去铁胺通过调控铁自噬减轻HT22细胞缺氧复氧损伤

Deferoxamine Alleviates HT22 Cell Damage Induced by Hypoxia/Reoxygenation Injury Through Regulating Ferritinophagy

目的探讨去铁胺(deferoxamine,DFO)通过调控核受体共激活因子4(nuclear receptor coactivator 4,NCOA4)介导的铁自噬在减轻小鼠海马神经元细胞系(HT22)缺氧复氧(hypoxia/reoxygenation,HR)损伤中的作用。方法采用随机数字表法将HT22细胞系随机分为对照组(Ctrl组)、缺氧复氧组(HR组)和缺氧复氧+DFO组(HR+DFO组)。HR组无糖缺氧培养6h后,复糖复氧继续培养24h,建立神经元HR模型。DFO组于HR前给予150μmol/L的DFO预处理24h。免疫荧光法检测细胞活性与毒性、活性氧物质(reactive oxygen species,ROS),酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)及亚铁离子,电镜检测自噬小体,Western blot法检测NCOA4、LC3B、Beclin-1和铁蛋白的表达。结果与Ctrl组比较,HR组细胞生存率、SOD降低,MDA、ROS、NCOA4、LC3B、Beclin-1、自噬小体、铁蛋白、亚铁离子增多(P<0.05);与HR组比较,HR+DFO组细胞生存率、SOD升高,MDA、ROS、NCOA4、LC3B、Beclin-1、自噬小体、铁蛋白、亚铁离子减少(P<0.05)。结论DFO通过降低NCOA4表达,下调氧化应激和亚铁离子含量从而有效减轻HT22细胞HR损伤。

Objective To investigate the role of deferoxamine(DFO)in alleviating hypoxia/reoxygenation(HR)injury in mouse hippocampal neuronal cells(HT22)by regulating ferritinophagy mediated by nuclear receptor coactivator 4(NCOA4).Methods HT22 cell lines was randomly divided into control group using a random number table(Ctrl group),hypoxia/reoxygenation group(HR group),hypoxia/reoxygenation+DFO group(HR+DFO group).The HR group was cultured under sugar-free hypoxia for 6hours,followed by sugar-reoxygenation for 24hours to establish the neuronal HR model.The DFO group received a pre-treatment of 15Oμmol/L DFO for 24hours before HR.Immunofluorescence was used to detect live-dead cell staining and reactive oxygen species(ROS)levels.Enzyme-linked immunosorbent assay(ELISA)was used to detect the superoxide dismutase(SOD),malondialdehyde(MDA)and fer-rous ion levels.Autophagosomes were observed by electron microscopy.The expression levels of NCOA4,LC3B,Beclin-1 and ferritin was detectd by Western blot.Results Compared with the Ctrl group,the cell survival rate and SOD levels were decreased in HR group,MDA,ROS,NCOA4,LC3B,Beclin-1,autophagosomes,ferritin and ferrous ion content were increased(P<0.05).Compared with the HR group,the cell survival rate and SOD levels were increased in HR+DFO group,MDA,ROS,NCOA4,LC3B,Beclin-1,auto-phagosomes,ferritin and ferrous ion content were decreased(P<0.05).Conclusion DFO downregulates oxidative stress levels and fer-rous ion content by decreasing the expression of NCOA4,effectively alleviating HT22 cell damage caused by HR injury.