激动素对谷氨酸诱导的HT22细胞氧化损伤干预作用及相关机制研究
发布时间：2019-05-15 23:51
【摘要】：背景:阿尔茨海默病(Alzheimer’s disease,AD)是一种严重危害老年人身心健康和生活质量的神经退行性疾病。越来越多的证据表明,氧化应激很可能在AD发病和发展过程中发挥着极其重要的作用。激动素是腺嘌呤的甲基呋喃衍生物,研究表明其能够在动物个体、组织和细胞水平上发挥显著的抗氧化应激和抗衰老作用。HT22细胞是来源于小鼠海马神经元的永生化细胞系,谷氨酸能诱导其发生氧化应激和细胞死亡,因此它是研究神经元氧化损伤的良好细胞模型。本研究探索了激动素对谷氨酸诱导的HT22细胞氧化损伤的保护作用,对抗AD药物的筛选具有重要意义。目的:研究激动素对谷氨酸诱导的HT22细胞氧化损伤的保护作用及其相关机制。方法:首先,通过DPPH清除试验、三价铁还原试验、羟自由基抑制试验、超氧阴离子抑制试验和二价铁离子螯合试验,对激动素体外抗氧化能力予以评估,同时通过CCK8细胞活力试验对激动素的安全浓度范围和最佳处理时间进行了筛选。然后,通过CCK8细胞活力试验和乳酸脱氢酶(Lactate dehydrogenase,LDH)细胞毒性试验研究了激动素对谷氨酸处理的HT22细胞活力下降及LDH泄露的影响;通过流式细胞术研究了激动素对细胞内活性氧(Reactive oxygen species,ROS)和Ca~(2+)水平的影响;通过相关试剂盒研究了激动素对细胞内谷胱甘肽(Glutathione,GSH)水平、抗氧化酶活性和细胞抗氧化能力的影响;通过流式细胞术评估了激动素对细胞凋亡的影响;通过ATP试剂盒检测细胞内ATP含量,流式细胞术检测细胞线粒体膜电位(Mitochondrial membrane potential,MMP)以及Western Blot(WB)方法检测凋亡诱导因子(Apoptosis inducing factor,AIF)核转移,评估了激动素对线粒体功能的影响;通过WB方法研究了激动素对谷氨酸处理的HT22细胞中凋亡信号调节激-1(Apoptosis signal-regulating kinase 1,ASK-1)、c-jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和丝裂原激活蛋白激酶p38(Mitogen activated protein kinase p38,p38)活化的影响。最后,通过WB、实时定量PCR(Quantitative real-time polymerase chain reaction,qRT-PCR)研究了激动素对Nrf2核转移和血红素加氧酶-1(Heme oxygenase-1,HO-1)表达影响,通过siRNA沉默和CCK8细胞活力试验研究了Nrf2和HO-1对激动素细胞保护作用的影响。结果:(1)和阳性对照相比,激动素自身几乎没有抗氧化能力;(2)激动素的安全浓度范围在5mg/L之下,最佳预处理时间为8h;(3)激动素能够抑制谷氨酸诱导的HT22细胞活力下降和LDH泄露增加;(4)激动素能够抑制细胞内ROS和Ca~(2+)水平上升,提高细胞内抗氧化酶的活性和GSH水平,增强细胞抗氧化能力;(5)相对于谷氨酸处理组,激动素能部分恢复细胞内ATP水平,抑制MMP的去极化和AIF的核转移,同时能够抑制谷氨酸诱导的HT22细胞凋亡;(6)激动素能够抑制谷氨酸诱导的ASK-1、JNK和p38的磷酸化;(7)激动素能够显著促进Nrf2的核转移,且具有时间依赖性;能够显著促进Nrf2下游基因HO-1在转录和翻译水平上的表达,且呈现出时间和剂量依赖性。此外,使用Nrf2 siRNA靶向沉默Nrf2几乎完全消除了激动素的细胞保护作用,而使用HO-1siRNA靶向沉默HO-1只能部分消除激动素的细胞保护作用。结论:激动素自身基本不具备抗氧化能力,但其能够抑制谷氨酸诱导的HT22细胞毒性,抑制细胞内ROS聚集和Ca~(2+)水平升高,抑制细胞内抗氧化酶活性的下降,且激动素自身对细胞抗氧化酶系统具有活化作用。同时,激动素能够抑制线粒体功能障碍和细胞凋亡,抑制谷氨酸诱导的HT22细胞中ASK-1、JNK和p38的活化。此外,激动素能够激活Nrf2信号通路,并能增强其下游抗氧化基因HO-1的表达,而且激动素细胞保护作用的发挥依赖Nrf2的活化和HO-1的表达。这些结果表明激动素具有神经保护作用,可能在预防和治疗AD的研究和实践中具有应用潜力。
[Abstract]:BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects the physical and mental health and quality of life of the elderly. An increasing number of evidence suggests that oxidative stress is likely to play a very important role in the pathogenesis and development of AD. Akinetin is a methorphan derivative of the adenoid, which shows that it can play a significant anti-oxidative stress and anti-aging effect on the individual, tissue and cellular level of the animal. HT22 cells are immortalized cell lines derived from mouse hippocampal neurons, and glutamate can induce oxidative stress and cell death, so it is a good cell model for the study of neuronal oxidative damage. This study explored the protective effect of the agonist on the oxidative damage of the HT22 cell induced by glutamate, which is of great significance to the screening of AD drugs. Objective: To study the protective effect and related mechanism of agonist on glutamate-induced oxidative damage of HT22 cells. The method comprises the following steps of: firstly, performing a DPPH clearance test, a trivalent iron reduction test, a hydroxyl radical inhibition test, a super-oxygen anion inhibition test and a divalent iron ion agglutination test, and evaluating the in vitro antioxidant capacity of the agonist, At the same time, the safe concentration range and the optimal treatment time of the agonist were selected through the cell viability test of the CCK8 cell. Then, the effects of the activation of the agonist on the activity of the HT22 cells and the leakage of the LDH were studied by the cell viability test of the CCK8 and the cell toxicity of the lactate dehydrogenase (LDH), and the reactive oxygen species (reactive oxygen species) in the cells were studied by flow cytometry. The effects of kinetin on the level of Glutathione (GSH), the activity of anti-oxidation enzyme and the anti-oxidation ability of the cells were studied by the related reagent kit, and the effect of the agonist on the apoptosis of the cells was assessed by flow cytometry. The intracellular