Abstract：
Our previous study found that receptor interacting protein 3 (RIP3) and apoptosis-inducing factor (AIF) were involved in neuronal programmed necrosis during global cerebral ischemia-reperfusion (I/R) injury. Here, we further studied its downstream mechanisms and the role of the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BAF). A 20-min global cerebral I/R injury model was constructed using the 4-vessel occlusion (4-VO) method in male rats. 3-MA and BAF were injected into the lateral ventricle 1 h before ischemia. Spatial and activation changes of proteins were detected by immunofluorescence (IF), and protein interaction was determined by immunoprecipitation (IP). The phosphorylation of H2AX (γ-H2AX) and activation of mixed lineage kinase domain-like protein (p-MLKL) occurred as early as 6 h after reperfusion. RIP3, AIF, and cyclophilin A (CypA) in the neurons after I/R injury were spatially overlapped around and within the nucleus and combined with each other after reperfusion. The survival rate of CA1 neurons in the 3-MA and BAF groups was significantly higher than that in the I/R group. Autophagy was activated significantly after I/R injury, which was partially inhibited by 3-MA and BAF. Pretreatment with both 3-MA and BAF almost completely inhibited nuclear translocation, spatial overlap, and combination of RIP3, AIF, and CypA proteins. These findings suggest that after global cerebral I/R injury, RIP3, AIF, and CypA translocated into the nuclei and formed the DNA degradation complex RIP3/AIF/CypA in hippocampal CA1 neurons. Pretreatment with autophagy inhibitors could reduce neuronal necroptosis by preventing the formation of the RIP3/AIF/CypA complex and its nuclear translocation.
摘要：
我们先前的研究发现，受体相互作用蛋白3（RIP3）和凋亡诱导因子（AIF）参与了全脑缺血再灌注（I/R）损伤中的神经元程序性坏死。这里,我们进一步研究了其下游机制以及自噬抑制剂3-甲基腺嘌呤(3-MA)和巴弗洛霉素A1(BAF)的作用.在雄性大鼠中使用4血管闭塞（4-VO）方法构建了20分钟的全脑I/R损伤模型。缺血前1小时将3-MA和BAF注入侧脑室。通过免疫荧光（IF）检测蛋白质的空间和活化变化，通过免疫沉淀（IP）确定蛋白质相互作用。H2AX（γ-H2AX）的磷酸化和混合谱系激酶结构域样蛋白（p-MLKL）的激活最早发生在再灌注后6小时。RIP3,AIF,I/R损伤后神经元中的亲环蛋白A（CypA）在核周围和内部空间重叠，并在再灌注后相互结合。3-MA和BAF组的CA1神经元存活率显著高于I/R组。I/R损伤后自噬显著激活，3-MA和BAF部分抑制。3-MA和BAF预处理几乎完全抑制了核易位，空间重叠，以及RIP3、AIF、和CypA蛋白。这些发现表明，在全脑I/R损伤后，RIP3,AIF,和CypA转位到细胞核中并在海马CA1神经元中形成DNA降解复合物RIP3/AIF/CypA。用自噬抑制剂预处理可以通过阻止RIP3/AIF/CypA复合物的形成及其核易位来减少神经元坏死。
