Intensive Care Unit of the Affiliated Huai'an Hospital of Xuzhou Medical University, Huai'an 223001, Jiangsu, China.
Laboratory of Emergency Medicine, Second Clinical Medical College of Xuzhou Medical University, Xuzhou, 221004, China.
J Stroke Cerebrovasc Dis. 2022 Jul;31(7):106516. doi: 10.1016/j.jstrokecerebrovasdis.2022.106516. Epub 2022 Apr 28.
Our preliminary experiments indicate that receptor-interacting protein 3 (RIP3) is S-nitrosylated and contributes to its autophosphorylation (activation) after 3 h of rat brain ischemia/reperfusion mediated by activation of the N-methyl-D-aspartate receptor (NMDAR)-dependent neuronal NO synthase (nNOS) and is involved in the process of neuronal injury. Here, we will to demonstrate whether S-nitrosylation of RIP3 facilitates the activation of the downstream signaling pathway and finally exacerbates ischemic neuron death.
Adult male Sprague-Dawley rat transient brain ischemia/reperfusion and cortical neurons oxygen and glucose deprivation (OGD)/reoxygenation models were performed. The hippocampal CA1 regions or cultured cells were homogenized and the cytosolic fraction were collected as tissue samples. Coimmunoprecipitation and western blot analysis were carried out for detecting phosphorylation of RIP1 and mixed lineage kinase-like domains (MLKL) and the Cleaved-Caspase8 (Cl-Caspase8). The activities of Glycogen phosphorylase (PYGL), Glutamate-ammonia ligase (GLUL) and Glutamate dehydrogenase (GLUD1) were detected with ultraviolet absorption method.
This study showed that active RIP3 could phosphorylate RIP1 and MLKL through its kinase activity, promote the conversion of Caspase8 to active Cl-Caspase8, enhance the activities of PYGL, GLUL and GLUD1, and finally aggravate neuronal injury in cerebral ischemia/reperfusion. The inhibition of RIP3 S-nitrosylation inhibited the phosphorylation of RIP1 and MLKL, inhibited the activities of Caspase8, PYGL, GLUL, and GLUD1, and alleviated neuronal damage in cerebral ischemia/reperfusion.
S-nitrosylation of RIP3 increased RIP1 and MLKL phosphorylation levels, Cl-Caspase8 content and PYGL, GLUL and GLUD1 activities and aggravated neuronal damage during cerebral ischemia/reperfusion and regulating the S-nitrosylation of RIP3 and its downstream signaling pathway might be a therapeutic target for stroke.
我们的初步实验表明，受体相互作用蛋白 3（RIP3）在大鼠脑缺血/再灌注 3 小时后被 S-亚硝基化，并通过 N-甲基-D-天冬氨酸受体（NMDAR）依赖性神经元一氧化氮合酶（nNOS）的激活而被激活，这与神经元损伤过程有关。在这里，我们将证明 RIP3 的 S-亚硝基化是否促进下游信号通路的激活，最终加剧缺血性神经元死亡。
采用成年雄性 Sprague-Dawley 大鼠短暂性脑缺血/再灌注和皮质神经元氧葡萄糖剥夺（OGD）/复氧模型。将海马 CA1 区或培养细胞匀浆，收集胞质部分作为组织样本。进行免疫共沉淀和 Western blot 分析，以检测 RIP1 和混合谱系激酶样结构域（MLKL）的磷酸化以及 Cleaved-Caspase8（Cl-Caspase8）的含量。采用紫外吸收法检测糖原磷酸化酶（PYGL）、谷氨酸-氨连接酶（GLUL）和谷氨酸脱氢酶（GLUD1）的活性。
本研究表明，活性 RIP3 可以通过其激酶活性磷酸化 RIP1 和 MLKL，促进 Caspase8 向活性 Cl-Caspase8 的转化，增强 PYGL、GLUL 和 GLUD1 的活性，最终加重脑缺血/再灌注后的神经元损伤。RIP3 的 S-亚硝基化抑制可抑制 RIP1 和 MLKL 的磷酸化，抑制 Caspase8、PYGL、GLUL 和 GLUD1 的活性，减轻脑缺血/再灌注后的神经元损伤。
RIP3 的 S-亚硝基化增加了 RIP1 和 MLKL 的磷酸化水平、Cl-Caspase8 含量以及 PYGL、GLUL 和 GLUD1 的活性，加重了脑缺血/再灌注过程中的神经元损伤，调节 RIP3 的 S-亚硝基化及其下游信号通路可能是中风的治疗靶点。
