【作者】 杨申；

【导师】 杜怡峰；

【作者基本信息】 山东大学 ， 神经病学（专业学位）， 2013， 博士

【摘要】 研究背景：血管性痴呆(vascular dementia,VD)是由一系列脑血管因素(包括缺血性卒中、出血性卒中等)导致的脑组织损害引起的严重的认知功能障碍为特征的综合症,是仅次于阿尔茨海默病(AD)的第二大常见的痴呆类型。我国VD的患病率为1.1%～3.0%,年发病率在5-9/1000人,并且发病率呈逐年上升的趋势,VD不仅严重影响病人自身的生活质量,还对社会和家庭造成沉重的经济和社会负担,血管性痴呆的防治已成为当前急需解决的医学和社会问题。VD是目前老年期痴呆中可预防和有希望治疗的痴呆类型之一,但是目前血管性痴呆的具体发病机制尚不清楚,这方面的研究成为了目前的研究热点。大脑海马体是与人类的学习记忆等高级功能有密切关系的重要功能区,其对缺血低灌注损伤有易感性,海马区神经元凋亡可能在血管性痴呆的发病过程中具有重要作用。已有实验研究发现减少Bcl-2、增加Bax的表达可激活Caspase-3和p53,进一步诱发大鼠海马区神经元的凋亡。Bcl-2和Caspase-3是参与调节细胞凋亡的两个重要分子,Bcl-2的过量表达能有效地抑制Caspase-3激活,最终引起海马区细胞凋亡。p38MAPK (mitogen-activated protein kinase, MAPK)的激活是缺血低灌注损伤引起神经元凋亡的重要信号转导通路之一,P38MAPK可通过调节Bcl-2、Bax和Caspase-3的表达影响细胞凋亡。目前已有p38MAPK特异性的抑制剂SB202190减轻脑缺血再灌注损伤的报道,但SB202190对海马神经元凋亡因子表达和学习记忆能力的影响却少有报道,特别是SB202190对海马神经元凋亡和Bcl-2、Caspase-3表达影响的研究国内外未见有报道。本研究中我们通过观察血管性痴呆大鼠海马区p38MAPK磷酸化的变化,探讨p38MAPK在血管性痴呆发病机制中的作用,并利用p38MAPK抑制剂SB202190作为研究干预方式,血管性痴呆大鼠侧脑室注射SB202190抑制p38MAPK信号通路后,通过Western Blot蛋白印迹法、免疫荧光法和激光扫描共聚焦显微镜等技术观察血管性痴呆大鼠海马区神经元凋亡、Bcl-2和Caspase-3凋亡因子表达变化和大鼠学习记忆能力的改变,探讨p38MAPK信号通路对血管性痴呆大鼠学习记忆的影响,并初步探讨其作用机制。第一部分血管性痴呆大鼠模型的制备和P38MAPK抑制剂对血管性痴呆大鼠行为学测试的影响血管性痴呆(VD)是老年期痴呆的最常见重要病因之一,学习记忆功能作为大脑的高级神经生理活动,在临床及实验研究中成为了药物干预血管性痴呆动物的一项重要检测指标。在学习记忆的高级神经活动中,动物海马回是最重要的中枢结构,其对缺血缺氧等损伤很敏感,特别是慢性脑低灌注损伤,实验已证实大脑的慢性缺血-再灌注可引起海马结构损伤最终导致学习、记忆功能受损。慢性脑灌注不足是血管性痴呆发病的主要病因,故两血管法被公认是目前国内外进行血管性痴呆动物研究中较为经典的动物实验模型,假手术组只分离双侧颈总动脉并不结扎,作为两血管法VD模型的正常对照。Morris水迷宫实验广泛应用于实验动物行为学的研究,隐蔽平台实验中的逃避潜伏期反映了动物空间记忆的情况,即学习能力；探索实验是Morris水迷宫中学习记忆保持情况的测试方法,即测试动物的记忆能力。我们应用两血管法建立血管性痴呆大鼠模型,研究P38MAPK抑制剂对血管性痴呆大鼠学习记忆能力的影响。目的与方法本实验采用了双侧颈总动脉结扎法造成大鼠脑组织慢性缺血损伤,并经过进行行为学测试确定存在学习、记忆障碍,制作血管性痴呆大鼠模型,假手术组只分离出双侧颈总动脉并不结扎动脉,作为两血管法VD模型的正常对照。VD大鼠造模手术恢复后采用侧脑室注射给药方式给予P38MAPK抑制剂SB202190,注射药物2周后用Morris水迷宫法测试各组大鼠的学习记忆能力的变化。结果1.各组大鼠平台逃避潜伏期的比较在侧脑室注射结束后1周后以Morris水迷宫开始隐蔽平台实验观察大鼠学习能力,随着训练天数的增加,各组大鼠平台逃避潜伏期均缩短,从第1天起假手术组潜伏期为24.17±4.12Ss,模型组潜伏期明显延长,为82.71±8.27s,应用SB202190后潜伏期明显缩短为48.72±7.01s,假手术组和SB202190组与模型组比较差异有显著性意义(P<0.01),第2天和第3天假手术组和SB202190组与模型组比较仍有显著性差异2.各组小鼠探索实验结果比较在Morris水迷宫的第4天撤除隐蔽平台后,观察各组大鼠的空间探索能力,假手术组大鼠停留平台象限时间35.21±3.78s,SB202190组为26.45-4.66s,模型组18.67±5.39s,应用SB202190组与VD模型组比较原平台象限停留时间有显著性差异(P<0.01)；各组大鼠当天的速度比较无明显差异(P>0.05)；假手术组、模型组和SB202190组穿越原平台次数分别为3.2±1.4；1.3±0.5；1.8±0.9,各组大鼠间比较有显著性差异(P<0.01)。结论本实验记录了各组大鼠平台逃避潜伏期并进行比较,结果发现从第1天起连续3天观察发现模型组大鼠的逃避潜伏期明显长于假手术组,说明作为同一种系假手术组大鼠学习能力比模型组强,模型组大鼠结扎双侧颈总动脉后学习能力明显下降,同时也证实了VD模型的成功性。进一步结果显示给予SB202190治疗后,相比于模型组大鼠,SB202190组大鼠逃避潜伏期明显缩短,原平台象限停留时间明显延长,提示给予SB202190治疗后大鼠的学习能力得以改善。研究结果发现SB202190作为P38MAPK抑制剂之一,在改善血管性痴呆大鼠的学习记忆能力方面具有确切疗效。第二部分SB202190对血管性痴呆大鼠海马区P38MAPK磷酸化变化、细胞凋亡及Bcl-2和Caspase-3表达影响的研究细胞凋亡(Apoptosis)是细胞的一种基本的生物学现象,是一个多基因严格控制的过程,Bcl-2和Caspase-3分别是调节细胞凋亡分子家族中的两个重要成员之一,特别是Bcl-2和Caspase-3在调节脑细胞凋亡中的作用已得到广泛实验证实。研究表明Bcl-2表达与细胞存活关系非常密切,增加脑内Bcl-2的表达可减少脑梗死病灶的大小并对神经元有保护作用。Caspase-3为Bcl-2的下游调控蛋白,是引起凋亡的始发因子,Bcl-2的过量表达能有效地抑制Caspase-3激活和细胞凋亡的发生。p38MAPK信号转导通路在细胞凋亡中发挥着重要作用,p38MAPK可被磷酸化激活后调节基因转录,诱导一些特定基因的表达,引起细胞增殖、分化及各种细胞因子的合成等,诱导Bcl-2、Caspase-3介导的凋亡通路,引起一系列的生物学效应。SB202190抑制缺血后神经元凋亡可能是多途径多靶点的过程,可能是通过Bcl-2抑制Caspase3/Caspase2依赖的蛋白水解级联反应,从而抑制线粒体释放细胞色素C减少细胞凋亡的发生。有研究表明VD大鼠学习记忆能力与P38MAPK的表达密切相关,本研究不仅要证实上述学者的研究结果,并且还要再进一步证实P38MAPK通路可调控凋亡因子Bcl-2和Caspase-3的表达,这可能是P38MAPK通路影响VD大鼠学习记忆能力的途径。目的与方法应用两血管法(bilateral common carotid artery occlusion,2VO)建立血管性痴呆(VD)模型,将60只三月龄雄性Wistar大鼠随机分成假手术组、血管型痴呆模型组和P38MAPK抑制剂SB202190组。