Ferroptosis is a type of programmed cell death dependent on iron.It is different from other forms of cell death such as apoptosis,classic necrosis and autophagy.Ferroptosis is involved in many neurodegenerative diseas...Ferroptosis is a type of programmed cell death dependent on iron.It is different from other forms of cell death such as apoptosis,classic necrosis and autophagy.Ferroptosis is involved in many neurodegenerative diseases.The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood.To test its toxicity,glutamate(1.25–20 mM)was applied to HT-22 cells for 12 to 48 hours.The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate.Cells were cultured with 3–12μM ferrostatin-1,an inhibitor of ferroptosis,for 12 hours before exposure to glutamate.The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity.Damage to cell structures was observed under light and by transmission electron microscopy.The release of lactate dehydrogenase was detected by the commercial kit.Reactive oxygen species were measured by flow cytometry.Glutathione peroxidase activity,superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit.Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction.Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis.Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells,improving the survival rate,reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure.However,it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells.Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity.It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor(erythroid-derived)-like 2 in glutamate-injured HT-22 cells.Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone(2–8μM),autophagy inhibitor 3-methyladenine(100–400μM)or necrosis inhibitor necrostatin-1(10–40μM)had no effect on glutamate induced cell damage.However,the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death.Thus,the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.展开更多
以传统药用植物意大利牛舌草(Anchusa italica Retz.)的临床功效和现代药理学研究为基础,通过构建体外抑郁症模型,系统研究牛舌草的药理作用及物质基础.HPLC检测不同产地(新疆、巴基斯坦、海南)牛舌草总黄酮(TFAI)的化学成分;然后用海...以传统药用植物意大利牛舌草(Anchusa italica Retz.)的临床功效和现代药理学研究为基础,通过构建体外抑郁症模型,系统研究牛舌草的药理作用及物质基础.HPLC检测不同产地(新疆、巴基斯坦、海南)牛舌草总黄酮(TFAI)的化学成分;然后用海马细胞(HT-22)通过皮质酮诱导建立神经细胞损伤模型,MTT法检测了5种牛舌草对HT-22细胞活力的影响,并计算其EC_(50).结果表明不同产地TFAI在500µg/mL浓度下对HT-22细胞均未表现出明显的细胞毒性作用,且对HT-22细胞具有显著的增殖作用,对过量皮质酮诱导的海马细胞损伤具有明显的细胞保护作用,比较5批牛舌草中的总黄酮含量和EC_(50)值,海南产地的牛舌草中的黄酮类含量最高,且对HT-22细胞的增殖作用EC_(50)值最低,具有较高的效价.研究结果说明五批TFAI提取物对皮质酮诱导的HT-22海马细胞损伤均具有保护作用,可以不同程度地增加HT-22细胞活力,具有显著的神经保护作用.展开更多
文摘Ferroptosis is a type of programmed cell death dependent on iron.It is different from other forms of cell death such as apoptosis,classic necrosis and autophagy.Ferroptosis is involved in many neurodegenerative diseases.The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood.To test its toxicity,glutamate(1.25–20 mM)was applied to HT-22 cells for 12 to 48 hours.The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate.Cells were cultured with 3–12μM ferrostatin-1,an inhibitor of ferroptosis,for 12 hours before exposure to glutamate.The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity.Damage to cell structures was observed under light and by transmission electron microscopy.The release of lactate dehydrogenase was detected by the commercial kit.Reactive oxygen species were measured by flow cytometry.Glutathione peroxidase activity,superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit.Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction.Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis.Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells,improving the survival rate,reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure.However,it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells.Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity.It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor(erythroid-derived)-like 2 in glutamate-injured HT-22 cells.Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone(2–8μM),autophagy inhibitor 3-methyladenine(100–400μM)or necrosis inhibitor necrostatin-1(10–40μM)had no effect on glutamate induced cell damage.However,the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death.Thus,the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.
文摘以传统药用植物意大利牛舌草(Anchusa italica Retz.)的临床功效和现代药理学研究为基础,通过构建体外抑郁症模型,系统研究牛舌草的药理作用及物质基础.HPLC检测不同产地(新疆、巴基斯坦、海南)牛舌草总黄酮(TFAI)的化学成分;然后用海马细胞(HT-22)通过皮质酮诱导建立神经细胞损伤模型,MTT法检测了5种牛舌草对HT-22细胞活力的影响,并计算其EC_(50).结果表明不同产地TFAI在500µg/mL浓度下对HT-22细胞均未表现出明显的细胞毒性作用,且对HT-22细胞具有显著的增殖作用,对过量皮质酮诱导的海马细胞损伤具有明显的细胞保护作用,比较5批牛舌草中的总黄酮含量和EC_(50)值,海南产地的牛舌草中的黄酮类含量最高,且对HT-22细胞的增殖作用EC_(50)值最低,具有较高的效价.研究结果说明五批TFAI提取物对皮质酮诱导的HT-22海马细胞损伤均具有保护作用,可以不同程度地增加HT-22细胞活力,具有显著的神经保护作用.
