Ferrostatin-1保护谷氨酸诱导的HT-22细胞Ferroptosis机制研究
发布时间：2018-09-04 06:45
【摘要】：神经退行性疾病(Neurodegenerative disease)是由于神经细胞出现渐进性功能异常所造成的功能障碍和机体行为异常的常见病,如阿尔茨海默病、帕金森病、亨廷顿病等。这类疾病的发生涉及神经元之间一系列复杂的作用,包括氧化应激、遗传因素和兴奋性氨基酸作用等。谷氨酸,作为哺乳动物脑内最主要的兴奋性神经递质,它在神经元的生存、生长、分化和迁移过程中起着至关重要的作用。研究显示,谷氨酸是造成神经细胞损伤的重要因素,它参与神经元死亡的机制十分复杂,主要表现为谷氨酸的氧化性毒性和兴奋性毒性。其中,谷氨酸氧化毒性引起的一系列改变与新发现的细胞死亡形式Ferroptosis很相似。Ferroptosis是一种不同于自噬、凋亡、坏死等的氧化性细胞死亡方式,它具有独特的形态特征和复杂的生化反应。Ferroptosis的发展进程以活性氧(ROS)异常增多引起的氧化还原失衡为特点。因此,我们认为谷氨酸氧化毒性可能是诱导神经细胞发生Ferroptosis且影响机体的氧化平衡状态,最终导致神经退行性疾病发生的主要原因。那么谷氨酸影响神经细胞死亡的作用途径和机制值得研究。本课题拟通过建立的谷氨酸损伤HT-22细胞的模型,考察Ferroptosis在其中的角色与可能机制,为探索神经疾病的发生提供一些相应的理论依据。目的:(1)研究谷氨酸诱导的神经细胞模型中氧化性损伤的发生;(2)研究谷氨酸诱导的神经细胞死亡与Ferroptosis发生的关系;(3)探讨Ferrostatin-1对谷氨酸损伤的HT-22细胞的保护作用及其相关作用机制。方法:建立谷氨酸损伤HT-22细胞神经损伤模型,采用MTT法和Trypan blue法检测细胞活力并确立谷氨酸最佳造模浓度,同时筛选不同抑制剂(自噬抑制剂3-Methyladenine、凋亡抑制剂Z-VAD-FMK、坏死抑制剂Necrostatin-1、Ferroptosis特异性抑制剂Ferrostatin-1和铁离子螯合剂DFO)对谷氨酸神经损伤模型的影响;不同试剂盒分别检测细胞LDH漏出率、谷胱甘肽GSH、谷胱甘肽过氧化物酶GPX、丙二醛MDA和超氧化物歧化酶SOD各指标活性变化;倒置显微镜、透射电子显微镜观察细胞形态学变化;流式细胞术检测胞质活性氧(ROS)、脂质活性氧(ROS)、线粒体膜电位(△Ψm)、MDC水平;荧光显微镜观察DAPI染色后细胞形态学变化;Western blot法检测Ptgs2、Nrf2、GPX4、FP1相关蛋白变化;Real Time RT-PCR技术检测Ptgs2、GPX4mRNA水平变化。结果:1.MTT和Trypan blue实验结果表明,5m M谷氨酸作用HT-22细胞24h,细胞生长抑制率接近50%,选择此剂量和时间造模;2.细胞形态学观察结果显示,当5 mM谷氨酸作用HT-22细胞24h后,细胞变小变圆,折光度下降,细胞的贴壁状态差,出现损伤状态;3.MTT法结果显示,Ferroptosis特异性抑制剂Ferrostatin-1以及铁离子螯合剂DFO对谷氨酸损伤的HT-22细胞具有保护作用,与模型组相比,细胞活力明显升高(P0.01);同时,细胞形态学观察结果显示,3-12μM Ferrostatin-1预保护后细胞形态较模型组更接近正常细胞。自噬抑制剂3-Methyladenine、凋亡抑制剂Z-VAD-FMK和坏死抑制剂Necrostatin-1并未显著提高细胞活力;4.DAPI染光染色结果表明,Ferrostatin-1给药组的细胞凋亡率与谷氨酸损伤HT-22细胞组的凋亡率相比,无明显变化;5.透射电子显微镜观察发现,谷氨酸模型组与正常对照组相比,细胞线粒体变小、双层膜密度增加,线粒体数目减少;而3-12μM Ferrostatin-1预处理后,细胞形态结构逐渐接近于正常状态;6.MDC染色后,流式细胞术检测结果表明,与正常对照组相比,谷氨酸模型组峰出现微弱的右移,荧光强度增加(P0.05);与模型组比较,Ferrostatin-1给药组的MDC染色后荧光强度无明显降低(P0.05);同时,JC-1染色,流式细胞仪检测结果显示谷氨酸诱导线粒体膜电位的降低(P0.01),Ferrostatin-1预保护明显降低线粒体的损伤程度(P0.01);7.H2DCF-DA和BODIPY-C11荧光染色结果表明,与模型组相比较,Ferrostatin-1给药组明显抑制谷氨酸诱导的细胞胞质和脂质ROS的升高,差异具有显著性(P0.01);生化法检测发现Ferrostatin-1能够促进谷氨酸损伤的细胞内GSH含量及GPX酶活性增加,均有显著性差异(P0.01);8.Ferrostatin-1影响Ptgs2表达水平的改变。Western blot法结果表明,谷氨酸促进细胞Ptgs2蛋白的表达,同时RT-qPCR法从基因水平证实谷氨酸促进Ptgs2mRNA水平上调。而Ferrostatin-1剂量依赖性抑制Ptgs2上调,其蛋白水平和mRNA水平与模型组相比,具有统计学意义(P0.05);9.Western blot实验结果表明,Ferrostatin-1呈浓度依赖性诱导Nrf2/HO-1/GPX4、FP1蛋白的表达。与模型组比较,Fer-1给药组细胞的Nrf2、HO-1、GPX4和FP1蛋白的表达量明显上调,且呈一定的剂量依赖性,具有显著性差异(P0.01)。10.RT-qPCR法检测,与正常组相比,谷氨酸模型组GPX4mRNA的表达下调;不同浓度Ferrostatin-1预保护后,GPX4的mRNA表达水平均提高,有显著性差异(P0.01)。同时,与正常对照组相比,谷氨酸模型组Ptgs2mRNA的表达上升;不同浓度Ferrostatin-1预保护后,Ptgs2的m RNA表达均下降,有统计学意义(P0.05)。结论:谷氨酸可能是通过诱导细胞内胞质和脂质ROS积累、线粒体膜电位下降、GSH-GPX水平降低、SOD和MDA水平上升,上调Ferroptosis相关蛋白Ptgs2,抑制GPX4表达,诱导细胞Ferroptosis发生,引起细胞死亡。与其他细胞死亡抑制剂相比较,Ferrostatin-1更能有效的保护谷氨酸损伤的HT-22细胞,Ferrostatin-1可能是通过激活Nrf2/HO-1/GPX4途径,增强细胞清除氧自由基的能力,抑制Ferroptosis的发生,从而发挥对神经细胞的保护作用。
[Abstract]:Neurodegenerative disease is a common disorder caused by progressive dysfunction of nerve cells and abnormal behavior of the body, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and so on. Glutamate, as the most important excitatory neurotransmitter in mammalian brain, plays an important role in the survival, growth, differentiation and migration of neurons. Ferroptosis is an oxidative cell death mode different from autophagy, apoptosis and necrosis. It has unique morphological characteristics and complex biochemistry. The development of Ferroptosis is characterized by a redox imbalance caused by an abnormal increase in reactive oxygen species (ROS). Therefore, we believe that oxidative toxicity of glutamate may be the main reason for inducing Ferroptosis in nerve cells and affecting the body's oxidative balance and eventually leading to neurodegenerative diseases. The mechanism and pathway of cell death are worth studying. In this study, the role and possible mechanism of Ferroptosis in HT-22 cells injured by glutamate were investigated, and some theoretical basis was provided