各组大鼠均采用侧脑室注射给药方式给药,SB202190组大鼠应用微量注射器将10μmol/LSB202190溶液5μL进行侧脑室注射,模型组和假手术组大鼠给予侧脑室注射等量的1%DMSO溶媒液,注射药物后应用TUNEL法检测海马区神经元凋亡,应用蛋白印迹(Western blot)法检测大鼠海马区P38MAPK磷酸化变化,免疫荧光法和激光共聚焦显微镜观察大鼠海马区Bcl-2和Caspase-3表达。结果荧光强度检测结果显示,TUNEL阳性细胞以绿色荧光表示。假手术组切片偶见TUNEL阳性(绿色荧光)细胞,模型组、SB202190组阳性细胞明显增多,差异有显著性意义；SB202190组与模型组相比,TUNEL染色阳性细胞明显减少。以免疫印迹法观察各组大鼠海马p38MAPK磷酸化变化,三组大鼠海马组织内磷酸化p38MAPK的表达比较有显著性差异,模型组的表达在明显升高,而p-p38MAPK在假手术组仅有少量的表达,SB202190组的p-p38MAPK表达较模型组明显降低,有显著性差异。Bcl-2蛋白在本实验中标记为绿色荧光。激光共聚焦显微镜观察假手术组切片仅见极少量神经细胞免疫荧光表达,模型组可见Bcl-2免疫荧光强度增加,SB202190组明显增加。SB202190组与模型组比较,Bcl-2免疫荧光强度明显增加,有统计学意义。Bcl-2阳性细胞以绿色荧光表示,假手术组切片偶见阳性绿色荧光细胞,模型组可见Bcl-2阳性细胞数增加,SB202190组明显增加。SB202190组与模型组比较,Bcl-2阳性细胞数明显增多,有统计学意义。Caspase-3蛋白在本实验中也标记为绿色荧光。激光共聚焦显微镜观察假手术组切片仅见极少量免疫荧光表达,SB202190组可见Caspase-3免疫荧光强度增加,模型组明显增加。SB202190组与模型组比较,Caspase-3免疫荧光强度明显减少,有统计学意义。Caspase-3阳性细胞以绿色荧光表示,假手术组切片偶见阳性细胞,模型组可见Caspase-3阳性细胞数明显增加,SB202190组阳性细胞数增加。SB202190组与模型组比较,Bcl-2阳性细胞数明显减少,有统计学意义。结论本研究证实了P38MAPK通路可调控凋亡因子Bcl-2和Caspase-3的表达,这可能是P38MAPK通路影响VD大鼠学习记忆能力的途径。应用P38MAPK抑制剂SB202190后可抑制血管性痴呆大鼠海马区细胞凋亡,可能是通过Bcl-2抑制Caspase3依赖的蛋白水解级联反应,减少细胞凋亡的发生,从而改善大鼠的学习记忆能力,这些变化进一步证实了SB202190对大鼠海马神经元的独特保护作用。更多还原

【Abstract】 BackgroundsVascular dementia (VD) is the second most common form of dementia after Alzheimer’s disease (AD).VD is a syndrome includeing Severe cognitive impairment caused by a series of cerebral vascular factors(ischemic stroke, hemorrhagic stroke, etc). In China, the prevalence rate of VD was1.1%～3%, the annual incidence rate in5-9/1000, and the incidence rate increased year by year. VD not only seriously affects the life quality of the patients, but also caused heavy economic burden on society and family. The prevention and treatment of vascular dementia has become a serious medical and social problem.At present,VD is one of the dementia type which has the hope to get prevention and treatment,but the pathogenesis of vascular dementia is not clear,and the research in this area has become a research focus.Hippocampus is an important functional area which has close relationship with the human learning and memory, and it has susceptibility to ischemia hypoperfusion injury.The apoptosis of hippocampal neurons plays an important role in the pathogenesis of vascular dementia. The study found that decreased Bcl-2, increased the expression of Bax could activate Caspase-3and p53, consequently induce neuronal apoptosis in rat hippocampus.Bcl-2and Caspase-3are two important regulation molecular in apoptosis, overexpression of Bcl-2can effectively inhibit caspase-3activation and apoptosis.Activation of the p38MAPK (mitogen-activated protein kinase) signaling pathway by hypoxia may initiate neuronal apoptosis, leading to the functional deficits of VaD, P38MAPK signaling pathway can affect apoptosis by regulating the expression of Bcl-2, Bax and Caspase-3. At present, SB202190, the specific inhibitor of p38MAPK alleviate cerebral ischemia reperfusion injury were reported. No previous study has examined the effect of SB202190for sustaining hippocampus-dependent spatial memory or the effects of this inhibitor on the expression of apoptotic regulators under chronic ischemia, especially the expression of Bcl-2, Caspase-3.To these ends, we examined the effects of SB202190on hippocampal neuron apoptosis, Bcl-2and caspase-3expression, and p38MAPK phospho-activation in a rat model of VaD by Western blotting, immunofluorescence and confocal laser scanning microscopy, and tested