【作者】 刘瑜；

【导师】 梁江久；

【作者基本信息】 山东大学 ， 老年医学（专业学位）， 2016， 博士

【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P<0.05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(P<0.01),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(P<0.05)。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(P<O.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(P<0.05)。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(P<0.01)。根皮素处理各组脑水肿程度较I/R组明显下降(P<0.05)。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(P<0.01)。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(P<0.05)。I/R组丙二醛活力较假手术组明显升高(P<0.01)。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(P<0.05)。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(P<0.05)。根皮素处理组Nrf2的mRNA表达及蛋白水平较I／R组显著升高(P<0.05)。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。更多还原

【Abstract】 Ischemic cerebrovascular disease seriously affects the health of tens of millions of patients. Its morbidity and disability rate is very high. Yet it has been another difficulty of reperfusion injury when improve the cerebral blood supply in the intervention of ischemic cerebrovascular disease. Cerebral ischemia-reperfusion injury mechanism is unclear, there are many factors involved in the damage process. oxidative stress destroy the oxidative and antioxidative balance of tissues and cells,lead to too much free radical generation and insufficient removal, Therefore, the neural protection strategy for oxidative stress can significantly improve the effectiveness of the intervention of stroke.Recent discovery suggests that Antioxidant response element (ARE) exist in almost all antioxidant genes with protective effect. Various factors including oxidative stress can lead to Nrf2 nuclear translocation after action on ARE, Adjust the antioxidant gene expression to protect cells against oxidative stress damage, therefore the activation of Nrf2 pathway to start the body’s resistance to oxidative stress become feasible direction of reducing the cerebral ischemia reperfusion injury caused by oxidative stress.Nrf2/ARE pathway has been proved to be the most important way of antixidation.Some medicine can strengthen the capability of antioxidation by regulating the expression of Nrf2.Phloretin are dihydrochalcone which are members of the flavonoid family. We take SOD,GSH, GSH-PX and MDA as oxidative stress indicator to show the Oxidative stress injury and the capability of antioxidation. Phloretin is in apple and apple derived products are rich in content, a wide range of sources.Other researchs has proved that phloretin has Neuroprotective effect and antioxidation,and can protect tissues by regulate Nrf2.So it can be expected that phloretin has the substantial protective intervention on cerebral ischemia/reperfusion injury caused by oxidative stress. Therefore, we conducted this experiment to research the brain injury in the course of cerebral ischemia and reperfusion, the relationship between oxidative stress indicator enzymes and Nrf2 expression.,the index changes after phloretin intervention.The purpose of this study is to suggestthat phloretin probably play a protective role against oxidative stress after cerebral schemia/reperfusion by up-regulating the expression of Nrf2.Objective To investigate the protective effect and the mechanism of phloretin against oxidative stress on cerebral ischemia reperfusion injury in rats through up-regulation of Nrf2 pathway, so as to provide treatment of cerebral ischemia reperfusion injury in a new direction.Methods1. Healthy SD rats (weighing 220-280mg) were chosen as the research object, all animals fasted for 12 h before operation, all experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of LaboratoryAnimals. The protocol was approved by the Animal Ethics Committee of Shandong University.Rats were randomly assigned to five experimental groups:sham-surgery group, I/R group, and high-, medium-and low-dose phloretin treatment groups (20,40, and 80 mg·Kg·d-1 respectively). The phloretin groups were treated drugs with intragastric administration for 14 days and other groups were given the same amount of normal saline.1h after the last administration, the focal cerebral ischemia-reperfusion rat model was established with the method of intraluminal vascular occlusion as previously reported with slight modification.2. Experimental animals were anesthetized by intraperitoneal injection of 10% chloral hydrate of 35 mg/Kg,Rats in the I/R group and phloretin treatment group were buildedas cerebral ischemia reperfusion models by modified thread occluding right middle cerebral artery (MCA), the judgment standard of successful model:the rats were unstableafter they wake up from anesthesia, tumbling, left hemiparesis, left paw cannot extend,Chasing its own tail, walking along the right side, moving around a circle when grab the tails. After 2h of occlusion, withdraw the filament out carefully to achieve reperfusion.Twenty-four hours after the reperfusion, the following experiment was continued. The sham-surgery rats were subjected to the same surgical procedures,except that the insertion depth ofbrain line bolt was 8-10 mm,avoided to occludethe middle cerebral arteries,During thesurgical procedure, rectal temperature was monitored and maintained at 37-38℃ with a heating pad. The room temperature was controlled in the range of 25-27℃.3. Twenty four hours after reperfusion,accordingto the five-grade marking system blinding method of Runge et,rats were marked Neurological Severity Scores(NSS):no neurologic deficit mark 0 point; can not fully extend opposite forepaw mark 1 point; contralateral circling mark 2 points; contralateral dumping mark 3 points; no spontaneous walking with depressed consciousness level mark 4 points. The higher score, the more severe neurological damage.After finish Neurological Severity Scores, the rats were sacrificed and brain tissues were isolated and stained with 2,3,5-triphenyltetrazolium chloride (TTC), image was analysed with image analysis software, cerebral infarction area was calculated to evaluate the degree of cerebral infarction.4. After the rats were sacrificed, Determination of the degree of brain edema with wet dry method, remove and weight the two hemispheres of the brain (wet weight), dry tissue under 100 degrees celsius for 24 hours, determine dry weight. Formula:water