Ferrostatin-1保護谷氨酸誘導的HT-22細胞Ferroptosis機制研究
發(fā)布時間：2018-09-04 06:45
【摘要】：神經退行性疾病(Neurodegenerative disease)是由于神經細胞出現(xiàn)漸進性功能異常所造成的功能障礙和機體行為異常的常見病,如阿爾茨海默病、帕金森病、亨廷頓病等。這類疾病的發(fā)生涉及神經元之間一系列復雜的作用,包括氧化應激、遺傳因素和興奮性氨基酸作用等。谷氨酸,作為哺乳動物腦內最主要的興奮性神經遞質,它在神經元的生存、生長、分化和遷移過程中起著至關重要的作用。研究顯示,谷氨酸是造成神經細胞損傷的重要因素,它參與神經元死亡的機制十分復雜,主要表現(xiàn)為谷氨酸的氧化性毒性和興奮性毒性。其中,谷氨酸氧化毒性引起的一系列改變與新發(fā)現(xiàn)的細胞死亡形式Ferroptosis很相似。Ferroptosis是一種不同于自噬、凋亡、壞死等的氧化性細胞死亡方式,它具有獨特的形態(tài)特征和復雜的生化反應。Ferroptosis的發(fā)展進程以活性氧(ROS)異常增多引起的氧化還原失衡為特點。因此,我們認為谷氨酸氧化毒性可能是誘導神經細胞發(fā)生Ferroptosis且影響機體的氧化平衡狀態(tài),最終導致神經退行性疾病發(fā)生的主要原因。那么谷氨酸影響神經細胞死亡的作用途徑和機制值得研究。本課題擬通過建立的谷氨酸損傷HT-22細胞的模型,考察Ferroptosis在其中的角色與可能機制,為探索神經疾病的發(fā)生提供一些相應的理論依據。目的:(1)研究谷氨酸誘導的神經細胞模型中氧化性損傷的發(fā)生;(2)研究谷氨酸誘導的神經細胞死亡與Ferroptosis發(fā)生的關系;(3)探討Ferrostatin-1對谷氨酸損傷的HT-22細胞的保護作用及其相關作用機制。方法:建立谷氨酸損傷HT-22細胞神經損傷模型,采用MTT法和Trypan blue法檢測細胞活力并確立谷氨酸最佳造模濃度,同時篩選不同抑制劑(自噬抑制劑3-Methyladenine、凋亡抑制劑Z-VAD-FMK、壞死抑制劑Necrostatin-1、Ferroptosis特異性抑制劑Ferrostatin-1和鐵離子螯合劑DFO)對谷氨酸神經損傷模型的影響;不同試劑盒分別檢測細胞LDH漏出率、谷胱甘肽GSH、谷胱甘肽過氧化物酶GPX、丙二醛MDA和超氧化物歧化酶SOD各指標活性變化;倒置顯微鏡、透射電子顯微鏡觀察細胞形態(tài)學變化;流式細胞術檢測胞質活性氧(ROS)、脂質活性氧(ROS)、線粒體膜電位(△Ψm)、MDC水平;熒光顯微鏡觀察DAPI染色后細胞形態(tài)學變化;Western blot法檢測Ptgs2、Nrf2、GPX4、FP1相關蛋白變化;Real Time RT-PCR技術檢測Ptgs2、GPX4mRNA水平變化。結果:1.MTT和Trypan blue實驗結果表明,5m M谷氨酸作用HT-22細胞24h,細胞生長抑制率接近50%,選擇此劑量和時間造模;2.細胞形態(tài)學觀察結果顯示,當5 mM谷氨酸作用HT-22細胞24h后,細胞變小變圓,折光度下降,細胞的貼壁狀態(tài)差,出現(xiàn)損傷狀態(tài);3.MTT法結果顯示,Ferroptosis特異性抑制劑Ferrostatin-1以及鐵離子螯合劑DFO對谷氨酸損傷的HT-22細胞具有保護作用,與模型組相比,細胞活力明顯升高(P0.01);同時,細胞形態(tài)學觀察結果顯示,3-12μM Ferrostatin-1預保護后細胞形態(tài)較模型組更接近正常細胞。自噬抑制劑3-Methyladenine、凋亡抑制劑Z-VAD-FMK和壞死抑制劑Necrostatin-1并未顯著提高細胞活力;4.DAPI染光染色結果表明,Ferrostatin-1給藥組的細胞凋亡率與谷氨酸損傷HT-22細胞組的凋亡率相比,無明顯變化;5.