纤丝状Aβ_(1-42)与Aβ_(1-42)寡聚体诱导阿尔茨海默氏病模型大鼠行为学、海马超微结构变化、炎症因子表达及海风藤的干预研究
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摘要
研究背景：阿尔茨海默病(Alzheimer disease, AD)是中枢神经系统最常见的变性疾病,其典型的临床表现为进行性加重的记忆力减退、认知功能障碍、行为异常和社交障碍。主要的病理改变为神经纤维缠结(neurofibrillarytangle,NFT)、老年斑形成(senileplaque, SP)和神经元缺失。AD的发病机制复杂,病因至今不清,自20世纪80年代以来形成了各种假说,目前普遍公认的是β淀粉样蛋白β-amyloid protein, Aβ)假说,Aβ是SP的主要组成成分,由β淀粉样蛋白前体蛋白(Aβ precursor protein, APP)通过β、γ分泌酶水解产生。AD的发生、发展与Aβ因代谢障碍而在基底前脑异常沉积密切相关。而且近年来的研究发现,与纤丝状Aβ相比较Aβ寡聚体具有更高的神经毒性,是AD发病过程中的主要毒性物质。只是目前Aβ寡聚体的整体动物行为学实验结果还存在争议,不同聚集形式Aβ之间整体动物水平上的对比研究也较少。
目前关于AD发病过程的研究提示,Aβ激活胶质细胞诱发中枢神经系统慢性炎症损伤可能是导致AD的一个核心病理环节,参与该炎性反应的主要是小胶质细胞(microglia, Mi),位于Mi表面的Toll样受体(Toll—like receptors, TLRs)及其辅助受体CD14共同参与了Aβ激活Mi的过程,但是上述结论多来自纤丝状Aβ的研究,TLRs能否识别寡聚体的Aβ还没有定论。
此外,AD的基础研究过程中动物模型的建立一直是关键,但是目前各类模型都存不足,还没有任何一种动物模型能全面再现、模拟AD的病理、生化、行为学等方面的全部特征。本实验使用微渗泵将Aβ1-42聚合体和寡聚体持续灌注至大鼠侧脑室建立AD模型,从而使大量Aβ缓慢持续弥散性地分布到老龄大鼠脑内,较好地解决了同类模型Aβ短时在注射点局部聚集的问题,与AD的临床病理过程更接近。
中药海风藤是胡椒属植物风藤的滕的茎，中国药典记载其具有通经络、祛风湿和止痹痛的功效,现代医学研究发现其具有抗炎和神经保护作用,近年来开始应用于AD的治疗研究。
本研究应用纤丝状Aβ1-42和Aβ1-42寡聚体侧脑室持续灌注制备AD大鼠模型,观察两种不同聚集形式的Aβ对大鼠行为学、海马区神经细胞超微结构及Toll样受体4(Toll—like receptor4,TLR4)等炎症因子表达的影响,同时应用海风藤提取物干预治疗,观察其对上述指标的作用,以期进一步揭示AD的发病机制、探寻安全有效的治疗方法。
目的：探讨纤丝状Aβ1-42与Aβ1-42寡聚体对大鼠行为学、海马区神经细胞超微结构及TLR4、核因子-KB(nuclear factor-kappa B,NF-κB)、肿瘤坏死因子-α (tumor necrosisfactor-α,TNF-α)表达的影响；探讨应用海风藤干预后上述指标的变化。
方法：健康雄性SD大鼠60只,随机数字表法平均分为10组：正常对照组(Control组)、假手术组(Sham组)、纤丝状Aβ+载体1组(FAβ组,脑室内灌注纤丝状Aβ+乙腈和三氟乙酸造模)、纤丝状Aβ+海风藤提取物(Piper kadsura ohwi PKO)组(FAβ+PKO组,脑室内灌注纤丝状Aβ+乙腈和三氟乙酸造模,腹腔注射PKO治疗)、纤丝状Aβ+二甲基亚砜(Dimethyl sulfoxide DMSO)组(FAβ+DMSO组,脑室内灌注纤丝状Aβ+乙腈和三氟乙酸,腹腔注射DMSO).载体1组(Veh1组,脑室内不注射Aβ,仅灌注乙腈和三氟乙酸)、Aβ寡聚体+载体2组(AβO组，脑室内灌注Aβ寡聚体+高密度脂蛋白和羟乙基哌嗪乙磺酸造模)、Aβ寡聚体+PKO(A βO+PKO组,高密度脂蛋白和羟乙磺酸造模,腹腔注射PKO治疗)、Aβ寡聚体+DMSO组(AβO+DMSO(?)(?),脑室内灌注Aβ寡聚体+高密度脂蛋白和羟乙基哌嗪乙磺酸,腹腔注射DMSO)、载体2组(Veh2组,脑室内不注射Aβ,仅灌注高密度脂蛋白和羟乙基哌嗪乙磺酸)。采用微渗泵向侧脑室持续灌注纤丝状Aβ1-42或Aβ1-42寡聚体法制作动物模型。造模后每天给FAβ+PKO组和AβO+PKO组大鼠腹腔注射浓度10%含2.5%DMSO的PKO,按体重给药(1ml/100g),FAβ+DMSO组和AβO+DMSO组大鼠每天仅给予2.5%DMSO(?)腹腔注射(1ml/100g),连续给药35天。造模后第31天开始进行Morris水迷宫实验评定大鼠的学习记忆功能，喧闹验分为空间探索和定向航行两部分。空间探索实验时记录大鼠的逃避潜伏期,定向航行实验时录制大鼠在水迷宫游动视频，计算其穿越平台次数,在平台所在象限穿行的时间比率和路程比率。电镜观察海马区神经细胞超微结构,实时荧光定量聚合酶链式扩增反应(real-timepolymerasechainreactio,RT-PCR)法检测海马区TLR4mRNA表达,免疫印迹法(Western blot,WB)检测NF-κBP65、TLR4蛋白表达,酶标记免疫吸附法(ELISA)检测INF-α的表达。
结果：
(1)水迷宫实验结果：
Control组、Sham组、Veh1组和Veh2组之间比较逃避潜伏期、穿越平台的次数、时间比率和路程比率无显著性差异(P均>0.05)；FAβ组与FAβ+DMSO组比较、AβO组与AβO+DMSO组比较逃避潜伏期、穿越平台的次数、时间比率和路程比率无显著性差异(P均>0.05)；FAβ组、FA β+DMSO组和AβO组、A β O+DMSO组与Control组、Sham组、Veh1组和Veh2组比较逃避潜伏期显著延长、穿越平台的次数显著减少、时间比率和路程比率也显著减低(P均<0.05)；AβO组与FAβ组比较逃避潜伏期显著延长、穿越平台的次数显著减少、时间比率和路程比率也显著减低(P均<0.05)；FA β+PKO组和A β O+PKO组与Control组、Sham组、Veh1组和Veh2组比较逃避潜伏期显著延长、穿越平台的次数显著减少、时间比率和路程比率也显著减低(P均<0.05)；但与FAβ组、FAβ+DMSO组和AβO组、A β O+DMSO组比较逃避潜伏期显著缩短、穿越平台的次数显著增加、时间比率和路程比率也显著增高(P均<0.05)。