content=(wet weight -dry weight)/wet weight×100%.5. Superoxide dismutase, Glutathione, Glutathione Peroxidase is an important indicator enzyme to reflect oxidative stress, malondialdehyde can indirectly reflect the damage caused by oxidative stress, so we selected Superoxide dismutase,Glutathione, Glutathione Peroxidase,Malondialdehyde as the indicator enzyme of oxidative stress, we assayed Superoxide dismutase activity with the xanthine oxidase hydroxylamine method, checked the activity of GSH-PX and GSH with micro plate method measured Malondialdehyde activity with thiobarbituric acid method, and measured the protein concentration by Bradford.6. To detect mRNA expression and protein level of Nrf2 by Real-Time PCR and Western blot analysis.7. Statistics:Use SPSS 13.0 (Statistical Package statistics for the Social Sciences) statistical software (SPSS, Chicago, IL. USA) for statistical analysis.Experimental values were mean±standard deviation (mean±SD), was used for statistical analyses including one-way ANOVA and Student’s t-test. A probability (P)-value<0.05 was considered statistically significant.Results1.Twenty fourhours after reperfusion following a 2 hour MCA occlusion, rats’model showed that the reaction of rats in the sham operation group was normal, no physical activity obstacle, the gait was stable, No neurologic deficits symptoms; compared with sham operation group, rats in the ischemia/reperfusion groups stoodunstably, left hemiparesis, left claw cannot stretch, Chased its own tail along the left side, circled to the left side when grabbed the tail.The evidences means that cerebral ischemia reperfusion model had been constructed successfully.2.According to the five-grade marking system of neurologic deficits, neurological function score of rats in I/R group were significantly higher than sham operation group (P<0.01),it means that rats in 1/R group had a neurological impairment. Neurological function score of rats in all phloretin treatment groups were higher than sham-surgery group, but significantly lower than I/R group (P<0.05).3.Comparison of cerebral infarction area showed that in the sham-surgery group the red color was uniformly distributed in the brain tissue, we can not detect cerebral infarction, while in the I/R group extensive white infarction lesions was found (P<0.01).Cerebral infarction area in all phloretin treatment groups were reduced than those in I/R groups (P<0.05).4. The changes of brain edema:no cerebral edema in the sham operation group, brain edema was obvious in the I/R group, (P<0.01).Degree of cerebral edema in the phloretin treatment groups were decreased obviously than that in the I/R group (P<0.05).5. Superoxide dismutase (SOD), Glutathione (GSH), Glutathione Peroxidase (GSH-PX), Malondialdehyde (MDA) activity:24 hours after reperfusion, compared with sham operation group, the activities of SOD、GSH、GSH-PXS in the I/R group were significantly decreased while increased of MDA (P<0.01).t in each phloretin treatment group,they are decreased than those in sham operated group, but increased obviously than in I/R group(P<0.05), Malondialdehyde activity in I/R group increased obviously than sham operation group (P<0.01).MDA activity in each phloretin treatment group increased than sham operation group, but decreased significantly than in I/R group (P<0.05).6. Relative mRNA expression of Nrf2 was elevated in the I/R group than in the sham operation group (P<0.05). Treatments with phloretin significantly increased mRNA expression levels of Nrf2 in a dose-dependent manner (P<0.05). In accordance with RT-PCR results, western blot analysis showed that Nrf2 protein level was upregulated in the I/R group, and significantly increased in phloretin pretreatment groups(P<0.05). (Figure 4).Conclusions1. Cerebral ischemia/reperfusion can cause significant neurological damage and brain edema, cerebral infarction.2.Superoxide dismutase,Glutathione and Glutathione Peroxidase induced by cerebral ischemia/reperfusionwere significantly decreased, while MDA level increased significantly, differences in the level of representation was statistically significant, it suggests that ischemia/reperfusion caused obvious oxidative stress injury to the brain tissue.3. Compared with I/R group,the score of the rats’ brain function decreased obviously after phloretin intervention,it suggests that phloretin can reduce neurological damage induced by cerebral ischemia/reperfusion.4.Compared with model group, brain edema was significantly relieved after phloretin intervention. It suggests that phloretin intervention significantly reduce cerebral edema induced by cerebral ischemia/reperfusion in rats.5.After Phloretin intervention, the increaseof Superoxide dismutase, Glutathione and Glutathione Peroxidase levels is obvious than the shaml group.The comparison was statistically significant, it suggests that phloretin can improve the antioxidant capacity of brain tissue and reduce oxidative stress injuryin the ischemia/reperfusion,6.The level of Malondialdehyde decreased significantly when compared with model group after intervention of phloretin.The comparison was statistically significant; it suggests that phloretin can alleviate oxidative stress injury of cerebral ischemia/reperfusion.7.The Nrf2 mRNA expression and protein level of the model group significantly enhanced than the sham-surgery group, the difference was statistically significant, it indicates that cerebral ischemia/reperfusion activates Nrf2 gene expression.8.The Nrf2 mRNA expression and protein level significantly enhanced in the phloretin treatment group than the I/R group, the difference was statistically significant, it suggests that phloretin can enhance the expression of Nrf2 gene.Our results suggest that there is obvious oxidative stress damage in the process of cerebral ischemia/reperfusion, and the intervention of phloretin could inhibit the oxidativestress reaction during cerebral ischemia reperfusion injury, improve the cerebral infarction, cerebral edema and cerebral function score caused by cerebral ischemia reperfusion injury, in this process, Phloretin markedly enhanced the expression of Nrf2.Therefore, our study suggests that in the rats experiment,phloretin protect against oxidative stress injury during cerebral ischemia/reperfusion through the pathway of up-regulating Nrf2.更多还原