whether p38MAPK inhibition can rescue deficits in hippocampus-dependent Morris water maze spatial learning and memory. Our results indicate that blockade of the p38MAPK signaling pathway can indeed protect hippocampal neurons against chronic ischemic injury and rescue spatial learning and memory deficits, at least in part by suppressing caspase-3-dependent apoptosis. Part1The making of vascular dementia rat model and the effect of P38MAPK inhibitor on the behavior test of vascular dementia ratsVascular dementia (VD) is a common cause of senile dementia, learning and memory function as the advanced neural physiological activity in the brain, has become important in experimental vascular dementia animal model.The hippocampus involved in learning and memory function, the advanced nerve activity. The hippocampus is very sensitive to ischemia and hypoxia, especially the chronic cerebral hypoperfusion. Previous experiments have confirmed that the ischemia-reperfusion of brain could damage the hippocampus,and lead to learning and memory impairment.Chronic cerebral hypoperfusion is the main cause of vascular dementia, and the making of vascular dementia experiment animal model by two vessels method is recognized the classical method at home and abroad.In our experiment the rat in sham operation group only been isolated bilateral common carotid artery without ligation. Morris water maze was used in the experimental animal behavior experiments, the hidden platform in escape latency denote the animal’s spatial memory, learning ability; exploring experimental in Morris water maze is maintained to the test animal memory. We use two vessel method to establish vascular dementia rat model, the effects of P38MAPK inhibitors on the ability of learning and memory of vascular dementia rats.Objective and methodsThis experiment used the bilateral common carotid artery ligation caused by chronic ischemia injury of rat brain tissue, and through behavior tests were carried out to determine the existence of learning and memory disorders, make the model of vascular dementia rats, sham operation group only isolated bilateral common carotid artery ligation and normal control is not, as the two blood tube method VD model. The intracerebroventricular injection way to give P38MAPK inhibitor SB202190, affect learning and memory from the7day after the model was tested by Morris water maze in rats of each group.ResultOne week after ICV injection, hippocampus-dependent spatial learning was examined by the MWM hidden platform trail. On each of three testing days, the deficit in spatial learning as measured by escape latency was significantly longer in the model group than the sham-operated group (all P<0.01), suggesting successful induction of hippocampal deficits. Latencies were significantly lower in rats injected with SB202190compared to the model group on each day of testing (all P<0.01). indicating spatial learning, whilst latency in the VaD model group was not significantly reduced by training (from82.71±8.27s to77.74±6.33s, P>0.05). Escape latencies in the SB202190group decreased from48.72±7.01s to40.34±2.46s (P<0.05), indicating spatial learning improvementOn day4, the hidden platform was removed from the MWM in order to conduct a probe test of spatial memory. sham operation group rats remain platform quadrant