灯盏花乙素调控NOX2治疗脑缺血再灌注损伤的作用机制研究

发布时间：2018-06-11 19:26

本文选题：灯盏花乙素 + 脑缺血再灌注　；参考：《广州中医药大学》2017年博士论文

【摘要】：脑卒中目前在全球范围内,已成为被人们广泛关注的健康难题,包括缺血性卒中和出血性卒中。缺血性卒中是脑卒中的主要临床类型,约占60%-80%。氧化应激反应引起的过氧化损伤是缺血性脑损伤发生的重要机制,在脑组织缺血再灌注损伤的病理过程中,氧化应激产生大量活性氧,可导致DNA、蛋白质和脂质等的损伤,并可直接作用于生物膜上的不饱和脂肪酸,损伤生物膜。过量的活性氧会破坏细胞的正常功能,降低多种酶的活性及DNA和RNA的稳定性,诱发细胞分子(如蛋白质、DNA及脂质)过氧化损伤,促进细胞的凋亡。在脑缺血再灌注损伤的过程中,活性氧存在多种来源途径,其中,NADPH(还原型烟酰胺腺嘌呤二核苷酸磷酸)氧化酶(NOX)被认为是脑缺血再灌注后活性氧水平异常升高的一个关键因子。目前研究证明在NADPH氧化酶中,NOX2在缺血性卒中中扮演重要角色,与缺血性脑卒中等疾病密切相关。大量研究表明,上调缺血脑组织中NADPH氧化酶的表达会加重脑缺血再灌注损伤,增加神经元死亡,而抑制NOX2的活性可以减轻脑缺血再灌注损伤。"脉为血府,气为血帅",益气能够生血,祛瘀能够生新,活血能够生肌。生血、新、肌就包括生新脉和新络的意义。故脉络的新生无法离开气血,所以益气补血药可以发挥生成血管的功能。所以益气可以生脉。对于中风,古今医家均以促进脑络通畅为最终目的。灯盏生脉胶囊是临床上在治疗脑梗塞等缺血性脑损伤疾病中具有显著疗效的"益气生脉"中成药,我们承担了国际上首个探讨国产品牌中成药对缺血性卒中二级预防作用的大型临床研究-"灯盏生脉胶囊干预缺血中风二级预防的多中心、随机对照、双盲临床试验",结果显示:灯盏生脉胶囊可降低首发症状性脑梗死患者非致死性卒中再发率27.8%,其可有效改善缺血卒中患者残障程度,且不增加脑出血及其它出血性风险,在临床上已广泛用于缺血性心脑血管疾病的防治,灯盏生脉胶囊以灯盏细辛为主药,而灯盏花乙素(Scutellarin)是从灯盏细辛中提取的主要黄酮类活性成分之一,化学名为4',5,6三羟基黄酮-葡萄糖醛酸苷。近年来基础研究表明灯盏花乙素对脑血管的损伤具有保护作用。那么灯盏花乙素保护脑缺血再灌注损伤的作用机制是什么?本研究利用星形胶质细胞缺糖缺氧模型和大鼠大脑中动脉栓塞模型,探讨灯盏花乙素保护脑缺血再灌注损伤的可能作用机制。目的:通过细胞模型(星型胶质细胞缺氧/再复氧模型)及整体动物(大鼠大脑中动脉栓塞模型,tMCAO)研究灯盏花乙素处理对NOX2及过氧化损伤的改变,明确灯盏花乙素保护脑缺血再灌注后神经损伤的信号机制。方法:1.体外培养原代星形胶质细胞,制备缺糖缺氧/复糖复氧损伤的细胞模型。将星形胶质细胞分为正常对照组、缺糖缺氧/复糖复氧组、Scu低浓度(2 μM)+缺糖缺氧/复糖复氧组、Scu中浓度(10μM/L)+缺糖缺氧/复糖复氧组、Scu高浓度(50 μ M/L)+缺糖缺氧/复糖复氧组。分子对接技术研究灯盏花乙素和NOX2蛋白对接情况;Western Blot检测NOX2蛋白表达情况;活性氧试剂盒检测星形胶质细胞内活性氧含量。2.制备大鼠MCAO动物模型,采用简单随机分组法将SD大鼠随机分为以下几组:假手术组(sham)组、手术组、灯盏花乙素低浓度(50mg/kg)+手术组、灯盏花乙素高浓度(l00mg/kg)+手术组、不同浓度夹竹桃麻素+手术组。Bederson 5分法对大鼠进行神经行为学评分;TTC染色法检测大鼠脑梗死面积的变化;Western Blot法检测NOX2蛋白表达的变化情况;ELISA法检测氧化应激相关指标的变化;免疫组化检测大鼠脑组织中的细胞凋亡情况。结果:1.分子对接研究结果显示灯盏花乙素和NOX2蛋白的活性口袋形状相互吻合,二者的3D分子结构对接良好。原代培养的星形胶质细胞纯度98%。缺糖缺氧2 h复糖复氧24 h后,灯盏花乙素(10,50 μM)可以改善细胞存活率下降(P0.05)。Western Blot显示缺糖缺氧2 h后复糖复氧24 h的星形胶质细胞NOX2表达水平高于正常对照组,灯盏花乙素(2,10,50 μM)预处理的缺糖缺氧2 h后复糖复氧24 h的星形胶质细胞中NOX2表达水平与同时间点溶剂对照组相比下降(P0.05)。缺糖缺氧导致细胞内活性氧水平升高,而灯盏花乙素(10,50 μM)能降低细胞内活性氧的水平(P0.05)。2.大鼠神经行为学评分、脑梗死面积百分率的检测结果显示,与sham组相比,I/R组大鼠神经行为学评分,脑梗死面积百分率增高(P0.05),与I/R组相比,Scu(低、高浓度)+I/R组大鼠神经行为学评分,脑梗死面积百分率降低(P0.05)。3.大鼠神脑组织细胞凋亡及氧化应激相关指标(8-羟基脱氧鸟苷,8-OHdG;4-羟基壬烯醛,4-HNE;3-硝基酪氨酸,3-NT)的检测结果显示与sham组相比,I/R组大鼠免疫组化阳性细胞率和氧化应激相关指标增高(P0.05)。假手术组、I/R3d组的Caspase-3阳性细胞百分率分别为12.20±1.62%、55.45±4.99%。假手术组、I/R 3d 组的 8-OHdG 分别为 100%、177.2%±31.2%;4-HNE 分别为 100%、163.6%± 28.7%;3-NT 分别为 100%、159.3%±32.1%。与 I/R 组相比,Scu(低、高浓度)+I/R组大鼠免疫组化阳性细胞率和氧化应激相关指标降低(P0.05)。Scu低浓度组、Scu高浓度组的Caspase-3阳性细胞百分率分别为22.36±3.37%、11.70± 1.79%。Scu 低浓度组、Scu 高浓度组的 8-OHdG 分别为 113.1%±33.1%、99.9%± 11.3%;4-HNE 分别为 112.0%± 32.1%、95.2%± 18.9%;3-NT 分别为 85.6%±31.3%、90.0%±20.5%。4.Western Blot检测NOX2蛋白的结果显示,与sham组相比,I/R组大鼠3d的NOX2蛋白表达水平增高(P0.05)。与I/R组相比,Scu(低、高浓度)+I/R组大鼠3 d的NOX2蛋白表达水平下降(P0.05)。夹竹桃麻素+I/R组大鼠3 d的NOX2蛋白的表达水平也下降(P0.05)。结论:1.灯盏花乙素对缺糖缺氧/复糖复氧诱导的星形胶质细胞损伤具有保护作用。灯盏花乙素能够抑制缺糖缺氧星形胶质细胞NOX2蛋白的表达,降低缺糖缺氧星形胶质细胞内活性氧水平。2.灯盏花乙素能够保护脑缺血再灌注损伤,发挥NOX2蛋白抑制剂的作用。3.灯盏花乙素保护脑缺血再灌注损伤的作用机制与靶向调控NOX2引起的过氧化损伤有关。
