远志皂苷元对缺氧/复氧诱导的PC12细胞铁死亡的影响

Effects of senegenin on hypoxia/reoxygenation-induced ferroptosis in PC12 cells

目的:探讨远志皂苷元(Sen)对缺氧/复氧(H/R)损伤大鼠肾上腺嗜铬细胞瘤PC12细胞模型中铁死亡的影响及可能的机制。方法:培养高分化PC12细胞,构建H/R损伤细胞模型和erastin诱导铁死亡细胞模型。采用MTT法检测细胞活力;显微镜下观察细胞形态变化,微管相关蛋白2(MAP-2)荧光染色观察神经元突触的变化;检测细胞上清液乳酸脱氢酶(LDH)及细胞内亚铁离子(Fe2+)、总谷胱甘肽(GSH)和谷胱甘肽过氧化物酶(GSH-Px)水平;荧光探针DCFH-DA标记检测活性氧簇(ROS)水平;JC-1染色观察线粒体膜电位(ΔΨm)的变化;Western blot检测铁死亡相关蛋白(ACSL4、GPX4和SLC7A11)的表达。结果:确定H/R造模时间为缺氧12 h,复氧2 h,细胞活力降低约50%(P<0.05);铁死亡造模条件为10µmol/L铁死亡诱导剂erastin作用14 h,细胞活力降低约50%(P<0.05)。不同浓度Sen或铁死亡抑制剂ferrostatin-1(Fer-1)作用14 h后,细胞活力无显著变化(P>0.05)。Sen或Fer-1干预后,与模型组相比,细胞活力显著升高(P<0.05)。与对照组相比,H/R和erastin组细胞显著变小变圆,突触变短(P<0.05),细胞上清液LDH水平及细胞内Fe2+和ROS水平显著升高(P<0.05),细胞内GSH水平、GSH-Px活性和ΔΨm水平显著降低(P<0.05)。Sen或Fer-1干预后,与模型组相比,细胞形态接近正常,突触显著变长(P<0.05),细胞上清液LDH水平及细胞内Fe2+和ROS水平显著降低(P<0.05),细胞内GSH水平、GSH-Px活性和ΔΨm水平显著升高(P<0.05)。与对照组相比,H/R组和erastin组ACSL4蛋白表达显著升高,GPX4和SLC7A11蛋白表达显著降低(P<0.05);Sen或Fer-1干预后,与模型组相比,ACSL4蛋白表达显著降低,GPX4和SLC7A11蛋白表达显著升高(P<0.05)。结论:H/R损伤PC12细胞模型中存在铁死亡,且Sen能抑制铁死亡,发挥保护细胞的作用,其机制可能与Sen提高细胞清除氧自由基能力、降低细胞内Fe2+水平和上调GPX4蛋白表达等有关。

AIM:To explore the effect of senegenin(Sen)on ferroptosis in rat adrenal gland pheochromocytoma PC12 cell model of hypoxia/reoxygenation(H/R)injury and its possible mechanism.METHODS:Cultured well-differentiated PC12 cells were used to construct the cell models of H/R injury and erastin-induced ferroptosis.The cell viability was measured by MTT assay.The changes of cell morphology and microtubule-associated protein-2(MAP-2)fluorescence staining for neuron synapse were observed under microscope.The levels of lactate dehydrogenase(LDH),intracellular ferrous ion(Fe2+),total glutathione(GSH)and glutathione peroxidase(GSH-Px)were also measured.Fluorescent probe DCFH-DA labeling was used to detect the levels of reactive oxygen species(ROS).JC-1 staining was used to observe the changes of intracellular mitochondrial membrane potential(ΔΨm).The expression of ferroptosis-related proteins ACSL4,GPX4 and SLC7A11 was determined by Western blot.RESULTS:The optimal H/R modeling time was 12 h of hypoxia and 2 h of reoxygenation,and the cell viability was reduced by about 50%(P<0.05).The condition of ferroptosis modeling was exposure to ferroptosis inducer erastin at 10μmol/L for 14 h,and the cell viability was reduced by about 50%(P<0.05).Different concentrations of Sen and ferroptosis inhibitor ferrostatin-1(Fer-1)had no significant effect on the cell viability after 14 h of treatment(P>0.05).The cell viability was significantly increased after Sen or Fer-1 intervention compared with model group(P<0.05).Compared with control group,the cells in H/R and erastin groups were significantly smaller and rounder,the synapses were shorter(P<0.05),the LDH level in the cell supernatant and the intracellular Fe2+and ROS levels were significantly increased(P<0.05),while intracellular GSH level,GSH-Px activity andΔΨm level were significantly reduced(P<0.05).After Sen or Fer-1 intervention,the cell morphological change was close to normal,the synapses were significantly longer(P<0.05),the LDH level in the cell supernatant and the intracellular Fe2+and ROS levels were significantly reduced(P<0.05),while intracellular GSH level,GSH-Px activity andΔΨm level were significantly increased(P<0.05)compared with model group.Compared with control group,the protein expression of ACSL4 in H/R and erastin groups was increased,while the protein expression of GPX4 and SLC7A11 was decreased(P<0.05).After intervention with Sen or Fer-1,the protein expression of ACSL4 was decreased,while the protein expression of GPX4 and SLC7A11 was increased compared with model group(P<0.05).CONCLUSION:Ferroptosis exists in the H/R-injured PC12 cell model,while Sen protects the cells against ferroptosis.The mechanism may be related to the ability of Sen to scavenge oxygen free radicals,reduce intracellular Fe2+levels,and up-regulate GPX4 protein.