Neural Regeneration Research ›› 2020, Vol. 15 ›› Issue (5): 936-943.doi: 10.4103/1673-5374.268903

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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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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