Regulation of JNK signaling pathway and RIPK3/AIF in necroptosis-mediated global cerebral ischemia/reperfusion injury in rats
Regulation of JNK signaling pathway and RIPK3/AIF in necroptosis-mediated global cerebral ischemia/reperfusion injury in rats
复制标题
JNK信号通路和RIPK3/AIF在坏死性凋亡介导的大鼠全脑缺血/再灌注损伤中的调控
DOI：
10.1016/j.expneurol.2020.113374
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发表时间：
2020-09-01
影响因子：
5.3
通讯作者：
Xu, Yang
中科院分区：
文献类型：
--
作者：
Hu, Wenjie;Wu, Xiaodong;Xu, Yang
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。本研究的目的是探讨 RIPK3 与凋亡诱导因子 (AIF) 坏死性凋亡通路以及 JNK 介导的炎症通路之间相互作用的影响。对大鼠进行4血管闭塞和再灌注损伤。 I/R前脑室内注射RIPK3抑制剂GSK872、RIPk3重组腺相关病毒(rAAV)和JNK特异性抑制剂SP600125。获取海马CA1组织并通过Western blot分析测定RIPK3、AIF、p-JNK、IL-6。还通过免疫荧光和免疫沉淀分析了 RIPK3 和 AIF 相互作用。 I/R组海马CA1区内源性RIPK3、AIF、p-JNK、IL-6表达升高。此外，RIPK3的总蛋白和核蛋白均增加。 GSK872 给药减少了神经元死亡数量以及 RIPK3、p-JNK 和 IL-6 的表达。 GSK872还可以改善大鼠的神经行为。当使用RIPk3 rAAV治疗过度表达RIPK3时，出现神经元存活率降低。免疫荧光染色表明，RIPK3 和 AIF 首先在细胞质中形成新型复合物，然后发生核转位。 GSK872预处理减少了RIPK3阳性细胞的数量，并与核中RIPK3-AIF复合物的生成有关。此外，发现缺血再灌注后炎症因子的产生水平显着升高。我们进一步使用 SP600125 来减弱炎症级联反应。它不仅抑制炎症因子p-JNK和IL-6的表达，还抑制细胞质中的RIPK3和AIF。总的来说，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发生。
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.