Necrostatin-1 protects hippocampal neurons against ischemia/reperfusion injury via the RIP3/DAXX signaling pathway in rats.

Global cerebral ischemia/reperfusion (I/R) induces selective neuronal injury in CA1 region of hippocampus, leading to severe impairment in behavior, learning and memory functions. However, the molecular mechanism underlying the processes was not elucidated clearly. RIP3 is a key molecular switch connecting apoptosis, necrosis and necroptosis. DAXX, as a novel substrate of RIP3, plays a vital role in ischemia-induced neuronal death. The aim of this study is to investigate the role and mechanism of RIP3/DAXX signaling pathway on neurons in CA1 region of the rat hippocampus after cerebral I/R. Global cerebral ischemia was induced by the method of four-vessel occlusion. RIP1 specific inhibitor Necrostatin-1 was administered by intracerebroventricular injection 1h before ischemia. Open-field, closed-field, and Morris water maze tests were performed respectively to examine the anxiety and cognitive behavior in each group. Hematoxylin and eosinstaining was used to examine the survival of hippocampal CA1 pyramidal neurons. Western blot or immunoprecipitation were carried to detect protein expression, phosphorylation, and interaction. We found that pre-treatment with Nec-1 protected locomotive ability, relieved anxiety behavior, and improved cognitive ability in the rats subjected to cerebral I/R. In addition Moreover, Nec-1 decreased significantly the dead rate of neurons in hippocampal CA1 region after cerebral I/R through suppressing RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction, and then blocked DAXX translocation from nucleaus to cytoplasm, which resulted in the inactiviation of DAXX. We concluded that pre-treatment with Nec-1 can protect neurons in the hippocampal CA1 region against ischemic damage through the RIP3-DAXX signaling pathway.