【作者】 张静；

【导师】 刘雪平；

【作者基本信息】 山东大学 ， 老年医学（专业学位）， 2015， 博士

【摘要】 目的慢性脑灌注不足(CCH)可能导致各种神经系统疾病或精神疾病,CCH与学习和记忆功能障碍密切相关,尤其是血管性痴呆,表现为认知障碍和行为不良,CCH动物模型可以由中断两侧颈动脉主干的大鼠制备(称作2VO大鼠),2VO大鼠脑血流量不足导致大脑皮层、海马体和白质慢性低灌注,从而发生认知功能障碍和不良的新陈代谢,最终导致神经元损伤,可发生血管性痴呆。据报道称肾素-血管紧张素系统(RAS)为内环境平衡的重要调控系统,并被认为是治疗慢性脑灌注不足CCH诱发的认知功能障碍的潜在靶点。血管性痴呆主要病因之一是脑血流量减少诱发的活性氧(ROS)的过度表达。活性氧的超表达作为RAS过程一部分,可作为氧化应激开始的标志,这被定义为活性氧ROS和与抗氧化反应间平衡被打破的标志。过量的ROS和自由基会导致严重的细胞损伤,包括膜脂质过氧化作用,DNA断裂及损伤和蛋白质损失,在大脑中,活性氧的过量产生能够导致神经细胞及星形胶质细胞的死亡及细胞凋亡,从而导致永久性神经损伤。ROS生成和其有害影响不仅在血管性痴呆存在,也见于中枢神经系统缺血性、出血性及再灌注条件下的病变。就这一点而言,ROS清除化合物在各种疾病模型中被广泛测试以减少活性氧水平。葛根素,一种异黄酮,可以在许多植物和草药中发现,如葛根的根。葛根素也是常用于治疗发烧和腹泻的中药。事实证明,葛根素具有清除活性氧和减少脂质过氧化反应的能力。葛根素也被证明在不同种类的神经系统疾病中有神经保护作用,如阿尔茨海默氏症,帕金森氏病和脑缺血。关于葛根素的保护作用,其清除活性氧的能力是在大部分的研究中被重点提及的。一些研究还关注到葛根素在特殊的细胞类型中具有抗凋亡的活力。然而关于葛根素对血管性痴呆的保护作用目前尚无此类研究。在本研究中,我们试图通过2 VO大鼠模型验证葛根素对慢性缺血引起的血管性痴呆具有保护作用。进一步验证葛根素能够减轻血管性痴呆所致的认知和行为障碍。推论葛根素在2 V0大鼠模型中的保护作用可能是通过清除ROS以减少氧化应激反应达到的。方法6到8周成年雄性Wistar大鼠作为实验动物,经由中断两侧颈动脉主干制备2VO大鼠模型,给予或不给予葛根素治疗。采用莫里斯水迷宫测试大鼠的学习和记忆功能。丙二醇(MDA),谷胱甘肽过氧化物酶和总硫醇评估用于反映脑组织的氧化应激。SH-SY5Y细胞培养后给予过氧化氢模拟氧化应激,给予或不给予葛根素治疗,通过细胞计数Kit-8(CCK8)和流式细胞术(FCM)进行检查细胞不同时间点及给予不同浓度葛根素治疗后的生存率和凋亡率。活性氧(ROS)的产生由2’,7’二氯荧光素二乙酸(DCFH-DA)检验。采用qPCR和Western blot (WB)方法测试Nrf2、FoxO1 FoxO3和FoxO4.基因表达的变化,探讨葛根素的分子作用机制。结果1、莫里斯水迷宫试验中2VO组动物逃脱时间比假手术组呈现显著延迟(P<0.05)。在2VO加葛根素干预组中逃脱时间较2VO组明显缩短,改变显著(P<0.05)。假手术组动物给予葛根素干预后逃脱时间未见明显改变。在目标象限花费的时间测试中,2VO模型组在目标象限区域花费的平均时间较短,与假手术组差异显著(P<0.05)。在2VO加葛根素干预组在目标象限区域花费的平均时间有所延长,与2VO差异显著(P<0.05)。假手术组动物给予葛根素干预后在目标象限区域花费的平均时间没有任何改变。四组动物的游泳速度无显著差异。2、在2VO组海马和额叶皮质区的丙二醛含量较假手术组明显提高(P<0.05)；葛根素干预组可以显著下调海马和额叶皮层升高的MDA水平(P<0.05)；假手术组给予葛根素干预对丙二醛含量没有任何影响。2VO组的谷胱甘肽过氧化物酶浓度较假手术组较低,具有统计学差异(P<0.05)；葛根素干预组显示了葛根素对谷胱甘肽过氧化物酶浓度较大部分修复作用(P<0.05)；假手术组给予葛根素干预与假手术组比较未显示任何差异。2VO组的总硫醇浓度较假手术组显著减少；葛根素干预组可以部分提高2V0组减少的总硫醇浓度；假手术组给予葛根素干预与假手术组比较未显示任何有统计学意义的变化。3、以过氧化氢作为氧化应激物刺激SH-SY5Y细胞后,给予或不给予葛根素治疗均评估细胞的存活率。葛根素治疗在三个不同浓度:0.1umol/1,0.5umol/l和1umol/l。分别在12小时,24小时和48小时检测细胞存活率。结果表明,过氧化氢诱发的氧化应激在三个时间点均下调细胞的存活率。葛根素治疗上调SH-SY5Y细胞的细胞存活率呈剂量依赖性。1umol/1葛根素治疗组改善效果最好,几乎达到对照组的相同水平。然后我们检测了氧化应激后的细胞凋亡率,结果与前相反,葛根素治疗降低SH-SY5Y细胞的细胞凋亡率呈剂量依赖性,1umol/1葛根素治疗组改善效果最好,几乎达到对照组相同的水平。4、为了进一步探索葛根素对活性氧的清除能力,我们在SH-SY5Y细胞中通过DCFH-DA测定做ROS清除能力试验。细胞内ROS水平通过DCFH-DA来分析,因为DCFH-DA在细胞间具有渗透性,且对氧化敏感。给予氧化氢诱导24小时后,培养基换做含有不同剂量葛根素的培养基。葛根素治疗12小时或24小时后,通过DCFH-DA分析测定细胞内ROS水平。DCF荧光强度的定量分析显示给予过氧化氢诱导的未治疗组显示荧光水平最高。葛根素治疗组的ROS阳性细胞数在12小时和24小时均显著下调。1 umol/1浓度葛根素组与其他组比较显示最好的活性氧清除能力(P<0.05)。葛根素减少SH-SY5Y细胞内DCF荧光存在明显剂量依赖性。5、我们通过qPCR和免疫印迹进行测试Nrf2、FoxO1 FoxO3和FoxO4基因表达的变化。结果,我们发现,虽然葛根素治疗组可以显著提高Nrf2 mRNA的表达,但过氧化氢对Nrf2的mRNA水平并没有影响。至于FoxO家族,通过氧化氢诱导FoxO1 FoxO3 FoxO4提高了在mRNA水平表达。葛根素治疗增强了过氧化氢的FoxO1 FoxO3 FoxO4在mRNA水平表达增高的效应(P<0.05)。这个结果与通过WB检测的蛋白水平的结果是一致的。WB图像的定量分析显示,这些抗氧化应激基因的表达是根据mRNA水平改变情况以不同的模式被上调的。结论1、我们的研究结果表明,无论是体内环境慢性缺血诱发的还是体外过氧化氢诱发的氧化应激反应,葛根素干预后均可以有明显逆转作用。对于2V0老鼠,葛根素干预也会提高学习能力和巩固记忆,我们发现葛根素的保护作用都与葛根素对ROS的清除能力密切相关,无论是慢性缺血诱发的还是体外过氧化氢诱发的反应,此现象也能解释葛根素体外试验的抗凋亡活力。在分子水平,我们发现葛根素的治疗作用与几种抗氧化蛋白表达相关,mRNA水平, Nrf2、FoxO1 Fox03和Fox04蛋白质水平均显著上调。还有一个有趣的发现,过氧化氢处理也可以上调Nrf2的表达。2、我们的研究中2V0老鼠被选为体内动物模型,双侧的颈总动脉结扎可能导致大脑全球缺血。两个目标区域,即海马和额叶皮质,几乎全部受到这个缺血影响,其中海马负责学习和记忆功能。然后我们使用莫里斯水迷宫测试每组的学习和记忆功能。所有结果表明葛根素对2V0老鼠受损的学习和记忆能力具有保护作用。3、已经证实氧化应激在缺血和缺氧性脑损伤中扮演着关键角色。氧化应激也与炎性细胞因子的过度表达和血管受损导致的认知功能障碍相关。活性氧能够氧化膜脂质,细胞蛋白质和细胞核内的核酸,最终导致细胞功能障碍。考虑到这个,我们重点关注2V0大鼠脑组织中ROS的过度生成和自由基的产生,我们测量了MDA的水平,这是脂质过氧化反应的一个标志。谷胱甘肽过氧化酶和硫醇含量分别测量体外试验中对抗活性氧生成的酶促和非酶促性防御反应。为了进一步解释葛根素在体外试验中对ROS的清除能力,选取SH-SY5Y细胞进行培养,给予过氧化氢作为刺激物施加刺激,模拟体内状态时缺血诱导的氧化应激状态。我们的结果指出葛根素治疗能够减少过氧化氢诱导的细胞凋亡。我们发现葛根素治疗部分逆转了过氧化氢诱导的氧化应激反应的不利影响,呈计量依赖性。4、为了探索其潜在机制,我们重点关注调控因子FOX O家族(FoxO)和Nrf2的表达。FoxO家族通过提高几种抗氧化酶的氧化还原信号的表达以减少ROS生成。FoxO家族蛋白质也是调控细胞增殖,分化,细胞凋亡,细胞周期逮捕和自噬的转录因子。另一个我们关注Nrf2基因,也调节对氧化损害有保护作用的抗氧化蛋白质。FoxO家族和Nrf2在中枢神经系统具有重要的生理功能和病理作用。我们首次报道了葛根素治疗作用与上调缺血大脑中的这些基因具有相关性。总之,我们猜测葛根素对慢性脑缺血诱发的血管性痴呆具有保护作用表现在减轻氧化应激和提高认知能力。更多还原

