Abstract：
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.
OBJECTIVE: 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.
OBJECTIVE: 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.
摘要：
背景：据报道，含有谷氨酸红藻氨酸受体亚基2（GluK2）亚基的谷氨酸受体的激活和随后的Fas配体（FasL）上调，caspase-3激活，导致脑缺血再灌注后海马CA1亚区迟发性凋亡样神经元死亡。一氧化氮介导的S-亚硝基化可能抑制胱天蛋白酶原激活，而反硝基化可能有助于蛋白酶原的裂解和活化。
目的：本研究旨在阐明海人酸（KA）诱导大鼠海马兴奋性毒性后，胱天蛋白酶原3反硝基化和活化的分子机制。
方法：用生物素开关法检测胱天蛋白酶原-3的S-亚硝基化。胱天蛋白酶原-3的活化显示为通过免疫印迹检测到的胱天蛋白酶原-3的切割。免疫印迹法检测FasL表达。甲酚紫罗兰和TdT介导的dUTP尼克末端标记（TUNEL）染色用于检测大鼠海马CA1和CA3亚区的凋亡样神经元死亡。
结果：KA以剂量和时间依赖性方式导致胱天蛋白酶原3的激活，并且激活被KA受体拮抗剂NS102抑制。红藻氨酸给药后3小时，Procaspase-3被脱氮糖基化，反硝基化被SNP和GSNO逆转。FasLASODN抑制胱天蛋白酶-3的脱硝基化和活化。此外，硫氧还蛋白还原酶（TrxR）抑制剂金诺芬可防止大鼠海马CA1和CA3亚区中反硝基化和激活procaspase-3。NS102、FasLAS-ODN、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导致脱氮糖基化和激活procaspase-3。金诺芬抑制胱天蛋白酶-3的反硝基化，SNP,GSNO对KA诱导的大鼠海马CA1和CA3亚区凋亡样神经元死亡具有保护作用。这些研究表明，胱天蛋白酶-3在被激活之前经历了初始的脱硝基过程，为兴奋性毒性的潜在机制和可能的治疗提供有价值的见解。
目的：本研究旨在阐明海人酸（KA）诱导大鼠海马兴奋性毒性后，胱天蛋白酶原3反硝基化和活化的分子机制。
方法：用生物素开关法检测胱天蛋白酶原-3的S-亚硝基化。胱天蛋白酶原-3的活化显示为通过免疫印迹检测到的胱天蛋白酶原-3的切割。免疫印迹法检测FasL表达。甲酚紫罗兰和TdT介导的dUTP尼克末端标记（TUNEL）染色用于检测大鼠海马CA1和CA3亚区的凋亡样神经元死亡。
结果：KA以剂量和时间依赖性方式导致胱天蛋白酶原3的激活，并且激活被KA受体拮抗剂NS102抑制。红藻氨酸给药后3小时，Procaspase-3被脱氮糖基化，反硝基化被SNP和GSNO逆转。FasLASODN抑制胱天蛋白酶-3的脱硝基化和活化。此外，硫氧还蛋白还原酶（TrxR）抑制剂金诺芬可防止大鼠海马CA1和CA3亚区中反硝基化和激活procaspase-3。NS102、FasLAS-ODN、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导致脱氮糖基化和激活procaspase-3。金诺芬抑制胱天蛋白酶-3的反硝基化，SNP,GSNO对KA诱导的大鼠海马CA1和CA3亚区凋亡样神经元死亡具有保护作用。这些研究表明，胱天蛋白酶-3在被激活之前经历了初始的脱硝基过程，为兴奋性毒性的潜在机制和可能的治疗提供有价值的见解。
