Rasd1促进少突胶质细胞铁死亡加重大鼠蛛网膜下腔出血后白质损伤

Rasd1 aggravates white matter injury after subarachnoid hemorrhage in rats by promoting ferroptosis in oligodendrocytes

目的 探索Rasd1(ras related dexamethasone induced 1)在SD大鼠蛛网膜下腔出血(subarachnoid hemorrhage, SAH)后通过神经元-少突胶质细胞间通讯参与白质损伤(white matter injury, WMI)的机制。方法 将90只雄性SD大鼠(体质量180~250 g)采用随机抽签的方法分为假手术组、SAH组、SAH+LV-NC组、SAH+LV-Rasd1组和SAH+sh-Rasd1组,最终每组12只。利用血管内穿刺法构建大鼠在体SAH模型,侧脑室注射Rasd1基因过表达/敲低慢病毒,神经行为学评分检测神经功能变化,透射电镜检测胼胝体区域髓鞘脱失情况和少突胶质细胞铁死亡的发生情况,普鲁士蓝染色检测铁沉积,试剂盒检测丙二醛(malonic dialdehyde, MDA)和谷胱甘肽(glutathione, GSH),PCR检测Rasd1 mRNA的表达情况,Western blot检测核受体辅激活蛋白4(nuclear receptor coactivator 4,NCOA4)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、2’,3’-环腺苷酸-3’-磷酸二酯酶(2’,3’-cyclic-nucleotide 3’-phosphodiesterase, CNPase)和铁蛋白(Ferritin)的表达。结果 SAH后,Rasd1升高(P<0.01)并主要表达于皮层神经元中,出现髓鞘脱失,少突胶质细胞铁死亡;过表达Rasd1后,大鼠神经行为学评分显著降低,髓鞘脱失、少突胶质细胞铁死亡和铁沉积明显加重,MDA升高(P<0.01)、GSH下降(P<0.01)、NCOA4升高(P<0.01)、Ferritin和GPX4下降(P<0.01);敲低Rasd1后,大鼠神经行为学评分明显升高,髓鞘脱失、少突胶质细胞铁死亡和铁沉积显著减轻,MDA下降(P<0.01)、GSH升高(P<0.01)、NCOA4下降(P<0.01)、Ferritin和GPX4升高(P<0.01)。结论 Rasd1在蛛网膜下腔出血后通过铁自噬促进少突胶质细胞铁死亡,从而参与蛛网膜下腔出血后白质损伤。

Objective To explore the underlying mechanism in which Ras related dexamethasone induced 1(Rasd1)is involved in white matter injury through neuron-oligodendrocyte communication after subarachnoid hemorrhage(SAH)in SD rats.Methods Ninety male SD rats(180~250 g)were randomly divided into sham operation group,SAH group,SAH+LV-NC group,SAH+LV-Rasd1 group and SAH+sh-Rasd1 group,with 12 rats in each group.Intravascular puncture was used to construct the rat model of SAH.Rasd1 overexpression/knockdown lentivirus was injected into the lateral ventricle.Neurobehavioral rating scale was used to observe the changes in neural function,transmission electron microscopy was employed to detect the myelin detachment in callosum region and the occurrence of ferroptosis in oligodendrocytes,and Prussian blue staining was applied to measure iron deposition.Malondialdehyde(MDA)and glutathione(GSH)contents were detected with corresponding reagent kits,Rasd1 mRNA level was detected by PCR,and protein levels of nuclear receptor coactivator 4(NCOA4),2′,3′-cyclic-nucleotide 3′-phosphodiesterase(CNPase),glutathione peroxidase 4(GPX4)and ferritin were measured with Western blotting.Results SAH resulted in significantly highly expressed Rasd1,which was mainly distributed in the cortical neurons(P0.01),myelin loss,and ferroptosis in oligodendrocytes.Rasd1 overexpression significantly reduced the neurobehavioral scores,but increased myelin loss,ferroptosis and iron deposition in oligodendrocytes,elevated MDA content(P0.01)but decreased GSH content(P0.01),and up-regulation of NCOA4 expression(P0.01)and down-regulation of ferritin and GPX4(P0.01).However,Rasd1 knockdown reversed all above changes with statistical significances(all P0.01).Conclusion Rasd1 promotes ferroptosis in oligodendrocytes through ferritinophagy,and then is involved in white matter injury after SAH.