褪黑素通过Akt/Nrf2/Gpx4减轻新生大鼠海马神经元铁死亡的研究  

Melatonin attenuates ferroptosis in hippocampal neurons of neonatal rats through Akt/Nrf2/Gpx4 pathway

作　　者：孙梦雅[1] 赵亮[2] 姜红[1] 张沂洁[1] 王倩[1] 张璐璐 秦苗[1] Sun Mengya;Zhao Liang;Jiang Hong;Zhang Yijie;Wang Qian;Zhang Lulu;Qin Miao(Department of Neonatology,Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Ultrasound,Affiliated Hospital of Qingdao University,Qingdao 266000,China)

机构地区：[1]青岛大学附属医院新生儿科,青岛266000 [2]青岛大学附属医院超声科,青岛266000

出　　处：《中华新生儿科杂志（中英文）》2024年第8期486-492,共7页Chinese Journal of Neonatology

基　　金：山东省自然科学基金青年项目(ZR2020QH054)。

摘　　要：目的探讨褪黑素对早产儿脑损伤(brain injury in premature infants,BIPI)模型大鼠海马神经元损伤的保护作用及机制。方法选择3日龄雌性SD大鼠144只,随机分为假手术组、BIPI模型组、褪黑素干预组、褪黑素+MK-2206干预组各36只。BIPI模型组、褪黑素干预组和褪黑素+MK-2206干预组采用脂多糖预处理+缺氧缺血法制作BIPI模型,后两组在造模过程中分别应用褪黑素和褪黑素+MK-2206进行干预。各组于实验第7天处死30只大鼠,取右侧海马组织,采用苏木精-伊红染色观察海马病理损伤情况,透射电镜观察线粒体超微结构改变,实时荧光定量聚合酶链反应和Western blot检测海马神经元p-蛋白激酶B(protein kinase B,PKB或Akt)、核因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)mRNA和蛋白表达情况,活性氧(reactive oxygen species,ROS)、谷胱甘肽(glutathione,GSH)和组织铁含量检测试剂盒检测ROS、GSH和铁水平。各组剩余6只大鼠于日龄21 d时进行Morris水迷宫实验,检测大鼠学习记忆能力。结果与假手术组相比,BIPI模型组大鼠海马CA1区神经元细胞排列散乱、数量减少、细胞核变小;线粒体体积缩小、基质溶解、嵴消失,呈明显的铁死亡改变;海马组织ROS和铁含量升高,GSH水平下降;学习和记忆能力降低;p-Akt/Akt、Nrf2 mRNA和蛋白表达升高,但GPX4 mRNA和蛋白表达降低。与BIPI模型组相比,褪黑素干预组大鼠海马CA1区的病理改变和线粒体病变减轻,ROS和铁含量降低,GSH水平升高,学习记忆能力改善,p-Akt/Akt、Nrf2和GPX4 mRNA和蛋白表达升高。与褪黑素干预组相比,褪黑素+MK-2206干预组p-Akt/Akt、Nrf2和GPX4 mRNA和蛋白表达均下降。结论BIPI模型大鼠海马神经元存在铁死亡,褪黑素可通过调节Akt/Nrf2/Gpx4通路抑制海马神经元铁死亡的发生,改善BIPI模型大鼠的学习记忆功能。ObjectiveTo study the protective effects and mechanisms of melatonin on hippocampal injury in neonatal rat models of brain injury in premature infants(BIPI).MethodsA total of 144 neonatal female SD rats postnatal day 3 were randomly assigned into sham operation group,BIPI group,melatonin group and melatonin+MK-2206 group(n=36 for each group).BIPI model was established using lipopolysaccharide injection,right carotid artery ligation and hypoxia-ischemia treatment.Melatonin group and melatonin+MK-2206 group were further treated with melatonin and melatonin+MK-2206,respectively.30 rats in each group were sacrificed on d7 of the experiment and the right hippocampal tissues were harvested.Hematoxylin-eosinstaining was used to observe the pathological injuries of hippocampal neurons.The ultrastructural changes of mitochondria were studied using electron microscope.The mRNA and protein expressions of protein kinase B(PKB or Akt),nuclear factor-erythroid 2-related factor 2(Nrf2)and glutathione peroxidase 4(GPX4)were detected using RT-PCR and Western blot.The levels of reactive oxygen species(ROS),glutathione(GSH)and iron were detected using ROS,GSH and iron detection kits.The remaining 6 rats in each group received Morris water maze test for learning and memory abilities on d21.ResultsCompared with sham-operated group,neurons of CA1 area in BIPI group were scattered and decreased,with smaller nuclei;mitochondria shrinked,with matrix lysis and cristae disappearing,presenting ferroptosis alterations;hippocampal tissues had elevated levels of ROS and iron,and decreased levels of GSH;learning and memory abilities were reduced;and the mRNA and protein expressions of PKB/Akt and Nrf2 were elevated,but mRNA and protein expressions of GPX4 were decreased.Compared with BIPI group,the melatonin group showed reduced pathological changes and mitochondrial injuries in CA1 area of the hippocampus,decreased ROS and iron levels,increased GSH levels,improved learning and memory abilities and elevated p-Akt/Akt,Nrf2,and GPX4 mRNA and prot

关 键 词：早产儿脑损伤 褪黑素 海马神经元 铁死亡 Akt/Nrf2/Gpx4通路 

分 类 号：R722.1[医药卫生—儿科] R-332[医药卫生—临床医学]