time was35.21±3.78s, SB202190group was26.45±4.66s,18.67±5.39s in model group, there were significant differences between the SB202190group and sham operation group and model the residence time of the original platform quadrant group (P<0.01); rats crossing the original platform times had significant difference (P<0.01);there was no significant difference between the speed of the rats in each group (P>0.05).ConclusionIn this experiment, the platform escape latency of each rats was recorded and compared, in the first day, the escape latency of VDmodel group was significantly longer than that in sham operation group, indicate learning ability of fake operation group rats was better than the model group, the learning ability of VDmodel group by ligation of bilateral common carotid artery was significantly decreased, which confirmed the success of VD model. Furthermore after SB202190treatment, escape latent period of SB202190group rat is shortened obviously, the original platform quadrant time was significantly prolonged, indicate the learning ability of rats after SB202190treatment improved. SB202190, one of the P38MAPK inhibitors, which has definite effect on improve the learning memory ability in vascular dementia rats model. Part2The effect of P38MAPK inhibitor on P38MAPK phosphorylation、cell apoptosis、and the expression of Bcl-2、Caspase-3in hippocampus of vascular dementia ratApoptosis as a basic biological phenomena in cell, is a multi gene controlled process.Bcl-2and Caspase-3are two important member in of apoptosis molecules family, especially the function in the regulatiing on brain cell apoptosis has been widely confirmed. Studies have shown that the expression of Bcl-2is closely related to cell survival, and increase the expression of Bcl-2in brain may reduce cerebral infarct size and has protective effects on neural cells. Caspase-3is a downstream regulated protein of Bcl-2, also is the initial factor of apoptosis. The overexpression of Bcl-2can effectively inhibit the activation of Caspase-3and the apoptosis.P38MAPK signal transduction pathway plays an important role in apoptosis. Phosphorylated p38MAPK,the activated form can regulate gene transcription, induce the expression of specific genes, cause cell proliferation、differentiation、apoptosis and cytokine synthesis, and induce p38-Caspase-3apoptosis pathway. The inhibition of neuronal apoptosis after ischemia by SB202190may be the multi-channel and multi targets.lt can inhibite mitochondrial cytochrome C release and reduce apoptosis by Caspase3/Caspase2dependent proteolytic cascade which inhibited by Bcl-2. Studies showed that learning memory ability and the expression of P38MAPK was closely related to the VD rats. Our study want to confirme that the P38MAPK pathway can regulate the expression of apoptosis factor Bcl-2and Caspase-3, by which P38MAPK pathway can affect the learning and memory ability of VD rats model.Objective and methodsvascular dementia (VD) model was established by two vessels method (bilateral common carotid artery occlusion,2VO),60male Wistar rats3month old were randomly divided into sham operation group, vascular dementia model group and SB202190group. Rats were injected medication into the lateral ventricle, the rats of SB202190group injected5μL SB202190(10μmol/L)solution by a micro syringe