for exploring the occurrence of neurological diseases. (2) To study the relationship between glutamate-induced neuronal death and Ferroptosis; (3) To investigate the protective effect of Ferrostatin-1 on HT-22 cells injured by glutamate and its related mechanism. Methods: The neuronal injury model of HT-22 cells injured by glutamate was established, and the viability of HT-22 cells was detected by MTT and Trypan blue methods. The effects of different inhibitors (autophagy inhibitor 3-methyladenine, apoptosis inhibitor Z-VAD-FMK, necrosis inhibitor Necrostatin-1, Ferroptosis specific inhibitor Ferrostatin-1 and iron chelating agent DFO) on glutamate neuronal injury model were screened; LDH leakage rate and glutathione were detected by different kits. The activity changes of GSH, GPX, MDA and SOD were observed by inverted microscope, transmission electron microscope, flow cytometry, cytoplasmic reactive oxygen species (ROS), lipid reactive oxygen species (ROS), mitochondrial membrane potential (delta m), MDC levels, and DAPI staining were observed by fluorescence microscope. The expression of Ptgs2 and GPX4 mRNA was detected by Real Time RT-PCR. Results: 1. MTT and Trypan blue assay showed that 5 mM glutamate could inhibit the growth of HT-22 cells nearly 50% after 24 h treatment, and the cell morphology was modeled by this dose and time. The results showed that when 5 mM glutamate treated HT-22 cells for 24 hours, the cells became smaller and rounded, the refractive index decreased, the adherence state of cells was poor, and the damage state appeared. 3. MTT assay showed that Ferrostatin-1, a specific inhibitor of Ferroptosis, and DFO had protective effects on HT-22 cells damaged by glutamate, compared with the model group. Cell viability was significantly increased (P 0.01). Morphological observation showed that the morphology of the cells pretreated with 3-12 mu Ferrostatin-1 was closer to normal cells than that of the model group. Autophagy inhibitor 3-Methyladenine, apoptosis inhibitor Z-VAD-FMK and necrosis inhibitor Necrostatin-1 did not significantly increase cell viability. The apoptosis rate of HT-22 cells treated with Ferrostatin-1 was not significantly different from that of HT-22 cells treated with glutamate. 5. Transmission electron microscopy showed that the mitochondria of HT-22 cells treated with Ferrostatin-1 were smaller, the density of bilayer membranes increased and the number of mitochondria decreased compared with the control group. The results of flow cytometry showed that the peak of glutamate model group shifted slightly to the right and the fluorescence intensity increased (P 0.05) compared with the normal control group. Compared with the model group, the fluorescence intensity of Ferrostatin-1 treated group did not decrease significantly (P 0.05) after MDC staining. The results of cytometry showed that glutamate-induced decrease of mitochondrial membrane potential (P 0.01), Ferrostatin-1 preconditioning significantly decreased the degree of mitochondrial damage (P 0.01); 7. H2DCF-DA and BODIPY-C11 fluorescence staining showed that Ferrostatin-1 significantly inhibited the increase of glutamate-induced cytoplasmic and lipid ROS compared with the model group. Ferrostatin-1 was found to promote the increase of GSH content and GPX activity in glutamate-injured cells, with significant difference (P 0.01); 8. Ferrostatin-1 affected the change of Ptgs2 expression level. Western blot showed that glutamate promoted the expression of Ptgs2 protein, and RT-qPCR was used to detect the subunit of Ptgs2 protein. Ferrostatin-1 inhibited the up-regulation of Ptgs2 in a dose-dependent manner, and its protein and mRNA levels were significantly higher than those in the model group (P 0.05); 9. Western blot results showed that Ferrostatin-1 induced the expression of Nrf2/HO-1/GPX4 and FP1 in a concentration-dependent manner. The expression of Nrf2, HO-1, GPX4 and FP1 protein in Fer-1 treated group was significantly up-regulated in a dose-dependent manner (P 0.01). 