(2)电镜下海马CA1区神经细胞超微结构观察：
Control组、Sham组、Veh1组和Veh2组海马CA1区神经细胞超微结构未见明显异常；AβO组和A β O+DMSO组与FAβ组和FAβ+DMSO组均见到神经细胞大量凋亡和不同程度的损伤,以AβO组损伤最严重；FAβ+PKO组和AβO+PKO组均可见少量凋亡神经细胞,神经细胞损伤程度AβO组和AβO+DMSO组与FAβ组和FAβ+DMSO组较轻。
(3)电镜半薄切片光镜下海纪CA1区凋亡神经细胞观察：
Control组、Sham组、Veh1组Veh2组大鼠海马CA1均可见极少量凋亡神经元,但凋亡神经元数量间比较无显著性差异(P>0.05)； FAβ组、FAβ+DMSO组、AβO组和AβO+DMSO组大鼠海马CA1区均见大量凋亡神经元,且凋亡神经元数量较Control组、Sham组、Veh1和Veh2组显著增多(P均<0.05),但FAβ组与FAβ+DMSO组比较、AβO组与A β O+DMSO组比较凋亡神经元数量无显著性差异(P均>0.05)；AβO组大鼠海马CA1区凋亡神经元数量较FAβ组明显增多(P<0.05):FAβ+PKO组和AβO+PKO组凋亡神经元数量较Control组、Sham组、Veh1组、Veh2组增多(P均<0.05),但与FAβ组、FA β+DMSO组、AβO组和A β0+DMSO组比较明显减少(P均<0.05)。
(4)RT-PCR、WB、ELISA结果：
Comtrol组、Sham组、Veh1组和Veh2组之间比较TLR4、NF-κB、TNF-α表达无显著性差异(P均>0.05)；FAβ组与FAβ+DMSO组比较、AβO组与AβO+DMSO组比较TLR4、NF-κB、TNF-α表达无显著性差异(P均>0.05)；FAβ组、FAβ+DMSO组、AβO组和AβO+DMSO组与Control组、Sham组、Veh1组和Veh2组相比TLR4、 NF-κB、TNF-α表达均显著增强(P均<0.05)；AβO组TLR4、NF-κB、TNF-α表达较FAβ组增强(P<0.05)；FAβ+PKO组和AβO+PKO组TLR4、NF-κB、TNF-α表达较Control组、Sham组、Veh1组、Veh2组增多(P均<0.05),但与FAβ组、FAβ+DMSO组、AβO组和A β O+DMSO组比较TLR4、NF-κB、TNF-α表达明显减少(P均 (5)偏相关分析结果：
TLR4、NF-κB、TNF-α与穿越平台次数都呈现负相关(P均=0.000),TLR4、 NF-κB、TNF-α之间均为正相关(P均=0.000)。
结论：
(1)向一侧侧脑室持续灌注八Aβ1-42能成功建立AD动物模型；
(2)纤丝状Aβ1-42和Aβ1-42寡聚体均可诱导神经细胞凋亡、损伤和TLR4、 NF-κB、TNF-α表达增高,导致大鼠学习记忆功能障碍。
(3)Aβ1-42寡聚体可被TLR4识别,而且与纤丝状Aβ1-42相比较,其诱导的TLR4、NF-κB、TNF-α表达理高,细胞凋亡、损伤和大鼠学习记忆功能障碍更严重。
(4)海风藤干预能部分改善Aβ诱导的大鼠学习记忆功能障碍，其机制可能与减少炎症因子TLR4、NF-κB、TNF-α表达、抑制神经细胞亡有关。
Background:Alzheimer's disease is the most common degenerative disease of the central nervous system, its typical clinical manifestations are increasing memory loss, cognitive dysfunction, abnormal behavior and social barriers. Pathological changes of main are neurofibrillar tangles (NFT), senile plaque formation (SP) and neuron loss. The pathogenesis of AD is complex, so far it's cause is unclear, since in the1980of the20th century, forming a lot of hypotheses, now the widely recognized hypothesis is beta-amyloid protein hypothesis, the main composition of SP is Aβ, Aβ produced by β, γ apocrine enzyme hydrolysis Aβ precursor protein(APP).Occurrence, development of AD and Aβ's metabolic disorders due to abnormal deposition of basal forebrain are closely related. But the study found that in recent years, the Aβ oligomers compared with fibrillar Aβ with higher neurotoxicity, is the main toxic substances in the pathogenesis of AD. Currently The whole animal behavior of Aβ oligomer experiment results also controversial.comparative study of whole animal level between different forms of aggregation of Aβ on less.
