姜黄素对氧化损伤海马神经元HO-1、nNOS表达的影响研究

发布时间：2018-06-20 10:39

本文选题：氧化应激损伤 + 海马神经元　；参考：《泸州医学院》2014年硕士论文

【摘要】：研究背景血红素加氧酶(heme oxygenase, HO)是血红素分解代谢过程中的限速酶，人体内的CO主要是由血红素加氧酶(HO)代谢产生。HO有三种类型：诱导型(HO-1)、组成型(HO-2)及尚未明确的HO-3[1]。研究表明，HO-1不仅在机体生理状态下发挥作用，更主要是在机体其他非正常状态/应激状态下发挥作用。HO-1参与活性氧、活性氮、缺血、细菌脂多糖、血红素等诱导的氧化应激反应。研究显示HO-1表达升高可减轻应激损伤大鼠的脑组织形态与氧化应激损伤程度，其机制可能与Nrf2-Keap1［(NF-E2-relatedfactor2,转录因子NF-E2相关因子2)-(Kelch-likeECH－associated protein1,胞浆蛋白伴侣分子)］和PI3K/Akt/GSK-3β(phosphatidyllinositol-3-kinase/Akt/glycogen syntheses kinase-3β,磷脂酰肌醇激酶-3/丝苏氨酸蛋白激酶/糖原合成酶激酶-3β)通路有关[2-3]。血红素在HO-1作用下分解为胆红素、游离铁离子和CO，这些物质参与自由基的清除、炎症因子的抑制释放、转录激活因子的上调、tau蛋白的磷酸化和抑制HO-1的泛素化等病理生理过程[4-7]。HO-2主要分布在中枢神经系统及睾丸，它所产生的CO在神经信号传递中起重要作用，与CO发挥神经递质的作用密切相关；HO-2维持脑的正常生理功能，而HO-1与神经系统疾病密切相关。一氧化氮合酶(nitric oxide syntheses, NOS)有三种同工酶，包括主要存在于神经系统中的神经型一氧化氮合酶(nitric oxide syntheses, nNOS)，存在于巨噬细胞、肝细胞和神经胶质细胞中的诱导型一氧化氮合酶(nitric oxidesyntheses, eNOS)和主要存在于内皮细胞中的内皮型一氧化氮合酶(nitricoxide syntheses, iNOS)。在生物体内，NOS利用L-精氨酸和分子氧作为底物，NADPH辅酶作为辅助因子，经过一系列氧化反应生成一氧化氮(NO)和瓜氨酸。在神经系统中NO作为一种重要的信号分子，参与了学习与记忆等重要的神经生理活动，同时对脑部血流具有调节作用，并参与神经系统的免疫防御。另一方面过量的NO又生产细胞毒性氧化物质(如氮自由基、过氧亚硝酸盐等)，与脑缺血损伤、痴呆、帕金森病等疾病的发生、发展有密切关系。因此，在正常生理条件下，神经系统中存在着从时间与空间上精确调控NO产生、释放、扩散与灭活的机制，而这主要是通过调控nNOS的活化与去活化实现的。研究证明nNOS通过与CB2(type2cannabinoidreceptor,大麻素受体2)结合直接调节nNOS，进而与抗氧化蛋白Hsp70和抗凋亡蛋白Bcl-2共同完成CB2通路，，延迟神经退行性变[8]。一氧化氮合酶调节体内一氧化氮自由基和氧自由基平衡[9]，参与脑创伤后损伤及神经再生、缺血再灌注损伤、神经系统退行性疾病、脑卒中、精神分裂性疾病等病理生理过程[10-13]，其机制与脂质过氧化、蛋白质羰基化、蛋白硝基化和自由基清除有关[14-15]。Ríos R等[16]探讨低浓度砷中毒性大鼠中枢损害模型发现，不仅活性氧与脂质过氧化产物可作为氧化应激损伤的生物学标志，nNOS也可作为氧化应激损伤的生物学标志之一。姜黄素(curcumin, Cur)是姜科植物Curcuma longa根茎中分离得到的一种脂溶性多酚类化合物，具有抗氧化、抗炎症、抗肿瘤、抗纤维化、保护心血管系统和消化系统等功能[17-18]。近来研究发现在神经退行性疾病和各种脑功能紊乱疾病中有起到防治作用[19-20]，并证实Cur能保护多种细胞免受H2O2诱导的氧化损伤[21-22]。但关于Cur对神经细胞氧化应激损伤的保护作用机制的探讨相对较少[23-24]。如前所述，HO-1与nNOS在一些病理状态下与氧化应激损伤存在着密切关联，因此，本实验采用H2O2诱导海马神经元氧化应激的损伤模型，观察Cur对HO-1、nNOS的影响，探讨Cur抗氧化应激损伤的保护作用机制，为Cur的临床应用提供理论依据。