【Abstract】 ObjectiveChronic cerebral hypoperfusion (CCH) can be caused by different kinds of neurological diseases or psychiatric diseases. Studies have shown that CCH is closely related with learning and memory dysfunction within those diseases, especially in vascular dementiathe, characterized by cognitive impairment and bad behavior. The CCH animal models can be prepared by disruption of the bilateral common carotid arteries (2VO rats) as the pathological change occurred in vascular dementia. In the 2VO rats, cerebral blood flow was disrupted and cause cerebral chronic hypoperfusion in the cortex, hippocampus and white matter. Cognitive dysfunction and suboptimal metabolism thus occurred, which lead to final neuronal injury. The rennin-angiotensin system (RAS) was reported to be important regulatory systems in the circulatory homeostasis and was regarded as potential target in treating CCH induced cognitive defects.One of main cause of vascular dementia is reactive oxygen species (ROS) overexpression induced by cerebral blood flow decrease. Reactive oxygen species superexpression take part in the RAS process, it is the symbolled the beginning of oxidative stress, which is defined the symbol of unbalance between active oxygen ROS and anti-oxidative reaction. Excessive ROS and free radicals can cause serious cell damage, including the membrane lipid peroxidation, fracture and damage DNA and protein loss. In the brain excessive production of reactive oxygen species can lead to the death or apoptosis of neurocytes and astrocytesl, which can lead to permanent neurological damage. ROS generation and its harmful effects were found not only in the vascular dementia, but also in the ischemic, hemorrhagic desease and the reperfusion lesion of the central nervous system. In this regard, ROS-removal compounds were widely tested in all kinds of disease models in order to reduce the level of reactive oxygen species.Puerarin, one of isoflavones, can be found in a number of plants and herbs like the root of Pueraria. Puerarin is also commonly used in traditional Chinese medicine for symptoms like fever and diarrhea. It has been proved that puerarin has the ability of scavenging ROS and reducing lipid perioxidation. Puerarin was also proved to be neuroprotective in different kinds of neurological diseases like Alzheimer’s disease, Parkinson’s disease and brain ischemia. The ROS cavenging ability of puerarin was emphasized in most of the studies referring the protective effects of puerarin. Several studies also focused on the anti-apoptosis activity of puerarin on particular cell types. However, no study has yet been done considering the protective effects of puerarin on vascular dementia.In this study, we tried to validate that puerarin play a protective role in vascular dementia caused by chronic ischemia through 2VO rats model. At the same time, we also tried to validate that puerarin could reduce cognitive