The current research on the pathogenesis of AD tips, chronic inflammation of central nervous system injury induced by Aβ activation of glial cells may be a pathological key led to AD. involved in the inflammatory response is mainly microglial cells (Mi). located in Toll like receptor(TLRs) of the surface of Mi and its auxiliary receptor CD14are involved in the activation process of Aβ Mi,but the conclusion more from the research of fibril Aβ, TLRs can identify oligomers of AP still inconclusive.
In addition, on the process of AD's research, the animal model is always the key. But now all kinds of models are insufficient, still without a kind of animal model can fully reproduce simulation of AD pathology, biochemistry, behavior and other aspects of the full feature. This experiment use osmotic pump will Aβ1-42polymeric and oligomeric continuous perfusion to the lateral ventricle of the rat AD model was established, so that a large number of A βslowly diffuse distribution in aged rat brain, solve the similar model A short-term aggregation in local injection point better, closer to the clinical pathological process of AD.
Traditional Chinese medicine Piper Kadsura is the Piperaceae pepper plants of the genus wind vine stems, Chinese Pharmacopoeia records having through main and collateral channels, rheumatism and relieving pain efficacy, modern medical research has found its anti-inflammatory and neuroprotective effects, and has been applied in treatment and research for AD in recent years.
The research and application of fibrillar Aβ1-42and Aβ1-42oligomers intracerebroventricular continuous perfusion of rat model of AD preparation, observation of two different forms of aggregation of A f3on the behavior of rats, hippocampus nerve cell ultrastructure and Toll like receptor4(TLR4) and the expression of inflammatory factors the effects of simultaneous application of therapeutic intervention, futokadsura stem extract, observe its effect on these indexes, in order to further reveal the pathogenesis of AD, explore a safe and effective treatment method.
Objective:To investigate the fibrillar Aβ1-42and A β1-42oligomers on the behavior of rats, hippocampus nerve cell ultrastructure and TLR4nuclear factor-κ B (NF-κB). tumor necrosis factor alpha (TNF-α) expression changes of influence; application of Piper Kadsura stem prognostic index above.
Methods:60healthy male SD rats, were randomly divided into10groups:normal control group (Control group), sham operation group (Sham group), fibrillar Aβ+Vehicle group1(FAβ group, intracerebroventricular infusion of polymeric Aβ+ cetonitrile and three acetic acid fluoride die), polymeric Aβ+Piper Kadsura Ohwi (PKO) group (FAp+PKO group, intracerebroventricular infusion of polymeric Aβ+acetonitrile and three trifluoroacetic acid, molding, intraperitoneal injection of PKO treatment), polymeric Aβ+Dimethyl sulfoxide (DMSO)group (FAβ+DMSO group, intracerebroventricular infusion of polymeric Aβ+acetonitrile and three trifluoroacetic acid, intraperitoneal injection of DMSO), Vehicle1groups (group Vehl, intracerebroventricular injection of not Aβ, but only perfusion, acetonitrile and three trifluoroacetic acid), Aβ oligomer+Vehicle2group(ApO group, intracerebroventricular perfusion A P oligomer+high density lipoprotein and HEPES model), Aβ oligomer+PKO (APO+PKO group, high density lipoprotein and HEPES molding, intraperitoneal injection of PKO treatment), Ap oligomer+DMSO group (APO+DMSO group, ventricle instillation of Ap oligomer+high density lipoprotein and HEPES, intraperitoneal injection of DMSO), Vehicle2groups (Veh2group, ventricle no injection of Aβ, only perfusion of high density lipoprotein and HEPES). Using micro-osmotic pump infusion fibrillar A β1-42or A β1-42oligomer dimer into the lateral ventricle method of making animal model. A day after modeling for FAP+PKO and APO+PKO group rats were intraperitoneally injected with concentration of10%containing2.5%DMSO PKO, according to the weight of administration (1ml/100g), FAP+DMSO and ApO+DMSO rats every day only received intraperitoneal injection of2.5%DMSO (1ml/100g), continuous administration for35days. Thirty-first days after the model of learning and memory function in Morris water maze test evaluation in rats, the experiment was divided into two parts of space exploration and constant-bearing navigation. Space exploration experiment recording rat escape latent period, constant-bearing navigation test recording rats in the Morris water maze mobile video, calculating the times of crossing platform, time ratio and distance ratiothrough the quadrant in the platform. Electron microscopic observation on the ultrastructure of hippocampus neural cells, real-timepolymerasechainreactio,(RT-PCR) used to detect the expression of TLR4mRNA in the hippocampus, immunoblotting Western blot (WB) to detect the expression of NF-κBP65, TLR4 protein, enzyme labeled immunosorbent assay (ELISA) for the detection of the expression of TNF-α.
Result:
(1) the water maze experiment results:Control group, Sham group, Veh1group and Veh2group were compared between the escape latency, the times of crossing the platform, the time ratio and distance ratio had no significant difference (P>0.05); FAβ group compared with FAβ+DMSO group, A β O group compared with Aβ O+DMSO group in escape latency, the times of crossing the platform time and distance ratio, the ratio of no significant difference (P>0.05); the number of FAβ group, FAβ+DMSO and AβO group, AβO+DMSO group compared with Control group, Sham group, Vehl group and Veh2group had significantly longer escape latency, significantly reduces times of across the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); AβO and FAβ group had significantly longer escape latency, significantly decreased times of a cross the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); the number of FAβ+PKO and AβO+PKO group compared with Control group, Sham group, Vehl group and the Veh2group had significantly longer escape latency, significantly reduces times of across the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); but FAβ group, FAβ+DMSO compared with AβO group, AβO+DMSO escape latency significantly shortened significantly, cross platform Increase, time ratio and distance ratio also increased significantly (P <0.05).
