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.FeRhoNoxTM -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.
作者
宣文婷
杨泽勇
季雅茹
金卫林
李俊
李元海
XUAN Wen-ting;YANG Ze-yong;JI Ya-ru;JIN Wei-lin;LI Jun;LI Yuan-hai(Dept of Anesthesiology,the First Affiliated Hospital of Anhui Medical University,Hefei230022,China;Dept of Anesthesiology,International Peace Maternity & Child Health Hospital of China,Shanghai200030,China;School of Electronic Information and Electrical Engineering,Shanghai Jiao Tong University,Shanghai200240,China;School of Pharmacy,Anhui Medical University,Hefei230032,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2019年第5期654-660,共7页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81401279)
安徽省研究与开发计划项目(No 17041f0804021)