ATP content, the mitochondrial membrane potential (MMP) and the Western Blot (WB) method were used to detect the nuclear transfer of the apoptosis-inducing factor (AIF). The effects of agonist-1 (ASK-1), c-jun N-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38) on glutamate-treated HT22 cells were studied by WB method. Finally, by WB, real-time quantitative PCR (qRT-PCR), the effect of agonist on the nuclear transfer of Nrf2 and the expression of heme oxygenase-1 (HO-1) was studied. The effect of Nrf2 and HO-1 on the cytoprotective effect of Nrf2 and HO-1 was studied by siRNA silencing and cell viability test of CCK8. Results: (1) In contrast to the positive control, the agonist itself had little anti-oxidation ability; (2) the safe concentration of the agonist was below 5 mg/ L, the optimal pretreatment time was 8 h; (3) the agonist was able to inhibit the decrease of the activity of the HT22 cells induced by glutamate and the increase of LDH leakage; (4) The agonist can inhibit the level of ROS and Ca ~ (2 +) in the cells, increase the activity and GSH level of the antioxidant enzymes in the cells, enhance the anti-oxidation ability of the cells, and (5) restore the intracellular ATP level with respect to the glutamate treatment group. (6) AIF can inhibit the phosphorylation of TASK-1, JNK and p38 induced by glutamate, and (7) the agonist can significantly promote the nuclear transfer of Nrf2 and has time-dependence; The expression of the downstream gene HO-1 in the transcription and translation level of the Nrf2 downstream gene can be significantly promoted and time and dose-dependent. In addition, the use of the Nrf2 siRNA targeting silencing Nrf2 substantially completely eliminates the cytoprotective effect of the agonist, whereas the use of the HO-1 siRNA targeting silent HO-1 can only partially eliminate the cytoprotective effect of the agonist. Conclusion: The agonist itself does not possess the ability of anti-oxidation, but it can inhibit the toxicity of glutamate-induced HT22, inhibit the increase of ROS in the cells and increase the level of Ca ~ (2 +), and inhibit the decrease of the activity of antioxidant enzymes in the cells. And the agonist itself has an activating effect on the cell anti-oxidation enzyme system. In addition, the activation of ASK-1, JNK, and p38 in the HT22 cells induced by glutamate was inhibited by the inhibition of mitochondrial dysfunction and apoptosis. In addition, the agonist can activate the Nrf2 signal path and enhance the expression of the downstream anti-oxidation gene HO-1, and the activation of the agonist cell protection depends on the activation of Nrf2 and the expression of HO-1. These results suggest that the agonist has a neuroprotective effect and may have an application potential in the study and practice of prevention and treatment of AD.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16
本文编号：2477858
[Abstract]:BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects the physical and mental health and quality of life of the elderly. An increasing number of evidence suggests that oxidative stress is likely to play a very important role in the pathogenesis and development of AD. Akinetin is a methorphan derivative of the adenoid, which shows that it can play a significant anti-oxidative stress and anti-aging effect on the individual, tissue and cellular level of the animal. HT22 cells are immortalized cell lines derived from mouse hippocampal neurons, and glutamate can induce oxidative stress and cell death, so it is a good cell model for the study of neuronal oxidative damage. This study explored the protective effect of the agonist on the oxidative damage of the HT22 cell induced by glutamate, which is of great significance to the screening of AD drugs. Objective: To study the protective effect and related mechanism of agonist on glutamate-induced oxidative damage of HT22 cells. The method comprises the following steps of: firstly, performing