(2)to observe the ultrastructure of hippocampus neural cells of CA1region under electron microscope: Control group, Sham group. Vehl group and Veh2group in CA1area of hippocampus nerve cell ultrastructure had no obvious abnormalities; A βO group and the AβO+DMSO and FA β group and FAβ+DMSO were seen neural cell apoptosis and the damage of different degree, FAβ group+PKO group are in the most serious injury; Ap()+PKO group showed a small amount of AβOptosis of nerve cells, nerve cell damage of AβO group and the AβO+DMSO and FA Pgroup and FAβ+DMSO are lighter.
(3)electron microscopy semithin sections in CA1area of hippocampus neural cells apoptosis were observed under light microscope:Control group, Sham group, Vehl group and Veh2groupCA1of rat hippocampus were few of apoptotic neurons apoptosis of neurons, but there was no significant difference between groups (P>0.05); FAβ group, FAβ+DMSO group, ApO group and ApO+DMSO group in hippocampal CA1region of rats large number of apoptotic neurons were seen, and the number of apoptotic neurons compared with Control group, Sham group, Vehl group and Veh2group were significantly increased (P <0.05), but the FAβ group compared with FAβ+DMSO group, AβO group compared with the Aβ O+DMSO group apoptosis cells had no significant difference (P>0.05); hippocampal CA1region of AβO rats apoptosis neurons compared with FAβ group increased significantly (P<0.05);FAβ+PKO group and ApO+PKO group compared with Control group, Sham group,Vehl group, Veh2group the number of apoptotic neurons was increased (P <0.05), but compared with FAβ group, FA P+DMSO group, AβO and AβO+DMSO group decreased significantly (P <0.05).
(4) RT-PCR, WB, ELISA results:Between Control group, Sham group, Vehl group and Veh2group compared with TLR4, NF-kB, TNF-a expression had no significant difference (P>0.05); FAβ group and FAβ+DMSO group, AβO group and ApO+DMSO group compared with TLR4, NF-κB, TNF-a the expression of no significant difference (P>0.05); FAβ group, FAβ+DMSO group, AβO group and ApO+DMSO group compared with Control group, Sham group, Vehl group and Veh2group the expression of TLR4,NF-κB,TNF-α increased significantly (all P <0.05); AβO group in the expression of TLR4,NF-κB,TNF-α compared with FAβ group was increased (P<0.05); FAβ+PKO group and ApO+PKO group in the expression of TLR4, NF-kB, TNF-a compared with Control group, Sham group, Vehl group, Veh2group were increased (P <0.05). but compared with FAβ group, FAβ+DMSO group, ApO and AβO+DMSO groups expression in TLR4,NF-κB, TNF-a decreased obviously (P <0.05).
(5) the results of partial correlation analysis:TLR4, NF-κB, TNF-α with the times of crossing platform to present the negative correlation (P=0.000), TLR4, NF-κB, TNF-α were positive correlation (P=0.000).
Conclusion:
(1) infusion Aβ1-42into one side of the lateral ventricle constant can be successfully established the animal model of AD;
(2) fibrillar Aβ1-42and A β1-42oligomers can induce the apoptosis of nerve cells, injury and TLR4, NF-κB, TNF-α over-expression, resulting in impairment of learning and memory in rats.
(3) Aβ1-42oligomers can be recognized by the TLR4, but also compaired with fibrillar Aβ1-42, the induced TLR4, NF-κB, the expression of TNF-α is higher, apoptosis, damage and rat learning and memory dysfunction is more serious.
(4) the Piper Kadsura intervention can improve learning and memory dysfunction induced by A in rats, its mechanism may be associated with reduced inflammatory factors TLR4, NF-κB, the expression of TNF-α, inhibiting nerve cell apoptosis.