10. RT-qPCR assay showed that the expression of GPX4 mRNA was down-regulated in the glutamate model group compared with the normal group; the expression of GPX4 mRNA was up-regulated in the Ferrostatin-1 preconditioned group at different concentrations, with a significant difference (P 0.01). At the same time, compared with the normal control group, the expression of Ptgs2 m RNA increased in the glutamate model group, and the expression of Ptgs2 m RNA decreased after different concentrations of Ferrostatin-1 preconditioning, which was statistically significant (P 0.05). Conclusion: Glutamate may induce the accumulation of ROS in cytoplasm and lipid, decrease the mitochondrial membrane potential, decrease the level of GSH-GPX, and decrease the level of S-GPX. Compared with other cell death inhibitors, Ferrostatin-1 is more effective in protecting HT-22 cells from glutamate injury. Ferrostatin-1 may enhance cell clearance by activating Nrf2/HO-1/GPX4 pathway. The ability of oxygen free radicals to inhibit the occurrence of Ferroptosis can play a protective role in neurons.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741
本文编号：2221319
[Abstract]:Neurodegenerative disease is a common disorder caused by progressive dysfunction of nerve cells and abnormal behavior of the body, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and so on. Glutamate, as the most important excitatory neurotransmitter in mammalian brain, plays an important role in the survival, growth, differentiation and migration of neurons. Ferroptosis is an oxidative cell death mode different from autophagy, apoptosis and necrosis. It has unique morphological characteristics and complex biochemistry. The development of Ferroptosis is characterized by a redox imbalance caused by an abnormal increase in reactive oxygen species (ROS). Therefore, we believe that oxidative toxicity of glutamate may be the main reason for inducing Ferroptosis in nerve cells and affecting the body's oxidative balance and eventually leading to neurodegenerative diseases. The mechanism and pathway of cell death are worth studying. In this study, the role and possible mechanism of Ferroptosis in HT-22 cells injured by glutamate were investigated, and some theoretical basis was provided for exploring the occurrence of neurological diseases. (2) To study the relationship between glutamate-induced neuronal death and Ferroptosis; (3) To investigate the protective effect of Ferrostatin-1 on HT-22 cells injured by glutamate and its related mechanism. Methods: The neuronal injury model of HT-22 cells injured by glutamate was established, and the viability of HT-22 cells was detected by MTT and Trypan blue methods. The effects of different inhibitors (autophagy inhibitor 3-methyladenine, apoptosis inhibitor Z-VAD-FMK, necrosis inhibitor Necrostatin-1, Ferroptosis specific inhibitor Ferrostatin-1 and iron chelating agent DFO) on glutamate neuronal injury model were screened; LDH leakage rate and glutathione were detected by different kits. The activity changes of GSH, GPX, MDA and SOD were observed by inverted microscope, transmission electron microscope, flow cytometry, cytoplasmic reactive oxygen species (ROS), lipid reactive oxygen species (ROS), mitochondrial membrane potential (delta m), MDC levels, and DAPI staining were observed by fluorescence microscope. The expression of Ptgs2 and GPX4 mRNA was detected by Real Time RT-PCR. Results: 1. MTT and Trypan blue assay showed that 5 mM glutamate could inhibit the growth of HT-22 cells nearly 50% after 24 h treatment, and the cell morphology was modeled by this dose and time. The results showed that when 5 mM glutamate treated HT-22 cells for 24 hours, the cells became smaller and rounded, the refractive index decreased, the adherence state of cells was poor, and the damage state appeared. 