根皮素上调Nrf2途径对大鼠脑缺血再灌注氧化应激损伤的神经保护及机制研究

发布时间：2018-01-19 23:45

本文关键词： 脑缺血再灌注损伤氧化应激根皮素超氧化物歧化酶谷胱甘肽谷胱甘肽过氧化物酶丙二醛 Nrf2途径　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：缺血性脑血管病的高发病率、致残率严重影响千万患者的生命健康,但改善脑供血同时带来的脑缺血再灌注损伤也成为缺血性脑血管病治疗的难题之一。脑缺血再灌注损伤机制尚不明确,目前认为有多种因素参与损伤过程,氧化应激被认为是脑缺血再灌注损伤的一个主要因素。由于各种原因造成组织细胞的氧化和抗氧化平衡破坏,致使自由基生成过多或者清除不足,因而造成机体损伤结构和功能损伤,即氧化应激。因此,针对氧化应激的神经保护策略可以显著提高干预中风的成效。近年发现,几乎所有具有保护作用的抗氧化基因均存在抗氧化反应元件(ARE),包括氧化应激在内的各种因素可导致Nrf2易位细胞核内作用于ARE,激活抗氧化基因的表达,由此保护组织细胞免受氧化应激损伤,因此激活Nrf2途径,从而启动机体抗氧化应激反应成为改善氧化应激造成的脑缺血再灌注损伤的可行方向。Nrf2/ARE通路已经被证明是抗氧化应激最重要的一条通路,这条通路的激活及上调在脑缺血再灌注损伤中可以起到抗氧化应激及神经保护作用。氧化应激的发生可以激活Nrf2的表达,而某些药物可以通过上调Nrf2表达使机体增加抗氧化能力、减轻氧化损伤。但自由基本身十分活跃,加上检测自由基的方法复杂难行,实践中常以氧化应激指示酶水平的变化情况来反映氧化应激水平及损伤程度,故本实验选取SOD、GSH、GSH-PX、MDA作为指示机体氧化损伤程度及抗氧化应激能力的指示酶。根皮素属于二氢查耳酮的类黄酮家族,尤其在苹果和苹果的衍生产品中含量丰富,来源广泛。大量的证据表明,根皮素具有抗氧化及神经保护作用,在其他学者的多项实验中证实能够通过调控Nrf2抗氧化应激对组织功能起到保护作用,具有干预氧化应激损伤脑缺血再灌注损伤的潜在影响。因此,我们进行了本项实验,研究脑缺血再灌注损伤过程中的脑损伤、氧化应激指示酶变化与Nrf2表达的关系,以及经过根皮素干预后上述指标的变化对比,旨在揭示根皮素可以通过上调Nrf2表达对氧化应激引起的脑缺血再灌注损伤起到保护作用。目的探讨根皮素通过激活Nrf2途径对脑缺血再灌注由氧化应激导致的损伤的保护作用及相关机制,从而为脑缺血再灌注损伤提供新的干预方向。方法1.以体重在220-280mg的均龄清洁级SD健康人鼠为研究对象,所有动物术前12 h禁食,所有的实验程序都按照国立实验室动物的护理和使用健康指南进行。该方案经山东大学动物伦理委员会批准。将研究对象分为5组,分别为假手术组、I/R组(MCAO手术组)、根皮素干预组,根皮素干预组又分为三个亚组,分别为低剂量组、中剂量组、高剂量组。术前各根皮素组大鼠分别给予低剂量(20mg·Kg·D-1)、中剂量(40mg·Kg·D-1)、高剂量(80mg·Kg·D-1)艮皮素灌胃14天。末次给药后1小时开始造模,其余各组给予等量生理盐水灌胃。2.实验对象给予以10%水合氯醛35 mg/Kg腹腔注射麻醉,I/R组及根皮素组大鼠以改良后的线栓法阻断右侧大脑中动脉(MCA),建立局限性脑缺血再灌注模型,模型成功的判断标准：大鼠麻醉清醒后出现站立不稳,倾倒,左侧肢体瘫痪,左爪不能伸展,醒后追尾,向右侧行走,提尾时向一侧转圈。闭塞2小时后,拔出栓线实现血液再灌注,再灌注后24小时进行后续实验。假手术组大鼠线栓插入深度8-10mm避免闭塞大脑中动脉,其余操作与其他手术组相同。整个手术直肠温度维持在37-38℃,房间温度控制在25-27℃的范围内。3.再灌注24小时后,根据龙格等的5分制盲法对大鼠进行神经功能缺损评分：无明显神经损伤表现评0分；不能完全伸展对侧前爪评1分；向对侧转圈评评2分；向对侧倾倒评3分；不能自发行走,意识丧失评4分。得分越高,神经功能损害越严重。神经功能缺损评分后处死大鼠,分离脑组织,以2,3,5-三苯基四氮唑(TTC)染色,图像用图像分析软件进行分析,计算脑梗塞面积,对脑梗塞的程度进行评估。4.处死大鼠后,用湿一干法测定脑水肿程度,摘除大脑两个半球称重(湿重),然后将组织在100℃下干燥24小时,测定干重。公式计算：水含量=(湿重-干重)/湿重×重)测%。5.超氧化物歧化酶,谷胱甘肽、谷胱甘肽过氧化物酶可以反映机体抗氧化应激水平,丙二醛可以间接反映氧化应激导致的损伤,故选取超氧化物歧化酶,谷胱甘肽、谷胱甘肽过氧化物酶丙二醛为氧化应激指示酶,以黄嘌呤氧化酶-羟胺法测定超氧化物歧化酶活性,微板法查谷胱甘肽及谷胱甘肽过氧化物酶活性,硫代巴比妥酸法测定丙二醛活性,蛋白质浓度通过Bradford方法测定。6.采用RT-PCR和Western blot检测大鼠Nrf2基因表达及蛋白水平。7.统计：应用SPSS 13.0 (Statistical Package for the Social Sciences)统计软件(SPSS,芝加哥,IL,USA)进行统计分析,实验数据以均数士标准差(mean±SD)表示,采用单因素方差分析以及T检验,以P0.05视为差异有统计学意义。结果1.造模显示,MCA闭塞2小时后再灌注24 h,假手术组大鼠反应正常,肢体活动无障碍,步态平稳,无神经功能缺损症状；与假手术组相比,缺血／再灌注组大鼠出现站立不稳,左侧肢体瘫痪,行走时向左侧追尾,提尾时向左侧侧转圈,提示局限性脑缺血再灌注损伤模型构建成功。2.按照神经功能缺损5分制评分,FR组大鼠神经功能评分较假手术组明显增高(P0.01),说明I/R组大鼠出现明显神经功能损害。根皮素干预各组大鼠神经功能评分较假手术组大鼠增高,但较I/R组明显降低(P0.05)。3.脑梗塞面积对比结果示,假手术组中为均匀红色,没有检测到脑梗塞,而I/R组明显可见广泛白色梗塞病变,较假手术组梗塞面积明显扩大(PO.Q1)。根皮素各组梗塞面积与I/R组相比显著减少,差异有统计学意义(P0.05)。4.脑水肿变化情况：假手术组脑含水量正常,I/R较假手术组脑水肿明显(P0.01)。根皮素处理各组脑水肿程度较I/R组明显下降(P0.05)。5.超氧化物歧化酶(SOD),谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-PX).丙二醛(MDA)活力比较：灌注24小时后,与假手术组相比,I/R组超氧化物歧化酶,谷胱甘肽和谷胱甘肽过氧化物酶水平显著降低(P0.01)。根皮素处理组超氧化物歧化酶,谷胱甘肽和谷胱甘肽过氧化物酶水平虽然较假手术组下降,但较I/R组明显升高(P0.05)。I/R组丙二醛活力较假手术组明显升高(P0.01)。根皮素干预组丙二醛水平较假手术组增高,但较相比I/R组显著下降(P0.05)。6.RT-PCR及Western blot结果显示,I／R组Nrf2的mRNA表达及蛋白水平较假手术组明显升高(P0.05)。根皮素处理组Nrf2的mRNA表达及蛋白水平较I／R组显著升高(P0.05)。结论1.脑缺血/再灌注可以造成明显的脑神经功能损伤和脑水肿、脑梗塞。2.脑缺血/再灌注导致超氧化物歧化酶,谷胱甘肽和谷胱甘肽过氧化物酶水平明显下降,而丙二醛水平明显升高,水平差异有统计学意义,表示缺血/再灌注导致脑组织抗氧化能力下降及明显的氧化应激损伤。3.各组根皮素干预后较1dR组大鼠脑神经功能评分减少明显,差异有统计学意义,说明根皮素能够减轻脑缺血/再灌注导致神经功能损害。4.根皮素组大鼠脑水肿及脑梗塞面积程度较I/R组明显减轻,说明根皮素干预大鼠脑缺血/再灌注能够明显减轻脑水肿、脑梗塞。5.根皮素干预后超氧化物歧化酶,谷胱甘肽和谷胱甘肽过氧化物酶水平明显较I/R组明显升高,说明根皮素可以提高脑组织在缺血/再灌注时的抗氧化能力。6.根皮素干预后丙二醛水平较I/R组明显下降,对比有统计学意义,说明根皮素能够减轻脑缺血/再灌注的氧化应激损伤。7.I/R组Nrf2的mRNA及蛋白表达较假手术组明显增强,差异有统计学意义,说明脑缺血/再灌注作为刺激源激活了Nrf2的基因表达。8.根皮素干预组的Nrf2的mRNA及蛋白表达较I/R组明显增强,差异有统计学意义,说明根皮素能够上调Nrf2的基因表达。以上结果分析可以看出,脑缺血/再灌注过程存在明显的氧化应激损伤,而根皮素的干预可以明显抑制脑缺血再灌注损伤过程中的氧化应激反应,提高抗氧化水平,从而改善脑缺血再灌注损伤导致的脑组织损害以及脑功能评分。而Nrf2表达的上调与氧化应激指示酶的变化及神经功能评分下降、组织损害减轻同步,因此我们认为,根皮素有可能通过上调Nrf2途径起到抗氧化应激作用从而在大鼠脑缺血／再灌注损伤中的抗氧化应激从而对脑的起到神经保护作用。