[Abstract]:Cerebral apoplexy has become a worldwide concern, including ischemic stroke and hemorrhagic stroke. Ischemic stroke is the main clinical type of stroke. The oxidative damage caused by 60%-80%. oxidative stress is an important mechanism for ischemic brain injury, and it is in cerebral ischemia reperfusion injury. During the pathological process of injury, oxidative stress produces a large amount of active oxygen, which can cause damage to DNA, protein and lipid, and can directly affect the unsaturated fatty acids on the biofilm and damage the biofilm. Excessive active oxygen can destroy the normal function of the cells, reduce the viability of various enzymes and the stability of DNA and RNA, and induce cell molecules (such as protein). In the process of cerebral ischemia reperfusion injury, there are many sources of reactive oxygen species in the process of cerebral ischemia-reperfusion injury, in which NADPH (NOX) is considered to be a key factor in the abnormal increase of active oxygen level after cerebral ischemia reperfusion. In NADPH oxidase, NOX2 plays an important role in ischemic stroke and is closely related to ischemic stroke secondary disease. A large number of studies have shown that the up-regulated expression of NADPH oxidase in ischemic brain can aggravate cerebral ischemia reperfusion injury and increase neuron death, while inhibiting the activity of NOX2 can reduce cerebral ischemia reperfusion injury. "Pulse Blood can produce blood, Qi can produce blood, blood stasis can be born new, blood circulation can produce muscle. Blood, new, new, the meaning of new veins and new collateral. Therefore, the newborn can not leave Qi and blood, so Qi enriching blood medicine can play the function of generating blood vessels. So Qi Qi can produce pulse. For stroke, ancient and modern doctors all promote brain collaterals patency. End objective. Breviscapine Shengmai capsule is a clinical medicine which is clinically effective in the treatment of cerebral infarction and other ischemic brain injury diseases. We undertake the first international study of the first large-scale clinical research on the preventive effect of Chinese traditional Chinese patent medicine on the two stage of ischemic stroke - "breviscapine Shengmai capsule" in the intervention of two levels of ischemic stroke. A multi center, randomized controlled, double blind clinical trial, "the results showed: breviscapine Shengmai capsule can reduce the recurrence rate of non fatal stroke in patients with first onset symptomatic cerebral infarction by 27.8%. It can effectively improve the degree of disability in patients with ischemic stroke, without increasing cerebral hemorrhage and other hemorrhagic risk, and has been widely used in ischemic cardio cerebral vascular disease." The prevention and control of the disease, breviscapine Shengmai capsule with Breviscapine as the main drug, and