p53抑制铁死亡抵抗HT22细胞谷氨酸神经毒性
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摘要
目的探讨凋亡诱导因子p53抑制HT22细胞(小鼠海马神经元细胞)谷氨酸毒性的相关机制。方法 CCK-8法和PI/Hoechst荧光双染法检测HT22细胞存活率;Western blot检测p53以及胱氨酸/谷氨酸反向转运系统xCT(cystine/glutamate antiporter或system Xc~-, xCT或SLC7A11)蛋白表达;DHE荧光探针检测HT22细胞内活性氧簇(reactive oxygen species, ROS);BODIPY 581/591 C11脂质氧化探针和4-HNE免疫荧光染色,检测细胞内脂质氧化;FeRhoNox~(TM)-1荧光探针检测细胞内铁离子。结果 1μmol·L~(-1) Tenovin-1作用6 h后,p53蛋白的表达水平相较于对照组明显上升。在p53高表达后,经谷氨酸盐(glutamate, Glu)和铁死亡诱导剂Erastin处理8 h后,相较于仅Glu和Erastin处理组细胞死亡率明显下降,xCT蛋白表达水平明显上升。p53高表达后的Glu处理组细胞内ROS、脂质氧化以及Fe~(2+)的水平明显低于单纯Glu处理组。结论 p53可能通过调节xCT表达来抑制铁死亡,进而保护神经不受谷氨酸的损伤。
Aim To investigate the mechanism of the inhibitory effect of apoptosis inducible factor p53 on the toxicity of glutamate in HT22 cell lines.Methods CCK-8 and PI/Hoechst fluorescence double staining were used to detect the survival rate of HT22 cells. Western blot was applied to determine the protein expression levels of p53 and xCT. DHE fluorescence staining technique was employed to detect the intracellular reactive oxygen species(ROS), and BODIPY 581/591 C11 lipid peroxidation sensor was utilized to confirm intracellular lipid oxidant situation. FeRhoNox~(TM)-1 fluorescent probe was used to assess intracellular ferrous ions.Results After 6 h treatment of 1 μmol·L~(-1) Tenovin-1, the protein expression levels of p53 obviously increased. After high expression of p53, treatment with Glu and erastin(ferroptosis inducer) for 8 h(Glu-p53, Era-p53 groups), cell death rate decreased significantly, and the expression levels of xCT increased significantly, compared with only Glu and erastin treated groups(Glu, Era groups). Intracelluar ROS levels, lipid oxidant situations, ferrous ions declined in Glu-p53 groups compared to those of Glu group.Conclusions p53 inhibits the neurotoxicity induced by Glu, which may be related to the inhibition of ferroptosis by regulating xCT expression.
Aim To investigate the mechanism of the inhibitory effect of apoptosis inducible factor p53 on the toxicity of glutamate in HT22 cell lines.Methods CCK-8 and PI/Hoechst fluorescence double staining were used to detect the survival rate of HT22 cells. Western blot was applied to determine the protein expression levels of p53 and xCT. DHE fluorescence staining technique was employed to detect the intracellular reactive oxygen species(ROS), and BODIPY 581/591 C11 lipid peroxidation sensor was utilized to confirm intracellular lipid oxidant situation. FeRhoNox~(TM)-1 fluorescent probe was used to assess intracellular ferrous ions.Results After 6 h treatment of 1 μmol·L~(-1) Tenovin-1, the protein expression levels of p53 obviously increased. After high expression of p53, treatment with Glu and erastin(ferroptosis inducer) for 8 h(Glu-p53, Era-p53 groups), cell death rate decreased significantly, and the expression levels of xCT increased significantly, compared with only Glu and erastin treated groups(Glu, Era groups). Intracelluar ROS levels, lipid oxidant situations, ferrous ions declined in Glu-p53 groups compared to those of Glu group.Conclusions p53 inhibits the neurotoxicity induced by Glu, which may be related to the inhibition of ferroptosis by regulating xCT expression.
引文
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[3] Kang Y Y,Tiziani S,Park G,et al.Cellular protection using Flt3 and PI3Kα inhibitors demonstrates multiple mechanisms of oxidative glutamate toxicity[J].Nat Commun,2014,5(1):3672.
[4] Xu X S,Chua C C,Kong J M,et al.Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells[J].J Neurochem,2007,103(5):2004-14.
[5] Landshamer S,Hoehn M,Barth N,et al.Bid-induced release of AIF from mitochondria causes immediate neuronal cell death[J].Cell Death Differ,2008,15(10):1553-63.
[6] Checler F,Costa C A.p53 in neurodegenerative diseases and brain cancers[J].Pharmacol Ther,2014,142(1):99-113.
[7] Jiang L,Kong N,Li T Y,et al.Ferroptosis as a p53-mediated activity during tumour suppression[J].Nature,2015,520(7545):57-62.
[8] Hambright W S,Fonseca R S,Chen L J,et al.Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration[J].Redox Biol,2017,12:8-17.
[9] Chen L J,Hambright W S,Na R,et al.Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis[J].J Biol Chem,2015,290(47):28097-106.
[10] Van D B,Gouel F,Jonneaux A,et al.Ferroptosis,a newly characterized form of cell death in Parkinson?s disease that is regulated by PKC[J].Neurobiol Dis,2016,94:169-78.
[11] 姜懿纳,娄钰霞,张钊,等.Parkin相关疾病的研究进展[J].中国药理学通报,2016,32(4):455-8.[11] Jiang Y N,Lou Y X,Zhang Z,et al.Some diseases caused by Parkin[J].Chin Pharmacol Bull,2016,32(4):455-8.
[12] Wang S J,Li D W,Ou Y,et al.Acetylation is crucial for p53-mediated ferroptosis and tumor suppression[J].Cell Rep,2016,17(2):366-73.
[13] Stockwell B R,Friedmann Angeli J P,Bayir H,et al.Ferroptosis:a regulated cell death nexus linking metabolism.Redox biology,and disease[J].Cell,2017,171(2):273-85.