目的：探讨姜黄素对氧化应激损伤海马神经元的保护作用机制，为姜黄素的临床应用提供一些理论依据。方法：分离新生SD大鼠(≤24h)的海马，采用Neurobasal+2％B27无血清原代培养海马神经元，倒置相差显微镜观察海马神经元生长情况、检测神经元纯度，MTT法检测神经元活性，NeuN、NF-200进行神经元鉴定。培养至10d，将细胞分为6组：空白对照组、损伤组、姜黄素处理组(2.5μmol/L、5.0μmol/L、10.0μmol/L、20.0μmol/L)。等体积H2O2(1mmol/L)处理海马神经元1h，建立氧化应激损伤模型；用DMEM/F12轻洗2次，姜黄素各处理组分别加入浓度为2.5μmol/L、5.0μmol/L、10.0μmol/L、20.0μmol/L的姜黄素培养液300μl，空白对照组和损伤组分别加入无血清培养基300μl，培养孵育6h后利用倒置相差显微镜观察细胞变化，MTT法测定各组神经元活性，乳酸脱氢酶比色法和硫代巴比妥酸法测定LDH活力和MDA浓度，免疫组化和免疫荧光测定海马神经元HO-1、nNOS的表达，RT-PCR测定HO-1、nNOS的mRNA表达。结果：1.海马神经元接种在培养板上3~4h细胞贴壁，第1d细胞长出突起，第7~8d细胞突触生长迅速，第9~11d细胞交织成网状，细胞生长旺盛；20d后细胞轴突缩短、断裂、胞体无光晕。利用无血清培养基和差速贴壁方法培养的神经元纯度可达92%。MTT法检测较其他各时间点的神经元活性，9~11d的海马神经元活性较高，P 0.05，利于海马神经元氧化应激损伤模型建立。2.倒置相差显微镜观察海马神经元氧化应激损伤模型(1h)，细胞突触粘附力降低，部分细胞漂浮，轴突粗糙、断裂，细胞内可见空泡。3.6h后倒置相差显微镜下观察各组细胞，5.0μmol/L组、10.0μmol/L组细胞形态与1h时比较无明显变化；2.5μmol/L组、20.0μmol/L组轴突缩短比1h时明显，但轴突无断裂、胞体仍有光晕；损伤组细胞轴突缩短、断裂、胞体光晕减低比1h时更明显、数量更多；空白对照组细胞轴突完整、光滑、交织成网状、胞体光晕强度与1h时无变化。4. MTT检测5.0μmol/L组和10.0μmol/L组较损伤组神经元活力明显增高，P 0.05；较损伤组，空白对照组神经元活力更强，P 0.05；5.0μmol/L组和10.0μmol/L组神经元活力与2.5μmol/L组、20.0μmol/L组神经元活力比较，差异有统计学意义，P 0.05；2.5μmol/L组、20.0μmol/L组较损伤组神经元活力比较，差异无统计学意义，P0.05。5. LDH活力检测5μmol/L组和10μmol/L组与损伤组比较LDH活力降低，P 0.05；损伤组与空白对照组比较LDH活力增强，P 0.05；5.0μmol/L组和10.0μmol/L组LDH活力与2.5μmol/L组、20.0μmol/L组LDH活力比较，差异有统计学意义，P 0.05；2.5μmol/L组和20.0μmol/L组与空白对照组比较LDH活性降低，P 0.05。6. MDA浓度检测5.0μmol/L组和10.0μmol/L组与损伤组比较MDA浓度降低，P 0.05；损伤组与空白对照组比较MDA浓度升高，P 0.05；5.0μmol/L组和10.0μmol/L组MDA浓度与2.5μmol/L组、20.0μmol/L组MDA浓度比较，差异有统计学意义，P 0.05；2.5μmol/L组和20.0μmol/L组与空白对照组比较MDA浓度升高，P 0.05。7.免疫组化、免疫荧光、PCR检测与损伤对照组比较，5.0μmol/L组、10.0μmol/L组HO-1表达升高，nNOS表达降低，P 0.05；损伤组与空白对照组比较HO-1表达下降，nNOS表达升高，P 0.05；5.0μmol/L组和10.0μmol/L组HO-1、nNOS的表达与2.5μmol/L组、20.0μmol/L组比较差异有统计学意义，P 0.05；2.5μmol/L组、20.0μmol/L组与损伤组比较HO-1、nNOS的表达，差异无统计学意义，P0.05。结论：1.浓度为5μmol/L和10μmol/L的姜黄素可升高氧化应激损伤海马神经元HO-1的表达、降低nNOS的表达，表现出对神经元一定程度的保护作用；2.脂质过氧化参与了H2O2诱导的海马神经元的氧化应激性损伤，姜黄素处理组5μmol/L和10μmol/L可降低神经元的脂质过氧化，减少神经元LDH的释放，反映出对神经元细胞膜的保护作用。