3. MTT assay showed that Ferrostatin-1, a specific inhibitor of Ferroptosis, and DFO had protective effects on HT-22 cells damaged by glutamate, compared with the model group. Cell viability was significantly increased (P 0.01). Morphological observation showed that the morphology of the cells pretreated with 3-12 mu Ferrostatin-1 was closer to normal cells than that of the model group. Autophagy inhibitor 3-Methyladenine, apoptosis inhibitor Z-VAD-FMK and necrosis inhibitor Necrostatin-1 did not significantly increase cell viability. The apoptosis rate of HT-22 cells treated with Ferrostatin-1 was not significantly different from that of HT-22 cells treated with glutamate. 5. Transmission electron microscopy showed that the mitochondria of HT-22 cells treated with Ferrostatin-1 were smaller, the density of bilayer membranes increased and the number of mitochondria decreased compared with the control group. The results of flow cytometry showed that the peak of glutamate model group shifted slightly to the right and the fluorescence intensity increased (P 0.05) compared with the normal control group. Compared with the model group, the fluorescence intensity of Ferrostatin-1 treated group did not decrease significantly (P 0.05) after MDC staining. The results of cytometry showed that glutamate-induced decrease of mitochondrial membrane potential (P 0.01), Ferrostatin-1 preconditioning significantly decreased the degree of mitochondrial damage (P 0.01); 7. H2DCF-DA and BODIPY-C11 fluorescence staining showed that Ferrostatin-1 significantly inhibited the increase of glutamate-induced cytoplasmic and lipid ROS compared with the model group. Ferrostatin-1 was found to promote the increase of GSH content and GPX activity in glutamate-injured cells, with significant difference (P 0.01); 8. Ferrostatin-1 affected the change of Ptgs2 expression level. Western blot showed that glutamate promoted the expression of Ptgs2 protein, and RT-qPCR was used to detect the subunit of Ptgs2 protein. Ferrostatin-1 inhibited the up-regulation of Ptgs2 in a dose-dependent manner, and its protein and mRNA levels were significantly higher than those in the model group (P 0.05); 9. Western blot results showed that Ferrostatin-1 induced the expression of Nrf2/HO-1/GPX4 and FP1 in a concentration-dependent manner. The expression of Nrf2, HO-1, GPX4 and FP1 protein in Fer-1 treated group was significantly up-regulated in a dose-dependent manner (P 0.01). 10. RT-qPCR assay showed that the expression of GPX4 mRNA was down-regulated in the glutamate model group compared with the normal group; the expression of GPX4 mRNA was up-regulated in the Ferrostatin-1 preconditioned group at different concentrations, with a significant difference (P 0.01). At the same time, compared with the normal control group, the expression of Ptgs2 m RNA increased in the glutamate model group, and the expression of Ptgs2 m RNA decreased after different concentrations of Ferrostatin-1 preconditioning, which was statistically significant (P 0.05). Conclusion: Glutamate may induce the accumulation of ROS in cytoplasm and lipid, decrease the mitochondrial membrane potential, decrease the level of GSH-GPX, and decrease the level of S-GPX. Compared with other cell death inhibitors, Ferrostatin-1 is more effective in protecting HT-22 cells from glutamate injury. Ferrostatin-1 may enhance cell clearance by activating Nrf2/HO-1/GPX4 pathway. The ability of oxygen free radicals to inhibit the occurrence of Ferroptosis can play a protective role in neurons.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741
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