Regulation Of JNK Signaling Pathway And RIPK3/AIF In Necroptosis-mediated Global Cerebral Ischemia/reperfusion Injury In Rats

Objective: We used an in vivo model of rat global cerebral ischemia/reperfusion(I/R)injury dominated by necroptosis to explore the relationship between necroptosis signal pathway and neuroinflammatory signal after global cerebral I/R injury in rats,and to further reveal the feedback mechanism of neuroinflammatory signaling pathway mediated by c-Jun NH2-terminal kinase(JNK)on necroptosis after global cerebral I/R injury.Methods: 3-month-old stable male Sprague Dawley(SD)rats weighing 250-300 g were used for the preparation of a global cerebral I/R injury model.In this experiment,all SD rats were randomly divided into the following 5 groups: sham operation group(sham),Vehicle group(sham + NS),model group(I/R),GSK group(I/R + GSK872),SP group(I/R + SP600125),r AAV control group(sham + RIPK3 r AAV null),r AAV overexpression group(I/R + RIPK3 r AAV).The RIPK3 inhibitor GSK872 and the JNK inhibitor SP600125 were administered via the right lateral ventricle with a microinjection pump 1hour before the induction of global cerebral ischemia,and RIPK3 r AAV was administered via the right lateral ventricle 28 days before the induction of global cerebral ischemia.medicine.Hematoxylin/eosin staining was used to evaluate the neuron survival in the CA1 area of the rat hippocampus;Morris water maze experiment was used to evaluate the cognition and memory status of rats;Western blot was used to detect the expression level of RIPK3,AIF,p-JNK and IL-6.Co-localization of RIPK3 and AIF in the brain was analyzed by immunoprecipitation and immunofluorescence techniques.Results: Western blot found that the expression of AIF did not change significantly after I/R injury,but the expression of proteins were increased,such as RIPK3,p-JNK and IL-6.Both GSK872 and SP600125 reduced the expression of RIPK3,p-JNK and IL-6 after I/R injury,but the RIPK3 adeno-associated virus r AAV increased the expression of RIPK3,p-JNK and IL-6.HE staining showed that GSK872 and SP600125 had obvious protective effects on neuronal I/R injury.Morris Water Labyrinth Experiment shows that the sense of spatial location and orientation and learning and memory in rats after I/R injury have reduced,but GSK872 may boost the neurological deficit after I/R injury in rats.Immunoprecipitation and immunofluorescence further suggested that RIPK3 and AIF combined with each other in the cytoplasm after I/R injury to form a complex and shifted to the nucleus.Conclusion: The interaction between RIPK3 and AIF and its displacement into the nucleus is a key link in neuronal necroptosis mediated by global cerebral I/R injury.Necroptosis can activate JNK-mediated neuroinflammation.GSK872 can reduce the nerve damage and neuroinflammation caused by global cerebral I/R injury by inhibiting the combination of RIPK3 and AIF.SP600125 can reduce nerve damage caused by global cerebral I/R injury by inhibiting neuroinflammation.