[Abstract]:Ischemic cerebrovascular disease with high incidence, disability rate must seriously affect the life and health of patients, but also bring about improving the blood supply to the brain of cerebral ischemia reperfusion injury has become one of the difficulties in the treatment of ischemic cerebrovascular disease. The mechanism of cerebral ischemia reperfusion injury is not clear, now that there are many factors involved in the process of oxidative stress injury. Is considered a major factor in cerebral ischemia reperfusion injury. Due to various reasons, the balance of oxidation and antioxidant causing tissue damage, resulting in the generation of free radicals, excessive or inadequate removal, which caused the injury can damage the structure and function, namely oxidative stress. Therefore, the neuroprotective strategies of oxidative stress can significantly improve the intervention of stroke results. In recent years, almost all have the protective effects of antioxidant genes have the antioxidant response element (ARE), including a variety of oxidative stress in the The role of factors can lead to translocation of Nrf2 nucleus in ARE, activate the expression of antioxidant genes, thereby protecting cells from oxidative stress injury, therefore the activation of the Nrf2 pathway, thereby initiating antioxidant stress become the improvement caused by oxidative stress in cerebral ischemia reperfusion injury in the feasible direction of.Nrf2/ARE pathway has been shown to be a pathway of oxidative stress importantly, activation and upregulation of this pathway in cerebral ischemia reperfusion injury can play a neuroprotective effect against oxidative stress and oxidative stress. The expression of Nrf2 can be activated, and some drugs can make the body to increase the antioxidant capacity by up regulating Nrf2 expression, reduce oxidative damage. But the free basic body is very active, and method for detection of free radicals is complicated and difficult, changes in practice often with oxidative stress indicator enzyme levels to reflect the level of oxidative stress and The extent of the damage, so this experiment selected SOD, GSH, GSH-PX, MDA as the indicator enzyme indicating the oxidative damage degree and antioxidant ability. The flavonoid family phloretin belongs to two dihydrochalcone, especially content in apple and the derivative products in rich, wide range of sources. A large amount of evidence that root bark pigment has antioxidant and neuroprotective effects, in a number of other scholars in the experiment proved to be regulated by Nrf2 oxidative stress to protect the organization function, with intervention on oxidative stress injury of cerebral ischemia reperfusion injury. The potential impact because of this, we