and behavioral barriers caused by vascular dementia. We made a inference that puerarin protective effect in 2 VO rats model may be achieved through clearing ROS in order to reduce oxidative stress reaction.Methods6 to 8 week old male Wistar rats were adopted as experimental animals. The CCH animal models can be prepared by disruption of the bilateral common carotid arteries (2VO rats) as the pathological change occurred in vascular dementia. The rats were divided into defferent groups who accepted puerarin treatment or not. Morris water maze (MWM) test was adopted to test the learning and memory function of rats. MDA, glutathione peroxidase and total thiol assessment was done to reflect the oxidative stress in the brain tissue. Hydrogen peroxide is administered after SH-SY5Y cell culture-simulation of oxidative stress, give puerarin treatment or not. Cell Counting Kit-8 (CCK8) and flow cytometry (FCM) were performed to examine the cell viability and apoptosis rate. Reactive oxygen species (ROS) generation was determined by the 2’,7’-dichlorofluorescein diacetate (DCFH-DA) assay. qPCR and Western blot (WB) were adopted to test the change of gene expression including Nrf2、FoxO1 FoxO3 和 FoxO4, and investigate the molecular mechanism of puerarin treatment.Results1, In Morris water maze test 2VO animals spent more time on escaping time than control group, the delay is significant (P< 0.05). The escape time of 2VO followed puerarin-treat group is significantly shortened than 2VO group(P< 0.05). In the control with puerarin-treatment group the escape time had no obvious change with the control group. In Probe Trains, it took the average time in the target quadrant area by 2VO group model shorter than the control group, it’s significant difference (P< 0.05). 2VO with puerarin treatment group spent more average time in the target quadrant area than 2VO group, it’s significant difference (P< 0.05). Control with puerarin-treatment group spent almost the same average time in the target quadrant area as control group, there is no significant difference. There was no significant difference about swim speed among four groups of animals.2, The malondialdehyde(MDA) content of hippocampus and frontal cortex in 2VO group increased significantly compared with the control group (P< 0.05); Puerarin intervention group can significantly regulated-down the increased MDA level of hippocampus and frontal cortex (P< 0.05); The malondialdehyde content of control group could not be effected by the intervention of puerarin.The concentration of glutathione peroxidase in 2VO group was lower than the control group with statistical difference (P< 0.05); Puerarin intervention group shows the puerarin’s cure effect on glutathione peroxidase concentration compared with the 2VO group.(P< 0.05); The control with puerarin-treatment group did not show any difference compared with control group.2VO rats showed a significant decease in the level of total thiol compare to the sham group (P< 0.05). Administration of puerarin in 2VO rats could partial y improve the total thiol level decreased by 2VO procedure. Administration of puerarin in the sham+puerarin did not show statistical meaningful changes on the thiol level.3, To simulate the in vivo situation of vascular dementia and 2VO models, we stimulated