Autophagy Inhibitors 3-methyladenine And Bafilomycin A1 Attenuate Hippocampal Neuronal Necroptosis After Global Cerebral Ischemia-Reperfusion Injury In Rats By Inhibiting The Interaction Of RIP3/AIF/CypA Complex

Research objectivePrevious study found that the nuclear translocation of receptor interacting protein 3(RIP3)and apoptosis-inducing factor(AIF)and their interaction were involved in the hippocampal CA1 neuronal programmed necrosis during global cerebral ischemiareperfusion(I/R)injury.Here,we further studied its downstream mechanisms and the role of autophagy inhibitors 3-methyladenine(3-MA)and bafilomycin A1(BAF).Research methodGlobal cerebral ischemia-reperfusion injury was conducted on adult male SpragueDawley(SD)rats weighing 260~300 g using the four-vessel occlusion(4-VO)method.The rats were randomly divided into sham operation group,model group and administration intervention group(3-MA group,BAF group).In the model group,permanent electrocoagulation of bilateral vertebral arteries was performed,and blood perfusion was restored after 20 minutes of occlusion of bilateral common carotid arteries on the second day;For sham surgery,animals were subjected to the same anesthesia and surgical conditions,except that the vertebral and carotid arteries were not occluded;In 3-MA group and BAF group,3-MA(600 nmol)and BAF(0.1 μg)administered by intracerebroventricular injection at 1 h before ischemia,other operations were the same as model group.Sham operation group,model group and drug administration intervention group took brain tissue after formaldehyde perfusion and fixation at different reperfusion time points(6h,12 h,24h,48 h,72h,7d),and made pathological sections.In addition to 6 rats at 7 days of reperfusion,3 rats were taken at each time point.Hematoxylin-eosin staining(HE)was used to observe neuronal cell death;Immunofluorescence staining was used to observe RIP3,AIF,cyclophilin A(CypA),phosphorylation of histone H2AX(γ-H2AX)and phosphorylated mixed lineage kinase domain-like protein(p-MLKL)in spatial distribution;Western blot was used to detect the expression of microtubule-associated protein 1 light chain 3-Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ),RIP3,AIF,CypA proteins;The binding of RIP3,AIF and CypA was detected by immunoprecipitation.Research resultsImmunofluorescence staining showed that in sham operation group,AIF was distributed in the cytoplasm and axons of neurons,RIP3 and CypA were distributed in the cytoplasm of neurons,and H2 AX and MLKL were not significantly activated in neurons.In the model group,H2 AX began to enhance at 6 h of reperfusion at the edge of the nucleus,and the fluorescence of p-MLKL began to increase from 6 h of reperfusion in individual dying neurons with pyknotic nuclei;After reperfusion for 12 h to 24 h,RIP3,AIF and CypA increased greatly around the nucleus,and there was nuclear translocation in a few pyknotic neurons.H2AX(nucleus)and MLKL(cytoplasm)were significantly activated;After reperfusion for 48 hours,the translocation of RIP3,AIF and CypA increased,and the activation of H2 AX and MLKL reached the peak in neurons;After 72 h of reperfusion,the fusion of RIP3,AIF and CypA could still be seen in the pyknotic nucleus,The fluorescence of γ-H2 AX and pMLKL began to fade.Fluorescence double staining showed that RIP3,AIF and CypA fluorescence did not overlap in sham operation group;In the model group,AIF/RIP3,AIF/CypA,RIP3/CypA showed fluorescence superposition and co-staining around the nucleus of neurons at 24 h after reperfusion;The translocation of RIP3,AIF and CypA increased at 48 h after reperfusion,and there were fluorescence co-staining around and in the nucleus.The distribution of RIP3,AIF and CypA proteins in the CA1 region of hippocampus in the 3-MA and BAF pretreatment groups after 72 h reperfusion was similar to that in sham operation group,and the co-localization was not obvious.Immunocoprecipitation found that AIF interacted with RIP3 and CypA at 24 h after global cerebral ischemia reperfusion injury,while the results in 3-MA and BAF pretreatment groups were similar to that in sham operation group,and no interaction between them was detected.The results showed that 3-MA and BAF pretreatment could inhibit the nuclear translocation,spatial overlap and protein binding of RIP3,AIF and CypA.Western blot showed that the expression of LC3-II increased at 24 h after reperfusion after 20 minutes of ischemia,suggesting the activation of autophagy.HE staining showed that the survival rate of neurons in 3-MA group(87.2 ± 2.1%)and BAF group(83.3 ± 3.4%)was significantly higher than that in I/R group(8.8 ± 3.1%).Research conclusionAfter 20 minutes of global cerebral ischemia reperfusion injury in adult rats,autophagy was activated in neurons in the hippocampal CA1 region;DNA degradation complex RIP3/AIF/CypA was formed in the neuronal injury induced by ischemia,resulting in necroptosis of neurons;The pretreatment of autophagy inhibitor can inhibit the autophagy of neurons and the formation of the complex,thus reducing the necrosis of neurons.