Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institutes, The First Affiliated Hospital of Wannan Medical College, Wuhu 241000, Anhui, China; Department of Neurology, The First Affiliated Hospital of Wannan Medical College, Wuhu 241000, Anhui, China.
Department of Neurology, The Second Affiliated Hospital of Wannan Medical College, Wuhu 241000, Anhui, China.
Exp Neurol. 2020 Sep;331:113374. doi: 10.1016/j.expneurol.2020.113374. Epub 2020 Jun 2.
Receptor-interacting protein kinase 3 (RIPK3) regulates a newly discovered cell death form called necroptosis. RIPK3 nuclear translocation and inflammatory factor release are involved in necroptosis after rat global cerebral ischemia/reperfusion (I/R) injury. The purpose of this study was to investigate the effects of interactions between the RIPK3 and apoptosis-inducing factor (AIF) necroptosis pathway and the JNK-mediated inflammatory pathway. Rats were subjected to 4-vessel occlusion and reperfusion injury. RIPK3 inhibitor GSK872, RIPk3 recombinant adeno-associated virus (rAAV) and JNK-specific inhibitor SP600125 were intracerebroventricular injected before I/R. Hippocampus CA1 tissue were obtained and RIPK3, AIF, p-JNK, IL-6 were determined by western blot analysis. The RIPK3 and AIF interaction were also analyzed by immunofluorescence and immunoprecipitation. The expression of endogenous RIPK3, AIF, p-JNK and IL-6 was increased in hippocampus CA1 in I/R group. In addition, RIPK3 was increased in both the total protein and nuclear protein. GSK872 administration reduced the number of neuron deaths and the expression of RIPK3, p-JNK and IL-6. GSK872 also improve the rat neurobehavior. While use RIPk3 rAAV treatment to overexpress RIPK3, it appeared lower neuron survival. Immunofluorescence staining demonstrated that RIPK3 and AIF formed as a novel complex in the cytoplasm first, and then nuclear translocation. GSK872 pretreatment decreased the number of RIPK3-positive cells and related to the generation of RIPK3-AIF complex in nuclear. Moreover, the production of inflammatory factors levels was found to be significantly elevated after I/R. We further use SP600125 to attenuate inflammation cascade. It not only inhibits the expression of inflammatory factors p-JNK and IL-6, but also inhibits RIPK3 and AIF in the cytoplasm. Collectively, the results of our study indicate that RIPK3-mediated necroptosis interacts with the JNK-mediated inflammatory signaling pathway to participate in global cerebral I/R injury. JNK-regulated inflammatory mediators may promote the necroptosis initiation.
受体相互作用蛋白激酶 3（RIPK3）调节一种新发现的细胞死亡形式，称为坏死性凋亡。RIPK3 核转位和炎症因子释放参与大鼠全脑缺血再灌注（I/R）损伤后的坏死性凋亡。本研究旨在探讨 RIPK3 与凋亡诱导因子（AIF）坏死性凋亡途径以及 JNK 介导的炎症途径之间相互作用的影响。大鼠进行 4 血管闭塞再灌注损伤。在 I/R 前，通过侧脑室注射 RIPK3 抑制剂 GSK872、RIPK3 重组腺相关病毒（rAAV）和 JNK 特异性抑制剂 SP600125。通过 Western blot 分析检测海马 CA1 组织中 RIPK3、AIF、p-JNK、IL-6 的表达。还通过免疫荧光和免疫沉淀分析 RIPK3 和 AIF 的相互作用。I/R 组海马 CA1 区内源性 RIPK3、AIF、p-JNK 和 IL-6 的表达增加。此外，RIPK3 增加了总蛋白和核蛋白。GSK872 给药减少了神经元死亡数量和 RIPK3、p-JNK 和 IL-6 的表达。GSK872 还改善了大鼠的神经行为。而使用 RIPk3 rAAV 治疗过表达 RIPK3，则出现较低的神经元存活。免疫荧光染色表明，RIPK3 和 AIF 首先在细胞质中形成一种新的复合物，然后核转位。GSK872 预处理减少了 RIPK3 阳性细胞的数量，并与核内 RIPK3-AIF 复合物的产生有关。此外，I/R 后发现炎症因子水平显著升高。我们进一步使用 SP600125 来减轻炎症级联反应。它不仅抑制了炎症因子 p-JNK 和 IL-6 的表达，还抑制了细胞质中的 RIPK3 和 AIF。总之，我们的研究结果表明，RIPK3 介导的坏死性凋亡与 JNK 介导的炎症信号通路相互作用，参与全脑 I/R 损伤。JNK 调节的炎症介质可能促进坏死性凋亡的启动。
