Regulation of JNK Signaling Pathway and RIPK3/AIF in Necroptosis-mediated Global Cerebral Ischemia/reperfusion Injury in Rats

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.