[Abstract]:Research background
Heme oxygenase (HO) is a speed limiting enzyme in heme catabolism. The CO in human body is mainly produced by the metabolism of heme oxygenase (HO), which produces three types of.HO: inducible (HO-1), composition type (HO-2) and unspecified HO-3[1]. studies, and HO-1 not only plays a role in the physiological state of the body, but also mainly in the physiological state of the body. .HO-1 participates in the oxidative stress response induced by active oxygen, active nitrogen, ischemia, lipopolysaccharide and heme in other abnormal state / stress state. The study shows that the increase of HO-1 expression can reduce the brain tissue morphology and oxidative stress injury in rats with stress injury, and the mechanism may be associated with Nrf2-Keap1 (NF-E2-relatedfact Or2, transcription factor NF-E2 related factor 2) - (Kelch-likeECH associated protein1, Cytoplasmic Protein chaperone) and PI3K/Akt/GSK-3 beta (phosphatidyllinositol-3-kinase/Akt/glycogen syntheses kinase-3 beta, phosphatidyl inositol kinase -3/ silk threonine kinase / glycogen synthetase kinase -3 beta) pathway related to the transcription of the [2-3]. heme They are decomposed into bilirubin, free iron ions and CO. These substances are involved in the removal of free radicals, inhibition of inflammatory factors, up regulation of transcription activator, phosphorylation of tau protein, and inhibition of the ubiquitination of HO-1, [4-7].HO-2 is mainly distributed in the central nervous system and testis, and the CO produced by it is transmitted to nerve signals. It plays an important role, which is closely related to the role of CO in neurotransmitters. HO-2 maintains normal physiological functions of the brain, while HO-1 is closely related to nervous system diseases.