目前关于AD发病过程的研究提示,Aβ激活胶质细胞诱发中枢神经系统慢性炎症损伤可能是导致AD的一个核心病理环节,参与该炎性反应的主要是小胶质细胞(microglia, Mi),位于Mi表面的Toll样受体(Toll—like receptors, TLRs)及其辅助受体CD14共同参与了Aβ激活Mi的过程,但是上述结论多来自纤丝状Aβ的研究,TLRs能否识别寡聚体的Aβ还没有定论。
此外,AD的基础研究过程中动物模型的建立一直是关键,但是目前各类模型都存不足,还没有任何一种动物模型能全面再现、模拟AD的病理、生化、行为学等方面的全部特征。本实验使用微渗泵将Aβ1-42聚合体和寡聚体持续灌注至大鼠侧脑室建立AD模型,从而使大量Aβ缓慢持续弥散性地分布到老龄大鼠脑内,较好地解决了同类模型Aβ短时在注射点局部聚集的问题,与AD的临床病理过程更接近。
中药海风藤是胡椒属植物风藤的滕的茎，中国药典记载其具有通经络、祛风湿和止痹痛的功效,现代医学研究发现其具有抗炎和神经保护作用,近年来开始应用于AD的治疗研究。
本研究应用纤丝状Aβ1-42和Aβ1-42寡聚体侧脑室持续灌注制备AD大鼠模型,观察两种不同聚集形式的Aβ对大鼠行为学、海马区神经细胞超微结构及Toll样受体4(Toll—like receptor4,TLR4)等炎症因子表达的影响,同时应用海风藤提取物干预治疗,观察其对上述指标的作用,以期进一步揭示AD的发病机制、探寻安全有效的治疗方法。
目的：探讨纤丝状Aβ1-42与Aβ1-42寡聚体对大鼠行为学、海马区神经细胞超微结构及TLR4、核因子-KB(nuclear factor-kappa B,NF-κB)、肿瘤坏死因子-α (tumor necrosisfactor-α,TNF-α)表达的影响；探讨应用海风藤干预后上述指标的变化。
方法：健康雄性SD大鼠60只,随机数字表法平均分为10组：正常对照组(Control组)、假手术组(Sham组)、纤丝状Aβ+载体1组(FAβ组,脑室内灌注纤丝状Aβ+乙腈和三氟乙酸造模)、纤丝状Aβ+海风藤提取物(Piper kadsura ohwi PKO)组(FAβ+PKO组,脑室内灌注纤丝状Aβ+乙腈和三氟乙酸造模,腹腔注射PKO治疗)、纤丝状Aβ+二甲基亚砜(Dimethyl sulfoxide DMSO)组(FAβ+DMSO组,脑室内灌注纤丝状Aβ+乙腈和三氟乙酸,腹腔注射DMSO).载体1组(Veh1组,脑室内不注射Aβ,仅灌注乙腈和三氟乙酸)、Aβ寡聚体+载体2组(AβO组，脑室内灌注Aβ寡聚体+高密度脂蛋白和羟乙基哌嗪乙磺酸造模)、Aβ寡聚体+PKO(A βO+PKO组,高密度脂蛋白和羟乙磺酸造模,腹腔注射PKO治疗)、Aβ寡聚体+DMSO组(AβO+DMSO(?)(?),脑室内灌注Aβ寡聚体+高密度脂蛋白和羟乙基哌嗪乙磺酸,腹腔注射DMSO)、载体2组(Veh2组,脑室内不注射Aβ,仅灌注高密度脂蛋白和羟乙基哌嗪乙磺酸)。采用微渗泵向侧脑室持续灌注纤丝状Aβ1-42或Aβ1-42寡聚体法制作动物模型。造模后每天给FAβ+PKO组和AβO+PKO组大鼠腹腔注射浓度10%含2.5%DMSO的PKO,按体重给药(1ml/100g),FAβ+DMSO组和AβO+DMSO组大鼠每天仅给予2.5%DMSO(?)腹腔注射(1ml/100g),连续给药35天。造模后第31天开始进行Morris水迷宫实验评定大鼠的学习记忆功能，喧闹验分为空间探索和定向航行两部分。空间探索实验时记录大鼠的逃避潜伏期,定向航行实验时录制大鼠在水迷宫游动视频，计算其穿越平台次数,在平台所在象限穿行的时间比率和路程比率。电镜观察海马区神经细胞超微结构,实时荧光定量聚合酶链式扩增反应(real-timepolymerasechainreactio,RT-PCR)法检测海马区TLR4mRNA表达,免疫印迹法(Western blot,WB)检测NF-κBP65、TLR4蛋白表达,酶标记免疫吸附法(ELISA)检测INF-α的表达。
结果：
(1)水迷宫实验结果：
Control组、Sham组、Veh1组和Veh2组之间比较逃避潜伏期、穿越平台的次数、时间比率和路程比率无显著性差异(P均>0.05)；FAβ组与FAβ+DMSO组比较、AβO组与AβO+DMSO组比较逃避潜伏期、穿越平台的次数、时间比率和路程比率无显著性差异(P均>0.05)；FAβ组、FA β+DMSO组和AβO组、A β O+DMSO组与Control组、Sham组、Veh1组和Veh2组比较逃避潜伏期显著延长、穿越平台的次数显著减少、时间比率和路程比率也显著减低(P均<0.05)；AβO组与FAβ组比较逃避潜伏期显著延长、穿越平台的次数显著减少、时间比率和路程比率也显著减低(P均<0.05)；FA β+PKO组和A β O+PKO组与Control组、Sham组、Veh1组和Veh2组比较逃避潜伏期显著延长、穿越平台的次数显著减少、时间比率和路程比率也显著减低(P均<0.05)；但与FAβ组、FAβ+DMSO组和AβO组、A β O+DMSO组比较逃避潜伏期显著缩短、穿越平台的次数显著增加、时间比率和路程比率也显著增高(P均<0.05)。
(2)电镜下海马CA1区神经细胞超微结构观察：
Control组、Sham组、Veh1组和Veh2组海马CA1区神经细胞超微结构未见明显异常；AβO组和A β O+DMSO组与FAβ组和FAβ+DMSO组均见到神经细胞大量凋亡和不同程度的损伤,以AβO组损伤最严重；FAβ+PKO组和AβO+PKO组均可见少量凋亡神经细胞,神经细胞损伤程度AβO组和AβO+DMSO组与FAβ组和FAβ+DMSO组较轻。
(3)电镜半薄切片光镜下海纪CA1区凋亡神经细胞观察：
Control组、Sham组、Veh1组Veh2组大鼠海马CA1均可见极少量凋亡神经元,但凋亡神经元数量间比较无显著性差异(P>0.05)； FAβ组、FAβ+DMSO组、AβO组和AβO+DMSO组大鼠海马CA1区均见大量凋亡神经元,且凋亡神经元数量较Control组、Sham组、Veh1和Veh2组显著增多(P均<0.05),但FAβ组与FAβ+DMSO组比较、AβO组与A β O+DMSO组比较凋亡神经元数量无显著性差异(P均>0.05)；AβO组大鼠海马CA1区凋亡神经元数量较FAβ组明显增多(P<0.05):FAβ+PKO组和AβO+PKO组凋亡神经元数量较Control组、Sham组、Veh1组、Veh2组增多(P均<0.05),但与FAβ组、FA β+DMSO组、AβO组和A β0+DMSO组比较明显减少(P均<0.05)。
(4)RT-PCR、WB、ELISA结果：
Comtrol组、Sham组、Veh1组和Veh2组之间比较TLR4、NF-κB、TNF-α表达无显著性差异(P均>0.05)；FAβ组与FAβ+DMSO组比较、AβO组与AβO+DMSO组比较TLR4、NF-κB、TNF-α表达无显著性差异(P均>0.05)；FAβ组、FAβ+DMSO组、AβO组和AβO+DMSO组与Control组、Sham组、Veh1组和Veh2组相比TLR4、 NF-κB、TNF-α表达均显著增强(P均<0.05)；AβO组TLR4、NF-κB、TNF-α表达较FAβ组增强(P<0.05)；FAβ+PKO组和AβO+PKO组TLR4、NF-κB、TNF-α表达较Control组、Sham组、Veh1组、Veh2组增多(P均<0.05),但与FAβ组、FAβ+DMSO组、AβO组和A β O+DMSO组比较TLR4、NF-κB、TNF-α表达明显减少(P均
TLR4、NF-κB、TNF-α与穿越平台次数都呈现负相关(P均=0.000),TLR4、 NF-κB、TNF-α之间均为正相关(P均=0.000)。
结论：
(1)向一侧侧脑室持续灌注八Aβ1-42能成功建立AD动物模型；
(2)纤丝状Aβ1-42和Aβ1-42寡聚体均可诱导神经细胞凋亡、损伤和TLR4、 NF-κB、TNF-α表达增高,导致大鼠学习记忆功能障碍。
(3)Aβ1-42寡聚体可被TLR4识别,而且与纤丝状Aβ1-42相比较,其诱导的TLR4、NF-κB、TNF-α表达理高,细胞凋亡、损伤和大鼠学习记忆功能障碍更严重。
(4)海风藤干预能部分改善Aβ诱导的大鼠学习记忆功能障碍，其机制可能与减少炎症因子TLR4、NF-κB、TNF-α表达、抑制神经细胞亡有关。
