中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (5): 936-943.doi: 10.4103/1673-5374.268903

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

Necrostatin-1保护癫痫持续状态小鼠海马神经元损伤的最佳干预浓度

  

  • 出版日期:2020-05-15 发布日期:2020-06-01

Optimal concentration of necrostatin-1 for protecting against hippocampal neuronal damage in mice with status epilepticus

Dong-Qi Lin1, 2, Xin-Ying Cai3, Chun-Hua Wang2, Bin Yang2, Ri-Sheng Liang2   

  1. 1 Department of Cardiothoracic Surgery, the Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, China
    2 Department of Neurosurgery, Union Hospital, Fujian Medical University, Fuzhou, Fujian Province, China
    3 Clinical Research Center, Shantou Central Hospital, Affiliated Shantou Hospital of Sun Yat-sen University, Shantou, Guangdong Province, China
  • Online:2020-05-15 Published:2020-06-01
  • Contact: Ri-Sheng Liang, MD,doctorlr@126.com.
  • Supported by:
    This study was supported by the Key Discipline Construction Project of the Union Hospital of Fujian Province, China, No. Δ211002#.

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摘要：

癫痫持续状态发作后海马区神经细胞会出现不同类型的细胞死亡。有研究表明Necrostatin-1可特异性抑制受体相互作用蛋白激酶1，3(RIP1，RIP3)介导的细胞程序性坏死。然而目前国际上尚无小鼠癫痫持续状态模型在程序性坏死的报道。为此，实验旨在观察Necrostatin-1对癫痫小鼠海马神经元的影响，并探讨其有效的保护浓度。采用12 mg/kg 红藻氨酸盐对小鼠进行腹腔注射，建立癫痫持续状态小鼠模型，腹腔注射前15 min将不同浓度的Necrostatin-1 (10，20，40，80 μM)注入侧脑室。建模后24 h，苏木精-伊红染色检测小鼠海马组织损伤程度，采用TUNEL染色、Western blot和免疫组织化学检测凋亡相关通路和程序化坏死相关通路蛋白的表达，筛选出保护海马神经元最理想的Necrostatin-1浓度。结果发现：①Necrostatin-1可减轻模型小鼠海马组织损伤；②40 μM的Necrostatin-1干预后可显著降低模型小鼠海马CA1区凋亡阳性细胞数量；③Necrostatin-1可显著下调模型小鼠海马组织程序性坏死相关蛋白(MLKL、RIP1、RIP3)、促凋亡蛋白(Cleaved-caspase3、Bax)表达及免疫阳性细胞数量，并上调抗凋亡蛋白Bcl-2表达及免疫阳性细胞数量；④上述数据表明，Necrostatin-1可有效抑制癫痫持续状态模型小鼠海马组织神经元的程序性坏死及凋亡，40 μM Necrostatin-1干预效果最佳。实验于2016-11-9经福建医科大学动物伦理委员会批准，批准号：20160-32。

orcid: 0000-0002-1937-7188 (Ri-Sheng Liang) 

         0000-0002-9133-4502 (Dong-Qi Lin)

关键词: 癫痫, Necrostatin-1, 海马神经元, 凋亡, 程序性坏死, RIP1, MLKL, RIP3, Bcl-2, Bax, Cleaved-caspase 3

Abstract: Hippocampal neurons undergo various forms of cell death after status epilepticus. Necrostatin-1 specifically inhibits necroptosis mediated by receptor interacting protein kinase 1 (RIP1) and RIP3 receptors. However, there are no reports of necroptosis in mouse models of status epilepticus. Therefore, in this study, we investigated the effects of necrostatin-1 on hippocampal neurons in mice with status epilepticus, and, furthermore, we tested different amounts of the compound to identify the optimal concentration for inhibiting necroptosis and apoptosis. A mouse model of status epilepticus was produced by intraperitoneal injection of kainic acid, 12 mg/kg. Different concentrations of necrostatin- 1 (10, 20, 40, and 80 μM) were administered into the lateral ventricle 15 minutes before kainic acid injection. Hippocampal damage was assessed by hematoxylin-eosin staining 24 hours after the model was successfully produced. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, western blot assay and immunohistochemistry were used to evaluate the expression of apoptosis-related and necroptosis-related proteins. Necrostatin-1 alleviated damage to hippocampal tissue in the mouse model of epilepsy. The 40 μM concentration of necrostatin-1 significantly decreased the number of apoptotic cells in the hippocampal CA1 region. Furthermore, necrostatin-1 significantly downregulated necroptosis-related proteins (MLKL, RIP1, and RIP3) and apoptosis-related proteins (cleaved-Caspase-3, Bax), and it upregulated the expression of anti-apoptotic protein Bcl-2. Taken together, our findings show that necrostatin-1 effectively inhibits necroptosis and apoptosis in mice with status epilepticus, with the 40 μM concentration of the compound having an optimal effect. The experiments were approved by the Animal Ethics Committee of Fujian Medical University, China (approval No. 2016-032) on November 9, 2016.

Key words: apoptosis, Bax, Bcl-2, cleaved-Caspase-3, epilepsy, hippocampal neuron, MLKL, necroptosis, necrostatin-1, nerve regeneration, neural regeneration, RIP1, RIP3