天麻素抑制H2O2诱导的PC12细胞铁死亡

目的：研究天麻素对过氧化氢（H2O2）损伤的PC12细胞的保护作用及机制。方法：建立H2O2损伤PC12细胞神经损伤模型。MTT法检测细胞活力并确定H2O2最佳造模浓度，筛选和确定天麻素最佳处理浓度。试剂盒检测丙二醛（MDA）、总谷胱甘肽（GSH）、超氧化物歧化酶（SOD）水平和活力变化；流式细胞仪检测细胞脂质活性氧（ROS）水平；透射电子显微镜观察PC12细胞形态变化；蛋白免疫印迹法（Western blot）检测p53、谷胱甘肽过氧化物酶4（GPX4）蛋白表达变化。结果：MTT法实验结果显示，50 μmol/L H2O2处理PC12细胞48 h，细胞生长抑制率接近50%；而天麻素（1、5、25 μmol/L）预处理4 h可提高H2O2损伤细胞的存活率，且其作用呈浓度依赖性。MDA、GSH、SOD检测结果表明天麻素降低H2O2处理后的MDA水平，并提高SOD和GSH活力。流式细胞仪检测发现，H2O2损伤后细胞内ROS的含量升高，而天麻素预处理后有效降低H2O2损伤的细胞内ROS的含量。透射电子显微镜观察结果显示，与正常组相比，50 μmol/L H2O2处理后PC12细胞的线粒体脊变厚，线粒体体积变小；天麻素预保护处理后，PC12细胞的线粒体形态趋于正常状态。Western blot结果表明，天麻素预保护后可升高H2O2损伤后GPX4蛋白表达水平，并降低p53蛋白表达水平。 结论：H2O2可通过增加PC12细胞内氧化应激水平引起铁死亡；天麻素预保护后可上调细胞内GPX4蛋白表达和下调p53蛋白表达，从而减少细胞氧化应激产生，抑制细胞铁死亡发生而发挥保护PC12细胞的作用。

Gastrodin inhibits H2O2-induced ferroptosis of PC12 cells

AIM: To investigate the protective mechanism of gastrodin against hydrogen peroxide (H2O2) damage in PC12 cells. METHODS: PC12 cells were treated with H2O2 to establish injury model, MTT assay was used to detect cell viability to establish the best suitable concentration of the model and the optimal treatment concentration of gastrodin were determined. Biochemical methods were used to analyze MDA, GSH and SOD activity. Flow cytometry were used to detect the change of ROS. Inverted transmission electron microscopy was used to observe PC12 cell morphology. Western blot was used to analyze the expression of p53, GPX4 protein. RESULTS: MTT assay showed that 50 μmol/L H2O2 inhibited PC12 cells after 48 h of treatment, while 1, 5, 25 μmol/L gastrodin cells reduced the inhibition of H2O2 on PC12 cells. MDA, GSH and SOD results showed that gastrodin reduced MDA level and increased SOD and GSH level after H2O2 treatment. Flow cytometry detection showed that gastrodin pretreatment reduced the content of intracellular reactive oxygen species after H2O2 injury. Transmission electron microscopy showed that the mitochondrial ridge was thicker and the mitochondrial volume was smaller in the 50 μmol/L H2O2 group than in the normal group, while the mitochondrial morphology tend to normal after gastrodin preconditioned PC12 cells. Western blot results showed that gastrodin could increase GPX4 protein expression and decrease p53 protein expression after H2O2 injury. CONCLUSION: H2O2 can cause ferroptosis by increasing the level of oxidative stress in PC12 cells, and gastrodin pre-protection can reduce the generation of oxidative stress and prevent ferroptosis which may be related to the up-regulation of GPX4 protein expression and down-regulation of p53 protein expression.