carry out this experiment, brain injury of cerebral ischemia reperfusion injury in rats the relationship between oxidative stress, indicating enzymes and the expression of Nrf2, and after comparison of the change of root Pisugan prognosis of these indicators, in order to reveal phloretin can lead to oxidative stress by up regulating the expression of Nrf2 The cerebral ischemia reperfusion injury. Objective to investigate the protective effect of phloretin through activation of Nrf2 pathway on cerebral ischemia reperfusion injury caused by oxidative stress injury and the related mechanism, so as to provide the intervention of cerebral ischemia reperfusion injury. Methods 1. new directions to weight in 220-280mg were clean SD healthy people were as the research object, all animal preoperative fasting 12 h, all of the experimental procedures in accordance with the National Laboratory Animal Care and use of health guidelines. The plan is approved by the Shandong University animal ethics committee. The subjects were divided into 5 groups, namely sham operated group, I/R group (MCAO group), root Pisugan pretreatment group, root Pisugan pre group was divided into three subgroups, respectively, low dose group, middle dose group, high dose group. The preoperative phloretin group rats were given low dose (20mg - Kg - D-1), middle dose (40mg Kg D-1), high Dose (80mg Kg D-1) Gen meletin gavage for 14 days. 1 hours after the last administration began modeling, other groups were given normal saline.2. subjects given 10% chloral hydrate 35 mg/Kg intraperitoneal injection of anesthesia, I/R group and phloretin group rats were treated with modified thread occluding the right side of the brain artery (MCA), a limitation of the cerebral ischemia reperfusion model, criteria of success model: the rats were anesthetized awake after standing instability, dumping, left hemiparesis, left paw cannot extend, wake up to the right rear end, walking to the side. When the tail around the block 2 hours after the implementation of the blood reperfusion pull-out suture, 24 hours after reperfusion for subsequent experiments. The rats in the sham operation group the inserting depth of 8-10mm to avoid the middle cerebral artery occlusion, the same with other surgical group. The whole operation of rectal temperature at 37-38 degrees, the room temperature is controlled at 25-27 DEG C In the range of.3. after 24 hours of reperfusion, the neurological deficit scores of rats were divided into 5 blind method such as Runge system: no obvious neurological symptoms score of 0 points; can not fully extend the forepaw rated 1 points; to the contralateral circling judge 2 points; rated 3 points to the side of the dumping; not since the issuance, loss of consciousness, a score of 4 points. The higher the score, the more serious neurological damage. The neurological deficit score after the rats were sacrificed and brain tissues were isolated, with 2,3,5- three phenyl tetrazole (TTC) staining and image analysis using image analysis software, calculate the cerebral infarction area of cerebral infarction was performed to assess the degree of.4. the rats were sacrificed after the determination of the degree of brain edema by wet dry