Background:Alzheimer's disease is the most common degenerative disease of the central nervous system, its typical clinical manifestations are increasing memory loss, cognitive dysfunction, abnormal behavior and social barriers. Pathological changes of main are neurofibrillar tangles (NFT), senile plaque formation (SP) and neuron loss. The pathogenesis of AD is complex, so far it's cause is unclear, since in the1980of the20th century, forming a lot of hypotheses, now the widely recognized hypothesis is beta-amyloid protein hypothesis, the main composition of SP is Aβ, Aβ produced by β, γ apocrine enzyme hydrolysis Aβ precursor protein(APP).Occurrence, development of AD and Aβ's metabolic disorders due to abnormal deposition of basal forebrain are closely related. But the study found that in recent years, the Aβ oligomers compared with fibrillar Aβ with higher neurotoxicity, is the main toxic substances in the pathogenesis of AD. Currently The whole animal behavior of Aβ oligomer experiment results also controversial.comparative study of whole animal level between different forms of aggregation of Aβ on less.
The current research on the pathogenesis of AD tips, chronic inflammation of central nervous system injury induced by Aβ activation of glial cells may be a pathological key led to AD. involved in the inflammatory response is mainly microglial cells (Mi). located in Toll like receptor(TLRs) of the surface of Mi and its auxiliary receptor CD14are involved in the activation process of Aβ Mi,but the conclusion more from the research of fibril Aβ, TLRs can identify oligomers of AP still inconclusive.
In addition, on the process of AD's research, the animal model is always the key. But now all kinds of models are insufficient, still without a kind of animal model can fully reproduce simulation of AD pathology, biochemistry, behavior and other aspects of the full feature. This experiment use osmotic pump will Aβ1-42polymeric and oligomeric continuous perfusion to the lateral ventricle of the rat AD model was established, so that a large number of A βslowly diffuse distribution in aged rat brain, solve the similar model A short-term aggregation in local injection point better, closer to the clinical pathological process of AD.
Traditional Chinese medicine Piper Kadsura is the Piperaceae pepper plants of the genus wind vine stems, Chinese Pharmacopoeia records having through main and collateral channels, rheumatism and relieving pain efficacy, modern medical research has found its anti-inflammatory and neuroprotective effects, and has been applied in treatment and research for AD in recent years.
The research and application of fibrillar Aβ1-42and Aβ1-42oligomers intracerebroventricular continuous perfusion of rat model of AD preparation, observation of two different forms of aggregation of A f3on the behavior of rats, hippocampus nerve cell ultrastructure and Toll like receptor4(TLR4) and the expression of inflammatory factors the effects of simultaneous application of therapeutic intervention, futokadsura stem extract, observe its effect on these indexes, in order to further reveal the pathogenesis of AD, explore a safe and effective treatment method.
Objective:To investigate the fibrillar Aβ1-42and A β1-42oligomers on the behavior of rats, hippocampus nerve cell ultrastructure and TLR4nuclear factor-κ B (NF-κB). tumor necrosis factor alpha (TNF-α) expression changes of influence; application of Piper Kadsura stem prognostic index above.