the SH-SY5Y eel s with hydrogen peroxide for oxidative stress. Cell viability was evaluated with or without treatments of puerarin. Puerarin was treated under three different concentrations:0.1 uM,0.5 uM and 1 uM. Cell viability was measured at 12 h,24 h and 48 h respectively. The results showed that oxidative stress induced by hydrogen peroxide could significantly down-regulate the cell viability at the three time points. The cell viability of SH-SY5Y cells was improved by puerarin treatments in a dose-dependent way.1 uM of puerarin showed the best improving effects that almost reached to the same level of the control groups. We then tested the cell apoptosis rate induced by oxidative stress. The results went an opposite pattern showing that pueararin could decrease the apoptosis rate induced by hydrogen peroxide in a dose-dependent way.1 uM of puerarin showed the best anti-apoptosis effects compare to the other puerarin treatments groups.4, To further explore the ROS scavenging ability of puerarin, we performed ROS scavenging assay by DCFH-DA on SH-SY5Y cells. Intracellular ROS levels were analyzed by DCFH-DA, which is cell permeable and oxidation sensitive within cells. After 24h’s induction of hydrogen peroxide, culture medium was replaced with different dosages of puerarin. After 12 h or 24 h of puerarin treatment, intracelular ROS levels were then analyzed by DCFH-DA. The results remarkably showed that puerarin decrease the intensity of DCF fluorescence within SH-SY5Y cells in a dose-dependent way. Quantitative analysis of DCF fluorescence intensity showed that groups induced with hydrogen peroxide and no treatment of puerarin showed the highest fluorescence level. The ROS positive cell number was significantly down regulated by puerarin treatments at both 12 h and 24 h.1 uM of puerarin showed the best ROS scavenging ability compare to other groups (P< 0.05).5, We previously proved that puerarin could effectively scavenge intracellular ROS generation induced by hydrogen peroxide. To explore the underlying mechanisms, we hypothesized that the antioxidant activity of puerarin might be related with alterations of gene expressions. We then investigated several crucial genes related with antioxidant protein expression namely Nrf2, FoxO1. FoxO3 and FoxO4. Qpcr and western blot were performed to test the expression changes of these genes. From the results, we found that hydrogen peroxide is not affective on the mRNA level of Nrf2, while puerarin treatments could significantly improve the expression of Nrf2. As for the FoxO family, FoxO1, FoxO3 and FoxO4 were improved by hydrogen peroxide induction on the mRNA levels. Puerarin treatments could enhance the improving effects of hydrogen peroxide(P< 0.05). The results were consistent on the protein levels proved by WB. The quantitative analysis of WB images showed that these anti oxidative stress genes xpressions were up regulated in different patterns according to the mRNA level alterations.Conclusions1, Our results demonstrated that puerarin administration both in vivo and in vitro could significantly reverse the oxidative stress induced by ischemic conditions or hydrogen peroxide. Puerarin administration could also improve the learning ability and consolidate the memory in 2VO rats. We found that the protective