Nitric oxide syntheses (NOS) has three isozymes, including neural nitric oxide synthase (nitric oxide syntheses, nNOS), which mainly exists in the nervous system, and exists in macrophages, hepatocytes and glial cells, the inducible nitric oxide synthase (nitric oxidesyntheses, eNOS) and mainly in the inside. The endothelial nitric oxide synthase (nitricoxide syntheses, iNOS) in the skin cells. In vivo, NOS uses L- arginine and molecular oxygen as a substrate, NADPH coenzyme as an auxiliary factor, and produces nitric oxide (NO) and citrullinate through a series of oxidative reactions. In the nervous system, NO as an important signal molecule, participates in learning and Memory and other important neurophysiological activities also regulate the blood flow of the brain and participate in the immune defense of the nervous system. On the other hand, excessive NO produces cytotoxic oxidizing substances (such as nitrogen radical, peroxy nitrite, etc.), which are closely related to the occurrence and development of diseases such as cerebral ischemia, dementia, Parkinson's disease and so on. Under normal physiological conditions, there are mechanisms in the nervous system that regulate NO production, release, diffusion and inactivation from time and space, and this is mainly realized by regulating activation and deactivation of nNOS. It has been proved that nNOS directly regulates nNOS by combining with CB2 (type2cannabinoidreceptor, cannabinoid receptor 2) and then with antioxidant eggs. White Hsp70 and anti apoptotic protein Bcl-2 jointly complete the CB2 pathway, and delayed neurodegenerative [8]. nitric oxide synthase regulates the free radical of nitric oxide and oxygen free radical balance [9] in the body. It participates in post-traumatic injury and nerve regeneration, ischemia reperfusion injury, neurodegenerative disease, stroke, schizophrenia and other pathophysiological processes. [10-13], its mechanism is associated with lipid peroxidation, protein carbonylation, protein nitroylation and free radical scavenging related [14-15].R OS R and other [16] to explore the central damage model of low arsenic poisoning rats. Not only the reactive oxygen species and lipid peroxidation products can be used as the biological markers of oxidative stress damage, but nNOS can also be used as a result of oxidative stress injury. One of the symbols of physical science.
Curcumin (Cur) is a kind of fat soluble polyphenols isolated from the rhizomes of the ginger family Curcuma longa. It has antioxidative, anti-inflammatory, anti-tumor, anti fibrosis, the protection of cardiovascular system and digestive system and other functions of [17-18]. recently found in the neurodegenerative disease and various brain disorders to prevent the disease. The treatment of [19-20], and confirmed that Cur can protect a variety of cells from H2O2 induced oxidative damage [21-22]., but the mechanism of the protective action of Cur on oxidative stress injury of nerve cells is relatively less [23-24]. as mentioned earlier, HO-1 and nNOS are closely related to oxidative stress damage in some pathological conditions, therefore, H2 in this experiment is used in this experiment. O2 induced oxidative stress damage model of hippocampal neurons, observed the effect of Cur on HO-1 and nNOS, and explored the protective mechanism of Cur antioxidant stress injury, and provided a theoretical basis for the clinical application of Cur.
Objective: To investigate the protective mechanism of curcumin on hippocampal neurons damaged by oxidative stress, and to provide some theoretical basis for the clinical application of curcumin. Methods: the hippocampus of newborn SD rats (< < 24h) was isolated and hippocampal neurons were cultured with Neurobasal+2%B27 serum-free primary culture, and the growth of hippocampal neurons was observed by phase contrast microscope. The neuron purity was detected, the neuron activity was detected by MTT method, and the neuron was identified by NeuN and NF-200. The cells were cultured to 10d, and the cells were divided into 6 groups: blank control group, injury group, curcumin treatment group (2.5 mol/L, 5 mu mol/L, 10 mu mol/L, 20 Mu mol/L). 