Methods:60healthy male SD rats, were randomly divided into10groups:normal control group (Control group), sham operation group (Sham group), fibrillar Aβ+Vehicle group1(FAβ group, intracerebroventricular infusion of polymeric Aβ+ cetonitrile and three acetic acid fluoride die), polymeric Aβ+Piper Kadsura Ohwi (PKO) group (FAp+PKO group, intracerebroventricular infusion of polymeric Aβ+acetonitrile and three trifluoroacetic acid, molding, intraperitoneal injection of PKO treatment), polymeric Aβ+Dimethyl sulfoxide (DMSO)group (FAβ+DMSO group, intracerebroventricular infusion of polymeric Aβ+acetonitrile and three trifluoroacetic acid, intraperitoneal injection of DMSO), Vehicle1groups (group Vehl, intracerebroventricular injection of not Aβ, but only perfusion, acetonitrile and three trifluoroacetic acid), Aβ oligomer+Vehicle2group(ApO group, intracerebroventricular perfusion A P oligomer+high density lipoprotein and HEPES model), Aβ oligomer+PKO (APO+PKO group, high density lipoprotein and HEPES molding, intraperitoneal injection of PKO treatment), Ap oligomer+DMSO group (APO+DMSO group, ventricle instillation of Ap oligomer+high density lipoprotein and HEPES, intraperitoneal injection of DMSO), Vehicle2groups (Veh2group, ventricle no injection of Aβ, only perfusion of high density lipoprotein and HEPES). Using micro-osmotic pump infusion fibrillar A β1-42or A β1-42oligomer dimer into the lateral ventricle method of making animal model. A day after modeling for FAP+PKO and APO+PKO group rats were intraperitoneally injected with concentration of10%containing2.5%DMSO PKO, according to the weight of administration (1ml/100g), FAP+DMSO and ApO+DMSO rats every day only received intraperitoneal injection of2.5%DMSO (1ml/100g), continuous administration for35days. Thirty-first days after the model of learning and memory function in Morris water maze test evaluation in rats, the experiment was divided into two parts of space exploration and constant-bearing navigation. Space exploration experiment recording rat escape latent period, constant-bearing navigation test recording rats in the Morris water maze mobile video, calculating the times of crossing platform, time ratio and distance ratiothrough the quadrant in the platform. Electron microscopic observation on the ultrastructure of hippocampus neural cells, real-timepolymerasechainreactio,(RT-PCR) used to detect the expression of TLR4mRNA in the hippocampus, immunoblotting Western blot (WB) to detect the expression of NF-κBP65, TLR4 protein, enzyme labeled immunosorbent assay (ELISA) for the detection of the expression of TNF-α.
Result:
(1) the water maze experiment results:Control group, Sham group, Veh1group and Veh2group were compared between the escape latency, the times of crossing the platform, the time ratio and distance ratio had no significant difference (P>0.05); FAβ group compared with FAβ+DMSO group, A β O group compared with Aβ O+DMSO group in escape latency, the times of crossing the platform time and distance ratio, the ratio of no significant difference (P>0.05); the number of FAβ group, FAβ+DMSO and AβO group, AβO+DMSO group compared with Control group, Sham group, Vehl group and Veh2group had significantly longer escape latency, significantly reduces times of across the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); AβO and FAβ group had significantly longer escape latency, significantly decreased times of a cross the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); the number of FAβ+PKO and AβO+PKO group compared with Control group, Sham group, Vehl group and the Veh2group had significantly longer escape latency, significantly reduces times of across the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); but FAβ group, FAβ+DMSO compared with AβO group, AβO+DMSO escape latency significantly shortened significantly, cross platform Increase, time ratio and distance ratio also increased significantly (P <0.05).
(2)to observe the ultrastructure of hippocampus neural cells of CA1region under electron microscope: Control group, Sham group. Vehl group and Veh2group in CA1area of hippocampus nerve cell ultrastructure had no obvious abnormalities; A βO group and the AβO+DMSO and FA β group and FAβ+DMSO were seen neural cell apoptosis and the damage of different degree, FAβ group+PKO group are in the most serious injury; Ap()+PKO group showed a small amount of AβOptosis of nerve cells, nerve cell damage of AβO group and the AβO+DMSO and FA Pgroup and FAβ+DMSO are lighter.
(3)electron microscopy semithin sections in CA1area of hippocampus neural cells apoptosis were observed under light microscope:Control group, Sham group, Vehl group and Veh2groupCA1of rat hippocampus were few of apoptotic neurons apoptosis of neurons, but there was no significant difference between groups (P>0.05); FAβ group, FAβ+DMSO group, ApO group and ApO+DMSO group in hippocampal CA1region of rats large number of apoptotic neurons were seen, and the number of apoptotic neurons compared with Control group, Sham group, Vehl group and Veh2group were significantly increased (P <0.05), but the FAβ group compared with FAβ+DMSO group, AβO group compared with the Aβ O+DMSO group apoptosis cells had no significant difference (P>0.05); hippocampal CA1region of AβO rats apoptosis neurons compared with FAβ group increased significantly (P<0.05);FAβ+PKO group and ApO+PKO group compared with Control group, Sham group,Vehl group, Veh2group the number of apoptotic neurons was increased (P <0.05), but compared with FAβ group, FA P+DMSO group, AβO and AβO+DMSO group decreased significantly (P <0.05).
(4) RT-PCR, WB, ELISA results:Between Control group, Sham group, Vehl group and Veh2group compared with TLR4, NF-kB, TNF-a expression had no significant difference (P>0.05); FAβ group and FAβ+DMSO group, AβO group and ApO+DMSO group compared with TLR4, NF-κB, TNF-a the expression of no significant difference (P>0.05); FAβ group, FAβ+DMSO group, AβO group and ApO+DMSO group compared with Control group, Sham group, Vehl group and Veh2group the expression of TLR4,NF-κB,TNF-α increased significantly (all P <0.05); AβO group in the expression of TLR4,NF-κB,TNF-α compared with FAβ group was increased (P<0.05); FAβ+PKO group and ApO+PKO group in the expression of TLR4, NF-kB, TNF-a compared with Control group, Sham group, Vehl group, Veh2group were increased (P <0.05). but compared with FAβ group, FAβ+DMSO group, ApO and AβO+DMSO groups expression in TLR4,NF-κB, TNF-a decreased obviously (P <0.05).
