摘要
目的探究硫化氢(H_(2)S)对血红素诱导的小鼠海马神经元铁死亡的作用。方法取小鼠海马神经元细胞株HT-22,将其分为空白对照组和不同浓度(50、75、100、125μmol/L)的血红素组。血红素组细胞与血红素共培养24 h构建损伤模型。取HT-22细胞,将其分为75μmol/L血红素组和75μmol/L血红素+不同浓度(50、100、200μmol/L)硫氢化钠(NaHS)组,后者与血红素和NaHS共培养24 h。采用CCK-8法检测各组细胞的存活率,蛋白质免疫印迹(WB)法测定溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)的表达量;采用钙黄绿素/碘化丙啶(Calcein-AM/PI)双荧光染色法检测活细胞和死细胞,活性氧荧光探针(DCFH-DA)检测活性氧含量,透射电镜观察细胞的超微结构,亚铁离子(Fe^(2+))荧光探针检测Fe^(2+)含量,免疫荧光染色法检测GPX4的表达。结果CCK-8和WB结果显示,与空白对照组比较,不同浓度血红素组HT-22细胞的存活率下降、SLC7A11和GPX4的表达水平降低,且均随着血红素浓度的升高呈明显的下降趋势。差异均有统计学意义(均P<0.05)。CCK-8结果显示,75μmol/L血红素+不同浓度(50、100、200μmol/L)NaHS组较75μmol/L血红素组的细胞存活率均有不同程度增加。与75μmol/L血红素组比较,Calcein-AM/PI双荧光染色结果显示,75μmol/L血红素+100μmol/L NaHS组的PI阳性细胞减少;活性氧和Fe^(2+)检测结果显示,75μmol/L血红素+100μmol/L NaHS组的活性氧及Fe^(2+)水平下降;WB和免疫荧光染色法结果显示,75μmol/L血红素+100μmol/L NaHS组的SCL7A11、GPX4蛋白表达增加。上述数据差异均有统计学意义(均P<0.05);透射电镜显示,75μmol/L血红素+100μmol/L NaHS组的线粒体皱缩、嵴减少等细胞铁死亡特征性改变较75μmol/L血红素组减少。结论H_(2)S通过降低血红素诱导的氧化应激损伤及铁离子累积,提高血红素损伤的HT-22细胞的SLC7A11、GPX4活性,减轻铁死亡,发挥细胞保护作用。
Objective To investigate the effect of hydrogen sulfide(H_(2)S)on hemin-induced ferroptosis in mouse hippocampal neurons.Methods HT-22 cells were divided into normal group and hemin injury group with different concentrations(50,75,100 and 125μmol/L).The hemin injury group cells were injured by hemin for 24 h to establish the cell injury model.HT-22 cells were divided into hemin 75μmol/L group and hemin 75μmol/L+NaHS treatment group with different concentrations(50,100 and 200μmol/L).NaHS treatment group cells were treated with hemin and NaHS for 24 h.The cell viability was assessed by Cell Counting Kit-8(CCK-8).The expression of glutathione peroxidase 4(GPX4)and solute carrier family 7 member 11(SLC7A11)was detected by Western blotting.Necrotic cell death was measured by Calcein-AM/propidium iodide(Calcein-AM/PI)staining.The 2′,7′-dichlorodihydro-fluorescein diacetate(DCFH-DA)fluorescent probe was used to detect reactive oxygen species(ROS).Ultrastructural images were observed using a transmission electron microscope.Ferrous ion content was performed using the FerroOrange kit.Moreover,GPX4 was identified by immunofluorescence staining.Results Both CCK-8 and Western blotting results indicated that the survival rate and the expression levels of SLC7A11 and GPX4 decreased in different hemin concentration groups and showed a downward trend with the increase of hemin concentration;the differences were statistically significant(all P<0.05).CCK-8 assay results indicated that the survival rate in hemin 75μmol/L+NaHS(50,100 and 200μmol/L)groups was higher than that in hemin 75μmol/L group.Calcein-AM/PI assay results indicated that the PI positive cells in hemin 75μmol/L+NaHS 100μmol/L group was less than that in hemin 75μmol/L group.Both DCFH-DA and FerroOrange staining results indicated that the relative levels of ROS and Fe^(2+)in hemin 75μmol/L+100μmol/L NaHS group were lower than those in hemin 75μmol/L group.Both Western blotting and immunofluorescence staining indicated that hemin 75μmol/L+100μmol/L NaHS group had significantly increased SCL7A11 and GPX4 level as compared with hemin 75μmol/L group.The differences as above were all statistically significant(all P<0.05).Electron microscope investigation showed shrunken mitochondria and absence of mitochondria cristae in hemin 75μmol/L group and obvious improvement of mitochondrial morphology in hemin 75μmol/L+100μmol/L NaHS group.Conclusion H_(2)S treatment protects HT-22 mouse hippocampal neurons from hemin injury via decreasing oxidative stress,reducing iron accumulation,enhancing SLC7A11 and GPX4 level and inhibiting ferroptosis.
作者
鱼洋
李星慧
陈显金
李兴隆
魏嘉良
付利
张庆华
Yu Yang;Li Xinghui;Chen Xianjin;Li Xinglong;Wei Jialiang;Fu Li;Zhang Qinghua(Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging,National-Regional Key Technology Engineering Laboratory for Medical Ultrasound,School of Biomedical Engineering,Shenzhen University Medical School,Shenzhen 518060,China;Department of Neurosurgery,Huazhong University of Science and Technology Union Shenzhen Hospital,Shenzhen 518052,China;Colloge of Public Health,Ningxia Medical University,Yinchuan 750004,China;Department of Neurosurgery,The First Affiliated Hospital of Air Force Military Medical University,Xi′an 710032,China)
出处
《中华神经外科杂志》
CSCD
北大核心
2023年第4期401-408,共8页
Chinese Journal of Neurosurgery
基金
国家自然科学基金(82101374)
深圳市科创委自然资金(JCYJ20220530142001004)
深圳市南山区卫生健康局卫生科技项目(NS2022075)。
关键词
硫化氢
海马
神经元
体外研究
铁死亡
氧化应激
Hydrogen sulfide
Hippocampus
Neurons
In vitro
Ferroptosis
Oxidative stress