2 light washing for 2 times, the treatment groups of curcumin were treated with the concentration of 2.5 mu mol/L, 5 mu mol/L, 10 mu mol/L, 20 mol/L curcumin culture medium 300 mu L. The blank control group and the injured group were added to the serum-free culture medium 300 micron. After incubating 6h, the cell changes were observed by the inverted phase contrast microscope, and the activity of neurons in each group was determined by MTT method and lactic acid was removed. LDH activity and MDA concentration were measured by hydrogen enzyme colorimetric assay and thiobarbituric acid method. Immunohistochemistry and immunofluorescence were used to determine the expression of HO-1 and nNOS in hippocampal neurons. RT-PCR was used to determine the mRNA expression of HO-1 and nNOS. Results: 1. the hippocampal neurons were inoculated on the culture board, the 3~4h cells were adhered to the culture board, the 1D cells grew out, the 7~8d cell synapses grew rapidly, the 9~11d cell synapses were fast, the 9~11d cells were 9~11d. The cells were interwoven into a network, and the cells grew exuberant. After 20d, the cell axons were shortened, broken, and the cell body had no halo. The purity of the neurons cultured with the serum-free medium and the differential adherence method could reach the neuron activity of the 92%.MTT method compared with the other time points. The activity of the hippocampal neurons in the 9~11d was higher, and the P 0.05 was beneficial to the oxidative stress in the hippocampus neurons. The damage model established the.2. inverted phase contrast microscope to observe the oxidative stress damage model of hippocampal neurons (1H). The adhesion force of the synapse was reduced, some cells were floating, the axons were rough and fractured. The cells were observed under the inverted phase contrast microscope in the cell. The cell morphology of the group of 5 mu mol /L and the group of 10 mu mol/L had no obvious change. The axon shortening in the group of 2.5 mol/L and 20 mu mol/L was obviously shorter than that of 1H, but the axon was not fractured and the cell body still had halo, and the cell axons were shortened, broken and the cell body halo decreased more obviously than that of 1H, and the number of cell axons in the blank control group was more complete, smooth and interwoven into the network, and the cell body halo intensity was not changed by.4. MTT to detect 5 u Mo when 1H. The neuron vigor of the group l/L and the 10 mol/L group was significantly higher than that in the injured group, P 0.05. Compared with the injury group, the neuron vigor was stronger and the P 0.05. The neuron vigor of the 5 mu mol/L group and the 10 micron mol/L group was compared with the 2.5 mu mol/L group and the 20 u mol/L group. The difference was statistically significant, P 0.05, 2.5 mu mol/L group and 20 u mol/L group were more damaged. There was no significant difference in the activity of neuron in the injured group. The activity of P0.05.5. LDH was compared with the group of 5 mu mol/L and the 10 mol/L group, the activity of LDH was decreased and P 0.05 was compared with that of the injury group. The activity of LDH was enhanced and P 0.05 was compared with the blank control group, and the LDH vigor of the 5 mu group and the 10 mu mol/L group was compared with the 2.5 mu mol/L group, and the difference between the 20 mu group and the group was different. Statistical significance, P 0.05; 2.5 mu mol/L group and 20 mu mol/L group compared with blank control group, LDH activity decreased, P 0.05.6. MDA concentration was detected in 5 mu mol/L group and 10 micron group, and MDA concentration decreased and P 0.05 compared with injury group. The concentration of MDA in the injured group was higher than that in the blank control group, and 0.05, 5 mu and 10 micron groups were compared with 2.5 mu. In group ol/L, the concentration of MDA in group 20 mol/L was statistically significant, the difference was statistically significant, P 0.05; 2.5 mu mol/L group and 20 mol/L group were compared with the blank control group, MDA concentration increased, P 0.05.7. immunization, immunofluorescence, PCR detection compared with the damage control group, 5 mu mol/L group, 10 mu mol/L group increased expression, 0.05; injury group and space In the white control group, the expression of HO-1 decreased, the expression of nNOS increased, the expression of P 0.05, the 5 mu mol/L group and the 10 mu mol/L group HO-1, the nNOS expression was statistically significant compared with the 2.5 mu mol/L group, and the 20 micron mol/L group. The 20 micron mol/L group was compared with the injury group, and the difference was not statistically significant. 1. concentration was 5 mu. Mol/L and 10 mol/L curcumin can increase the expression of HO-1 in hippocampal neurons of oxidative stress, reduce the expression of nNOS, and show a certain protective effect on neurons; 2. lipid peroxidation is involved in oxidative stress injury induced by H2O2 induced hippocampal neurons. The lipid peroxidation in the group of curcumin can reduce the lipid of neurons. Mass peroxidation can reduce the release of LDH in neurons and reflect the protective effect on neuronal cell membrane.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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