透射電子顯微鏡觀察發(fā)現(xiàn),谷氨酸模型組與正常對照組相比,細胞線粒體變小、雙層膜密度增加,線粒體數(shù)目減少;而3-12μM Ferrostatin-1預處理后,細胞形態(tài)結構逐漸接近于正常狀態(tài);6.MDC染色后,流式細胞術檢測結果表明,與正常對照組相比,谷氨酸模型組峰出現(xiàn)微弱的右移,熒光強度增加(P0.05);與模型組比較,Ferrostatin-1給藥組的MDC染色后熒光強度無明顯降低(P0.05);同時,JC-1染色,流式細胞儀檢測結果顯示谷氨酸誘導線粒體膜電位的降低(P0.01),Ferrostatin-1預保護明顯降低線粒體的損傷程度(P0.01);7.H2DCF-DA和BODIPY-C11熒光染色結果表明,與模型組相比較,Ferrostatin-1給藥組明顯抑制谷氨酸誘導的細胞胞質和脂質ROS的升高,差異具有顯著性(P0.01);生化法檢測發(fā)現(xiàn)Ferrostatin-1能夠促進谷氨酸損傷的細胞內GSH含量及GPX酶活性增加,均有顯著性差異(P0.01);8.Ferrostatin-1影響Ptgs2表達水平的改變。Western blot法結果表明,谷氨酸促進細胞Ptgs2蛋白的表達,同時RT-qPCR法從基因水平證實谷氨酸促進Ptgs2mRNA水平上調。而Ferrostatin-1劑量依賴性抑制Ptgs2上調,其蛋白水平和mRNA水平與模型組相比,具有統(tǒng)計學意義(P0.05);9.Western blot實驗結果表明,Ferrostatin-1呈濃度依賴性誘導Nrf2/HO-1/GPX4、FP1蛋白的表達。與模型組比較,Fer-1給藥組細胞的Nrf2、HO-1、GPX4和FP1蛋白的表達量明顯上調,且呈一定的劑量依賴性,具有顯著性差異(P0.01)。10.RT-qPCR法檢測,與正常組相比,谷氨酸模型組GPX4mRNA的表達下調;不同濃度Ferrostatin-1預保護后,GPX4的mRNA表達水平均提高,有顯著性差異(P0.01)。同時,與正常對照組相比,谷氨酸模型組Ptgs2mRNA的表達上升;不同濃度Ferrostatin-1預保護后,Ptgs2的m RNA表達均下降,有統(tǒng)計學意義(P0.05)。結論:谷氨酸可能是通過誘導細胞內胞質和脂質ROS積累、線粒體膜電位下降、GSH-GPX水平降低、SOD和MDA水平上升,上調Ferroptosis相關蛋白Ptgs2,抑制GPX4表達,誘導細胞Ferroptosis發(fā)生,引起細胞死亡。與其他細胞死亡抑制劑相比較,Ferrostatin-1更能有效的保護谷氨酸損傷的HT-22細胞,Ferrostatin-1可能是通過激活Nrf2/HO-1/GPX4途徑,增強細胞清除氧自由基的能力,抑制Ferroptosis的發(fā)生,從而發(fā)揮對神經細胞的保護作用。
[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.
【學位授予單位】：安徽中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R741
本文編號：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.
【學位授予單位】：安徽中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R741
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相關期刊論文 前3條
1 劉晨旭;宋蕊;李慶林;;Ferrostatin-1通過抑制Ferroptosis保護谷氨酸誘導的HT-22細胞損傷[J];中國臨床藥理學與治療學;2016年10期
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