Ferrostatin-1通过抑制Ferroptosis保护谷氨酸诱导的HT-22细胞损伤

Protection of Ferrostatin-1 on HT-22 cells damage induced by glutamic acid via inhibition of Ferroptosis

目的:观察谷氨酸诱导HT-22细胞发生Ferroptosis的现象,以及初步探讨Ferrostatin-1对谷氨酸损伤的HT-22细胞保护作用机制。方法:建立谷氨酸损伤HT-22细胞研究模型,并采用MTT检测细胞存活率,同时观察3-Methyladenine、Z-VAD-FMK、Necrostatin-1、Ferrostatin-1对细胞存活率的影响;乳酸脱氢酶(LDH)释放法检测乳酸脱氢酶的漏出率;流式细胞术检测细胞内活性氧(ROS)的含量变化;生化法检测超氧化物歧化酶(SOD)、谷胱甘肽(GSH)变化。结果:5mmol/L谷氨酸作用HT-22细胞24 h后,能明显抑制细胞生长(P<0.01);增加细胞LDH的释放;ROS含量增加;SOD活性下降;GSH水平下降。Ferrostatin-1预处理后,谷氨酸诱导的HT-22细胞存活率显著增加(P<0.01),而3-Methyladenine、Z-VAD-FMK、Necrostatin-1不能显著阻止谷氨酸对HT-22细胞的生长抑制作用;同时,Ferrostatin-1能够阻止谷氨酸引起的细胞内LDH释放,减少ROS生成;SOD活性增加;促进GSH水平增加。结论:谷氨酸损伤的HT-22细胞可以被Ferroptosis特异性抑制剂Ferrostatin-1阻断,不能被其他死亡抑制剂阻断,表明谷氨酸诱导的HT-22细胞发生了Ferroptosis。谷氨酸通过引起ROS升高,降低细胞内GSH水平、SOD活性诱导HT-22细胞发生Ferroptosis。Ferrostatin-1对谷氨酸损伤的HT-22细胞具有保护作用,其作用机制与抗氧化的发生有关。

AIM： To investigate the identification of Ferroptosis in HT-22 cells induced by glutamic acid and study the protective effects of Ferrostatin-1 against glutamic acid-induced injury in HT-22 cells. METHODS： Glutamic acid-induced HT-22 cell injury model was established to detect the cell survival rate by MTT,the leakage rate of lactic dehydrogenases using LDH,the content of intracellular reactive oxygen species was detected by flow cytometry,and the level of glutathione and SOD by biochemical methods. RESULTS： Treatment of HT-22 mouse neuronal cell line with 5 mmol / L glutamic acid resulted in significantly cell death,which was inhibited by Ferrostatin-1 in a dose-dependent manner. In contrast,3-Methyladenine,Z-VAD-FMK,Necrostatin-1 cannot reverse glutamic acid-induced cell death. Ferrostatin-1 not only decreased glutamic acid-induced production of reactive oxygen species but also restored glutamic acid-induced depletion of glutathione and the level of SOD. CONCLUSION：Glutamic acid-induced HT-22 cell death was significantly inhibited by the Ferroptosis inhibitors Ferrostatin-1. And other inhibitors cannot reverse the cells death. We confirmed that the identification of Ferroptosis in HT-22 cells induced by glutamic acid.Glutamic acid induces Ferroptosis only by decreasing cellular GSH levels and SOD activity,but also increasing ROS levels. The protective mechanism may be related to preventing occurrence of Ferroptosis and oxidative stress in HT-22 cell.