into lateral ventricle. The model group and sham operation group rats were injected5μL1%DMSO solution The hippocampus cell apoptosis was detected by TUNEL method after the injection of drugs, western blot method was used to detect p-P38MAPK in the rat hippocampus, and mmunofluorescence and confocal laser scanning microscopy were used into observe Bcl-2and Caspase-3expression in hippocampus of rats.ResultThe results of the fluorescence intensity display, TUNEL positive cells expressed green fluorescence. The sham operation group slices express few TUNEL positive cells (green fluorescence), in the model group and the SB202190group positive cells increased obviously, there was significant difference; compared TUNEL positive cells decreased in SB202190group than in the model group.Observe the changes of p38MAPK phosphorylation in rats hippocampus by Western blot method, the expression of p-p38MAPK in different groups of rat hippocampus have obvious differences, the expression of p-p38MAPK increased significantly in the model group and a small amount of p-p38MAPK expression in sham operation group.the expression of p-p38MAPK in SB202190group decreased obviously compared with the model group, the difference was significant.Bcl-2protein in this experiment was marked as green fluorescence. The expressionof Bcl-2protein in the sham operation group was only few immunofluorescence, Bcl-2fluorescence intensity in the model group increased, and fluorescence intensity in SB202190group significantly.Bcl-2immune fluorescence intensity increased significantly in SB202190group compared with model group. The Bcl-2positive cells expressed green fluorescent, positive cells could be occasionally found in sham operation group, and increased in model group, significantly increased in SB202190group. the number of Bcl-2positive cells increased obviously in SB202190group compared with the model group, there was statistical significance.Caspase-3protein in this experiment was also marked as green fluorescence. only few immunofluorescence expressed in sham operation group, fluorescence intensity of Caspase-3in SB202190group increased, and significantly increased in the model group. The fluorescence intensity of Caspase-3decreased significantly in SB202190group compared with model group, and there was statistical significance. The Caspase-3positive cells expressed as green fluorescent, positive cells in sham operation group were occasionally observed, the number of Caspase-3positive cells in the model group increased significantly, the number in SB202190group increased. SB202190group compared with the model group, the number of Caspase-3positive cells decreased significantly, there was statistical significance.ConclusionThis study demonstrated that the P38MAPK pathway can regulate the expression of apoptosis factor Bcl-2and Caspase-3, which may be the way that P38MAPK pathway affect the learning and memory ability of VD rats. Application of P38MAPK inhibitor SB202190can inhibit the apoptosis of cells in hippocampus in vascular dementia rats model, may be a proteolytic cascade by the inhibition of Bcl-2on Caspase3, improving the ability of learning and memory in rats by the reduction of apoptosis.All of these changes confirmed the unique protective effect of SB202190on hippocampal neurons in VD rats model.更多还原