method, extraction of two hemispheres of the brain weight (wet weight), and then will be organized at 100 deg.c for 24 hours, the determination of dry weight. Formula: water content = (wet weight dry weight) / wet weight) *%.5. superoxide dismutase, glutathione Peptide, glutathione peroxidase can reflect the oxidative stress level of the body, MDA can indirectly reflect the damage caused by oxidative stress, so the selection of superoxide dismutase, glutathione, glutathione peroxidase as oxidative stress indicator enzyme, superoxide dismutase activity was determined by xanthine oxidase hydroxylamine method, micro plate method. Glutathione peroxidase activity determination of MDA and glutathione, thiobarbituric acid method, the protein concentration was determined by Bradford method using RT-PCR.6. and Western blot were detected Nrf2 gene expression and protein level of.7. statistics: application of SPSS 13 (Statistical Package for the Social Sciences) statistical software (SPSS, Chicago, IL, USA) for statistical analysis of experimental data. With the mean + standard deviation (mean + SD), using single factor analysis of variance and T test, with P0.05 as the difference Differences were statistically significant. Results 1. model showed that MCA occlusion 2 hours after reperfusion 24 h, rats in the sham operation group were normal reaction, disorder, no limb gait is smooth, no symptoms of neurological deficit; compared with sham operation group, ischemia / reperfusion group rats standing instability, left limb paralysis when walking to the left, rear end, tail to the left side when circling, reveals the limitation of cerebral ischemia reperfusion injury model was constructed successfully by.2. neural function defect score 5 points, FR group of rats neural function score was significantly higher than those in the sham operation group (P0.01), indicating significant neurological impairment in I rats in the /R group phloretin. Intervention rats neural function score compared with the sham operation group Shu Zenggao, but was lower than that of I/R group (P0.05).3. cerebral infarction area comparison showed that the sham group is uniform red, not detected in cerebral infarction, and I/R group was widely visible white The color of infarction lesions, compared with sham operation group, infarction area expanded significantly (PO.Q1). The infarct area of phloretin groups compared with I/R group were significantly decreased, the difference was statistically significant (P0.05) changes of cerebral edema in.4.: sham operation group, the brain water content of normal, I/R compared with the sham group, brain edema (P0.01) root bark. The degree of brain edema in each group was significantly decreased than I/R group (P0.05).5. superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX), malondialdehyde (MDA) activity: 24 hours after reperfusion, compared with sham operation group, I/R group, superoxide dismutase and glutathione. Glutathione peroxidase levels were significantly lower (P0.01). Phloretin treatment group of superoxide dismutase, glutathione and glutathione peroxidase levels while decreased than those of sham operated group, but significantly higher than that in I/R group (P0.05) in.I/R group