a DPPH clearance test, a trivalent iron reduction test, a hydroxyl radical inhibition test, a super-oxygen anion inhibition test and a divalent iron ion agglutination test, and evaluating the in vitro antioxidant capacity of the agonist, At the same time, the safe concentration range and the optimal treatment time of the agonist were selected through the cell viability test of the CCK8 cell. Then, the effects of the activation of the agonist on the activity of the HT22 cells and the leakage of the LDH were studied by the cell viability test of the CCK8 and the cell toxicity of the lactate dehydrogenase (LDH), and the reactive oxygen species (reactive oxygen species) in the cells were studied by flow cytometry. The effects of kinetin on the level of Glutathione (GSH), the activity of anti-oxidation enzyme and the anti-oxidation ability of the cells were studied by the related reagent kit, and the effect of the agonist on the apoptosis of the cells was assessed by flow cytometry. The intracellular ATP content, the mitochondrial membrane potential (MMP) and the Western Blot (WB) method were used to detect the nuclear transfer of the apoptosis-inducing factor (AIF). The effects of agonist-1 (ASK-1), c-jun N-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38) on glutamate-treated HT22 cells were studied by WB method. Finally, by WB, real-time quantitative PCR (qRT-PCR), the effect of agonist on the nuclear transfer of Nrf2 and the expression of heme oxygenase-1 (HO-1) was studied. The effect of Nrf2 and HO-1 on the cytoprotective effect of Nrf2 and HO-1 was studied by siRNA silencing and cell viability test of CCK8. Results: (1) In contrast to the positive control, the agonist itself had little anti-oxidation ability; (2) the safe concentration of the agonist was below 5 mg/ L, the optimal pretreatment time was 8 h; (3) the agonist was able to inhibit the decrease of the activity of the HT22 cells induced by glutamate and the increase of LDH leakage; (4) The agonist can inhibit the level of ROS and Ca ~ (2 +) in the cells, increase the activity and GSH level of the antioxidant enzymes in the cells, enhance the anti-oxidation ability of the cells, and (5) restore the intracellular ATP level with respect to the glutamate treatment group. (6) AIF can inhibit the phosphorylation of TASK-1, JNK and p38 induced by glutamate, and (7) the agonist can significantly promote the nuclear transfer of Nrf2 and has time-dependence; The expression of the downstream gene HO-1 in the transcription and translation level of the Nrf2 downstream gene can be significantly promoted and time and dose-dependent. In addition, the use of the Nrf2 siRNA targeting silencing Nrf2 substantially completely eliminates the cytoprotective effect of the agonist, whereas the use of the HO-1 siRNA targeting silent HO-1 can only partially eliminate the cytoprotective effect of the agonist. Conclusion: The agonist itself does not possess the ability of anti-oxidation, but it can inhibit the toxicity of glutamate-induced HT22, inhibit the increase of ROS in the cells and increase the level of Ca ~ (2 +), and inhibit the decrease of the activity of antioxidant enzymes in the cells. And the agonist itself has an activating effect on the cell anti-oxidation enzyme system. In addition, the activation of ASK-1, JNK, and p38 in the HT22 cells induced by glutamate was inhibited by the inhibition of mitochondrial dysfunction and apoptosis. In addition, the agonist can activate the Nrf2 signal path and enhance the expression of the downstream anti-oxidation gene HO-1, and the activation of the agonist cell protection depends on the activation of Nrf2 and the expression of HO-1. These results suggest that the agonist has a neuroprotective effect and may have an application potential in the study and practice of prevention and treatment of AD.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16
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