breviscapine (Scutellarin) is one of the main flavonoids extracted from Erigeron breviscapus, named 4', 5,6 three hydroxy flavonoid glucuronide. In recent years, the basic research shows that breviscapine has protective effect on the damage of cerebral blood vessels. What is the mechanism of the protection of cerebral ischemia reperfusion injury by acetic acid? This study uses the model of astrocyte glucose deficiency anoxia and rat middle cerebral artery embolization to explore the possible mechanism of the protective effect of erigerin on the protection of cerebral ischemia reperfusion injury. Objective: to use the cell model (astrocyte anoxia / reoxygenation model) and the whole Body animals (rat model of middle cerebral artery embolism, tMCAO) study the changes in NOX2 and peroxidation damage by breviscapine treatment, and make clear the signal mechanism of breviscapine to protect the nerve injury after cerebral ischemia and reperfusion. Method: 1. the primary astrocytes were cultured in vitro, and the cell model of oxygen deficiency anoxia / reoxygenation injury was prepared. The quality cells were divided into normal control group, hypoxic hypoxia / reoxygenation group, low concentration of Scu (2 mu M) + oxygen deficiency anoxia / reoxygenation group, concentration of Scu (10 M/L) + oxygen deficiency anoxia / reoxygenation group, high concentration of Scu (50 mu M/L) + oxygen deficiency anoxia / reoxygenation group. The docking of breviscapine and NOX2 protein by molecular docking technique; Western Blot detection NOX The expression of 2 protein; the MCAO animal model of rats was prepared by active oxygen kit to detect the active oxygen content in astrocytes. The SD rats were randomly divided into groups of the following groups: the sham operation group (sham) group, the operation group, the low concentration of breviscapine (50mg/kg) + operation group, the high concentration of breviscapine (l00mg/kg) + operation group, and the operation group. The neurobehavioral scores of rats were scored with the same concentration of.Bederson 5. The changes in the area of cerebral infarction in rats were detected by TTC staining; the changes of the expression of NOX2 protein were detected by Western Blot method; the changes of the related indexes of oxidative stress were detected by ELISA; the apoptosis in the brain tissue of rats was detected by immunohistochemistry. Results: the results of 1. molecular docking show that the shape of the active pocket of breviscapine and NOX2 protein is consistent with each other, and the 3D molecular structure of the two is well butted. The purity of the primary cultured astrocytes is 98%. deficient, 2 h h complex and 24 h, and breviscapine (10,50 M) can improve the decrease of cell survival rate (P0.05).Western Blot display The expression level of NOX2 in astrocytes was higher than that in the normal control