Background: It has been reported that activation of glutamate kainate receptor subunit 2 (GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation, caspase-3 activation, result in delayed apoptosis-like neuronal death in hippocampus CA1 subfield after cerebral ischemia and reperfusion. Nitric oxide-mediated S-nitrosylation might inhibit the procaspase activation, whereas denitrosylation might contribute to cleavage and activation of procaspases. Objectives: The study aimed to elucidate the molecular mechanisms underlying procaspase-3 denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat hippocampus. Methods: S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. Results: KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase (TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced apoptosis and cell death in hippocampal CA1 and CA3 subfields. Conclusions: KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR. Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process before becoming activated, providing valuable insights into the underlying mechanisms and possible treatment of excitotoxicity.

中文翻译：

---

KA 诱导的兴奋性毒性中 Procaspase-3 的激活和去亚硝基化

背景：据报道，含有谷氨酸受体的谷氨酸红藻氨酸受体亚基2（GluK2）亚基的激活和随后的Fas配体（FasL）的上调，caspase-3的激活，导致海马CA1中延迟的细胞凋亡样神经元死亡脑缺血再灌注后的亚场。一氧化氮介导的 S-亚硝基化可能会抑制 caspase 前体的激活，而去亚硝基化可能有助于 caspase 前体的裂解和激活。目的：本研究旨在阐明红藻氨酸 (KA) 诱导大鼠海马兴奋性毒性后 procaspase-3 去亚硝基化和激活的分子机制。方法：采用生物素开关法检测procaspase-3的S-亚硝基化。通过免疫印迹检测到 procaspase-3 的裂解，表明 procaspase-3 的激活。通过免疫印迹法检测FasL表达。使用甲酚紫和 TdT 介导的 dUTP 缺口末端标记 (TUNEL) 染色来检测大鼠海马 CA1 和 CA3 亚区中的细胞凋亡样神经元死亡。结果：KA以剂量和时间依赖性方式导致procaspase-3的激活，并且该激活被KA受体拮抗剂NS102抑制。Procaspase-3在红藻氨酸给药后3小时被去亚硝基化，并且该去亚硝基化被SNP和GSNO逆转。FasL ASODN 抑制 procaspase-3 去亚硝基化和激活。此外，硫氧还蛋白还原酶 (TrxR) 抑制剂金诺芬可阻止大鼠海马 CA1 和 CA3 亚区中 procaspase-3 的脱硝基化和激活。NS102、FasL AS-ODN 和金诺芬逆转了 KA 诱导的海马 CA1 和 CA3 亚区细胞凋亡和细胞死亡。结论：KA 通过 FasL 和 TrxR 导致 procaspase-3 的去亚硝基化和激活。金诺芬、SNP 和 GSNO 抑制 procaspase-3 去亚硝基化对大鼠海马 CA1 和 CA3 亚区中 KA 诱导的细胞凋亡样神经元死亡具有保护作用。这些研究表明，procaspase-3 在被激活之前经历了一个初始的去亚硝基化过程，为了解兴奋性毒性的潜在机制和可能的治疗提供了有价值的见解。