effects of puerarin on the chronic ischemic condition and oxidative stress induced by hydrogen peroxide in vitro is closely related with the ROS scavenging ability of puerarin, which also explained the anti-apoptosis activity of puerarin in vitro. On the molecular level, we discovered that treatments of puerarin is associated with the up regulation of several antioxidant protein expression. The mRNA level and protein levels of Nrf2, FoxOl, FoxO3 and FoxO4 were significantly up regulated by treatments of puerarin. It was also interesting to find that hydrogen peroxide treatment alone could also up regulate the expression of Nrf2.2,2VO rat model was chosen as the in vivo animal model in our study. The ligation of the bilateral common arteries could cause global ischemia of the brain. Two target areas were mostly affected from this ischemia namely hippocampus and frontal cortex in which hippocampus is in charge of the learning and memory function. We then performed the Morris Water Maze test for the evaluation of learning and memory function within each group. Al the results indicated protective effects of puerarin on the impaired function of learning and memory abilities in 2VO rats.3, It has already been proved that oxidative stress plays a crucial role in the ischemic and hypoxic brain damage. Oxidative stress is also related with over expression of inflammatory cytokines and cognitive dysfunction caused by vessels impairments. ROS is capable of oxidize membranous lipids, cell proteins and nuclear acids within the nuclei that eventually lead to cellular dysfunction. In this consideration, we focused on the ROS over generation and free radicals production in the brain tissue of 2VO rats. We measured the levels of MDA, which is a marker of lipid peroxidation. GSH-Px and thiol levels were measured as enzymatic and non-enzymatic defense of ROS generation in vitro respectively. To further explain the ROS scavenging ability of puerarin in vitro, SH-SY5Y cells were selected to be cultured in vitro and stimulated with hydrogen peroxide as simulation of ischemic induced oxidative stress in vivo. In our results, it was indicated that puerarin treatments were capable of reducing the hydrogen peroxide induced cell apoptosis. We found that puerarin treatments partially reversed the adverse effects of oxidative stress induced by hydrogen peroxide in a dose-dependent way.4, To explore the underlying mechanisms, we focused on the forkhead box O (FoxO) family and Nrf2 expression. The FoxO family reduces ROS production by increasing the expression of several antioxidant enzymes of redox signaling. FoxO family proteins are also transcription factors regulating cel proliferation, differentiation, apoptosis, cel cycle arrest and autophagy. Another gene we focused on is Nrf2, which also regulate antioxidant proteins protective on oxidative damages. Both FoxO family and Nrf2 have important roles in the physiological function and pathological conditions in the CNS. For the first time we reported that puerarin treatments is associated with the up regulation of both these genes in the ischemic brain. In conclusion, we hypothesized that puerarin is protective on the vascular dementia induced chronic brain ischemia by alleviating the oxidative stress and improving the cognitive functions.更多还原