group. The level of NOX2 expression in astrocytes after glucose deficiency and oxygen deficiency 2 h pretreated by breviscapine (2,10,50 mu M) was lower than that of the control group at the same time at the same time (P0.05). The level of intracellular reactive oxygen species caused by glucose deprivation (2,10,50 mu M) pretreated by breviscapine ethyl (2,10,50 mu M) was higher than that of the control group (P0.05). Increase, and breviscapine (10,50 mu M) can reduce the level of intracellular reactive oxygen level (P0.05).2. rats' neurobehavioral score, and the percentage of cerebral infarction area showed that compared with the sham group, the score of neurobehavioral score in the group I/R and the percentage of cerebral infarction area increased (P0.05), and compared with the I/R group, the +I/R group of Scu (low, high concentration) was the rat nerve. Behavioral score, the percentage of cerebral infarction area decreased (P0.05).3. rats' apoptosis and oxidative stress related indexes (8- hydroxy deoxy guanosine, 8-OHdG; 4- hydroxy nonylaldehyde, 4-HNE; 3- nitrotyrosine, 3-NT) detection results showed that compared with the sham group, the rate of immuno histochemical positive cells in the I/R group was higher than that of oxidative stress. (P0.05) in sham operation group, the percentage of Caspase-3 positive cells in group I/R3d was 12.20 + 1.62%, 55.45 + 4.99%. in sham operation group, 8-OHdG in group I/R 3D was 100%, 177.2% + 31.2%, 4-HNE was 100%, 163.6% + 28.7%, 3-NT was 100%, 159.3% + 32.1%. was compared with I/R group, Scu (low, high concentration) immunohistochemical positive rats The index of cell rate and oxidative stress decreased (P0.05).Scu low concentration group, the percentage of Caspase-3 positive cells in Scu high concentration group was 22.36 + 3.37%, 11.70 + 1.79%.Scu low concentration group, 8-OHdG in Scu high concentration group was 113.1% + 33.1%, 99.9% + 11.3%, 4-HNE was 112% + 32.1%, 95.2% +, respectively, 3-NT was 85.6%, respectively. 31.3%, 90% + 20.5%.4.Western Blot detection of NOX2 protein showed that the NOX2 protein expression level of 3D in the I/R group was higher than that in the sham group (P0.05). The expression level of 3 D in +I/R group was lower than that in the I/R group. Conclusion: 1. breviscapine B has protective effect on astrocyte damage induced by oxygen deficiency and hypoxia / reoxygenation. Breviscapine can inhibit the expression of NOX2 protein in hypoxia starlike astrocytes and reduce the level of reactive oxygen species (.2.) in starlike astrocytes, which can protect cerebral ischemia reperfusion injury and play NO. The role of X2 protein inhibitor.3. breviscapine in protecting cerebral ischemia-reperfusion injury is related to targeted regulation of NOX2 induced oxidative damage.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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