compared with the sham group, MDA activity of Ming Dynasty Xian Shenggao (P0.01). Phloretin intervention group compared with the sham group, MDA level increased, but less than the I/R group decreased significantly (P0.05).6.RT-PCR and Western blot showed that the expression of mRNA and protein levels of I / R group Nrf2 is higher than sham operated group (P0.05). Phloretin treatment group Nrf2 mRNA the mRNA and protein level of I / R group increased significantly (P0.05). The nerve function caused by brain injury and brain edema obviously 1. cerebral ischemia / reperfusion, superoxide dismutase.2. cerebral infarction cerebral ischemia / reperfusion resulted, glutathione and glutathione peroxidase levels were significantly decreased, while MDA levels were significantly increased. There are significant differences in the level of representation, ischemia / reperfusion resulted in decreased brain antioxidant capacity and oxidative stress in.3. group obviously phloretin after intervention compared with 1dR group rats brain nerve function score decreased significantly, the difference was statistically Meaning that phloretin can damage nerve function.4. phloretin rats cerebral edema and cerebral infarction area was significantly reduced compared with I/R group after ischemia / reperfusion resulted, that phloretin intervention significantly reduce cerebral edema after cerebral ischemia / reperfusion in rats, ultra dismutase cerebral infarction.5. root bark after the intervention of element oxides, glutathione and glutathione peroxidase levels were significantly higher than those in I/R group, that phloretin can improve the antioxidant capacity of brain tissue in the ischemia / reperfusion of the root bark of.6. in after the intervention of the content of MDA was significantly lower than that in I/R group, compared with statistical significance, that phloretin can alleviate cerebral ischemia / mRNA protein and reperfusion induced oxidative stress in.7.I/R group the expression of Nrf2 is stronger than the sham group, the difference was statistically significant, that stimulus activates Nrf2 gene expression.8. phloretin as cerebral ischemia / reperfusion The expression of mRNA protein and Nrf2 in the intervention group increased significantly compared with I/R group, the difference was statistically significant, that phloretin can upregulate the expression of Nrf2 gene. The above results can be seen, cerebral ischemia / reperfusion process significantly damaged by oxidative stress, and the intervention of phloretin can inhibit cerebral ischemia and oxidation the stress response during reperfusion injury, improve the antioxidant level, so as to improve the cerebral ischemia reperfusion injury caused by brain damage and brain function score. While regulation of oxidative stress and Nrf2 expression indicating function change and nerve enzyme score decreased, tissue damage to reduce synchronization, so we think, known as root bark may regulate Nrf2 pathway the oxidative stress resulting in rat cerebral ischemia / reperfusion injury and oxidative stress in the brain plays a neuroprotective role.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743

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