p38 MAPK信号通路的影响

doi:10.13481/j.1671-587x.20190520

摘要/Abstract

摘要： 目的：探讨丁苯酞对缺血性脑卒中大鼠海马神经元凋亡和p38丝裂原活化蛋白激酶（p38MAPK）信号通路的影响，阐明丁苯酞对缺血性脑卒中的作用机制。方法： 102只雄性SD大鼠随机分为假手术组、模型组和丁苯酞组，每组34只。模型组和丁苯酞组大鼠采用改良Zea-Longa法制备局灶性脑缺血大鼠模型，丁苯酞组大鼠建模后给予丁苯酞（4.5 mg·kg^-1）治疗，假手术组大鼠每天相同时间点腹腔注射等量生理盐水。采用HE染色观察大鼠海马神经元细胞形态表现，原位末端转移酶标记（TUNEL）染色观察大鼠海马神经元凋亡情况，Western blotting法测定大鼠海马组织中激活型caspase-3（cleaved caspase-3）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、p38、磷酸化p38（p-p38）和分裂原激活的蛋白激酶（MAPK）蛋白表达水平，逆转录-聚合酶链反应（RT-PCR）测定大鼠海马组织中cleaved caspase-3、Bax、Bcl-2、p38和MAPK mRNA表达水平。结果：假手术组大鼠海马CA1区神经元排列整齐，细胞核和细胞膜正常；模型组大鼠海马CA1区神经元排列紊乱，细胞肿胀破裂，细胞核固缩；丁苯酞组大鼠海马CA1区部分神经细胞结构恢复正常。与假手术组比较，模型组大鼠海马CA1区神经元数量明显降低（P<0.01），神经元凋亡指数明显增加（P<0.01）；与模型组比较，丁苯酞组大鼠海马CA1区神经元数量明显增加（P<0.01），神经元凋亡指数明显降低（P<0.01）。与假手术组比较，模型组大鼠海马组织中cleaved caspase-3、Bax、p-p38和MAPK蛋白表达水平明显升高（P<0.01），Bcl-2蛋白表达水平明显降低（P<0.01）；与模型组比较，丁苯酞组大鼠海马组织中cleaved caspase-3、Bax、p-p38和MAPK蛋白表达水平明显降低（P<0.01），Bcl-2蛋白表达水平明显升高（P<0.01）。与假手术组比较，模型组大鼠海马组织中cleaved caspase-3、Bax和MAPK mRNA表达水平明显升高（P<0.01），Bcl-2 mRNA表达水平明显降低（P<0.01）；与模型组比较，丁苯酞组大鼠海马组织中cleaved caspase-3、Bax和MAPK mRNA表达水平明显降低（P<0.01），Bcl-2 mRNA表达水平明显升高（P<0.01）。结论：丁苯酞可通过抑制海马组织p38 MAPK信号通路抑制缺血性脑卒中大鼠海马神经元凋亡。

关键词: 丁苯酞, 缺血性脑卒中, 海马, 细胞凋亡, p38丝裂原活化蛋白激酶, SD大鼠

Abstract: Objective:To investigate the effects of butylphthalide on the hippocampal neuron apoptosis and p38 mitogen-activated protein kinase (MAPK) signaling pathway in the rats with ischemic stroke, and to elucidate the mechanism of butylphthalide in ischemic stroke. Methods:A total of 102 male rats were randomly divided into sham operation group, model group and butylphthalide group;there were 34 rats in each group. The rats in model group and butylphthalide group were used to establish the focal cerebral ischemia models with modified Zea-Longa method. The rats in butylphthalide group were treated with butylphthalide (4.5 mg·kg^-1) after modeling.The rats in sham operation group were intraperitoneally injected with the same volume of normal saline at the same time points. The morphology of neurons was observed by HE staining. The apoptosis of hippocampal neurons was observed by in situ terminal transferase labeling (TUNEL) staining. The expression levels of activated caspase-3 (cleaved caspase-3), B lymphocyte tumor-2 (Bcl-2), Bcl-2 related X protein (Bax), p38, phosphorylation-p38 (p-P38) and MAPK proteins in hippocampus tissue of the rats in various groups were determined by Western blotting method. The expression levels of cleaved caspase-3, Bax, Bcl-2, p38, and MAPK mRNA in hippocampus tissue of the rats in various groups were determined by reverse transcription-polymerase chain reaction (RT-PCR). Results:The neurons in the hippocampal CA1 area of the rats in sham operation group were well aligned and the nuclei and cell membrane were normal. In model group, the neurons in the hippocampal CA1 area were disordered, the cells were swollen and burst, and the nuclei had pyknosis. In butylphthalide group, the structure of some neurons in the hippocampal CA1 area returned to normal. Compared with sham operation group, the number of neurons in the hippocampal CA1 area of the rats in model group was significantly reduced (P<0.01), and the apoptotic index of neurons was significantly increased (P<0.01).Compared with model group, the number of neurons in hippocampal CA1 area of the rats in butylphthalide group was significantly increased (P<0.01), and the apoptotic index of neurons was significantly decreased (P<0.01). Compared with sham operation group, the expression levels of cleaved caspase-3, Bax, p-p38 and MAPK proteins in hippocampal tissue of the rats in model group were significantly increased (P<0.01), and the expression level of Bcl-2 protein in hippocampal tissue of the rats in model group was significantly decreased (P<0.01); compared with model group, the expression levels of cleaved caspase-3, Bax, p-p38 and MAPK proteins in hippocampus tissue of the rats in butylphthalide group were significantly decreased (P<0.01),and the expression level of Bcl-2 protein in hippocampus tissue of the rats in butylphthalide group was significantly increased (P<0.01). Compared with sham operation group, the expression levels of cleaved caspase-3, Bax and MAPK mRNA in hippocampus tissue of the rats in model group were significantly increased (P<0.01), and the expression level of Bcl-2 mRNA in hippocampal tissue of the rats in model group was significantly decreased (P<0.01); compared with model group, the expression levels of cleaved caspase-3, Bax and MAPK mRNA in hippocampus tissue of the rats in butylphthalide group were significantly decreased (P<0.01),and the expression level of Bcl-2 mRNA in hippocampus tissue of the rats in butylphthalide group was significantly increased (P<0.01). Conclusion:Butylphthalide can inhibit the apoptosis of hippocampal neurons in the rats with ischemic stroke by inhibiting the p38 MAPK signaling pathway in the hippocampus tissue.

Key words: butylphthalide, ischemic stroke, hippocampus, apoptosis, p38 mitogen-activated protein kinase, SD rats

中图分类号:

R749.13

张晓璇, 马征, 于宁, 焦光美, 窦志杰, 赵亮, 高燕军. 丁苯酞对缺血性脑卒中大鼠海马神经元凋亡的抑制作用及其对p38 MAPK信号通路的影响[J]. 吉林大学学报(医学版), 2019, 45(05): 1086-1091.

ZHANG Xiaoxuan, MA Zheng, YU Ning, JIAO Guangmei, DOU Zhijie, ZHAO Liang, GAO Yanjun. Inhibitory effect of butylphthalide on hippocampal neuron apoptosis in ischemic stroke rats and itsinfluence in p38 MAPK signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1086-1091.

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