(5) the results of partial correlation analysis:TLR4, NF-κB, TNF-α with the times of crossing platform to present the negative correlation (P=0.000), TLR4, NF-κB, TNF-α were positive correlation (P=0.000).
Conclusion:
(1) infusion Aβ1-42into one side of the lateral ventricle constant can be successfully established the animal model of AD;
(2) fibrillar Aβ1-42and A β1-42oligomers can induce the apoptosis of nerve cells, injury and TLR4, NF-κB, TNF-α over-expression, resulting in impairment of learning and memory in rats.
(3) Aβ1-42oligomers can be recognized by the TLR4, but also compaired with fibrillar Aβ1-42, the induced TLR4, NF-κB, the expression of TNF-α is higher, apoptosis, damage and rat learning and memory dysfunction is more serious.
(4) the Piper Kadsura intervention can improve learning and memory dysfunction induced by A in rats, its mechanism may be associated with reduced inflammatory factors TLR4, NF-κB, the expression of TNF-α, inhibiting nerve cell apoptosis.
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42.Vehamas A.Kawas C,Stewart W,et al.Immune reactive cells in senile plaques and cognitive in Alzheiner,s disease.Neurobiol Aging,2003,24(2):321-31.
43.Casal C, Serratosa J, Tusell JM. Effects of beta-AP peptides on activation of the transcription factor NF-kappaB and in cellproliferation in glial cell cultures.Neurosci Res,2004,48(3):315-323.
44.Lee, S.J., Lee, S. Toll-like receptors and inflammation in the CNS. Curr. Drug Targets Inflamm. Allergy,2002,1,181-191.
45.Tu XK, Yang WZ, Shi SS, et al. Spatio-temporal distribution of inflammatory reaction and expression of TLR2/4 signaling pathway in rat brain following pemlaneut focal cerebralis chemia. Neurochem Res,2010,35(8):1147-1155.
46.Chen Z J.Ubiquitin signalling in the NF-kappaB pathway.Nat Cell Biol,2005,7(8):758-765.
47.KAWAI T,TAKEUCHI O,FUJITA T,et al.Lipopolysac—charide stimulates the MyD88-independent pathway and results in act ivation of IFN—regulatory factor 3 and the expression of a subset of lipopolysaccharide—induible genes.J Immunol,2001,167(10):5887—5894.
48.B.J. Ding, W.W. Ma, L.L. He, X. Zhou, L.H. Yuan, H.L. Yu, J.F. Feng, R. Xiao.2011.Soybean isoflavone alleviates-amyloid 1-42 induced inflammatory response to improve learning and memory ability by down regulation of Toll-likereceptor 4 expression and nuclear factor-B activity in rats. Devl Neuroscience.29.537-542.
49.韩恩吉,胡洪涛,何秀全,等.海风藤对β-APP基因农达的影响.山东大学学报(医学版),2003,41：87-89
50.Takashima A. GSK-3 is essential in the pathogenesis of Alzheimer's disease. J Alzheimers Dis, 2006;9:Suppl:309-17.
51.Akiyama H, Barger S, Barnum S, et al. Inflammation and Alzheimer's disease.Neurobiol Aging, 2000;21:383-421..
52.Cook DG, L 107. everenz JB, McMillan PJ,et al. Reduced hippocampal insulin-degrading enzyme in late-onset Alzheimer's disease is associated with the apolipoprotein E-epsilon4 allele[J]. Am J Pathol,2003; 162:313-9.
53.Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease --a double-edged sword. Neuron,2002;35:419-32.
54.邢华医,郑梅梅，杜怡峰,等.海风藤对活化的新生大鼠小胶质细胞的影响作用.中华行为医学与脑科学杂志,2011，20(9)：778-780.
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2. Dickstein D L, Biron K E, Ujiie M, et al. Abeta peptide immunization restores blood-brain barrier integrity in Alzheimer disease. Faseb J,2006,20:426-433
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42.Vehamas A.Kawas C,Stewart W,et al.Immune reactive cells in senile plaques and cognitive in Alzheiner,s disease.Neurobiol Aging,2003,24(2):321-31.
43.Casal C, Serratosa J, Tusell JM. Effects of beta-AP peptides on activation of the transcription factor NF-kappaB and in cellproliferation in glial cell cultures.Neurosci Res,2004,48(3):315-323.
44.Lee, S.J., Lee, S. Toll-like receptors and inflammation in the CNS. Curr. Drug Targets Inflamm. Allergy,2002,1,181-191.
45.Tu XK, Yang WZ, Shi SS, et al. Spatio-temporal distribution of inflammatory reaction and expression of TLR2/4 signaling pathway in rat brain following pemlaneut focal cerebralis chemia. Neurochem Res,2010,35(8):1147-1155.
46.Chen Z J.Ubiquitin signalling in the NF-kappaB pathway.Nat Cell Biol,2005,7(8):758-765.
47.KAWAI T,TAKEUCHI O,FUJITA T,et al.Lipopolysac—charide stimulates the MyD88-independent pathway and results in act ivation of IFN—regulatory factor 3 and the expression of a subset of lipopolysaccharide—induible genes.J Immunol,2001,167(10):5887—5894.
48.B.J. Ding, W.W. Ma, L.L. He, X. Zhou, L.H. Yuan, H.L. Yu, J.F. Feng, R. Xiao.2011.Soybean isoflavone alleviates-amyloid 1-42 induced inflammatory response to improve learning and memory ability by down regulation of Toll-likereceptor 4 expression and nuclear factor-B activity in rats. Devl Neuroscience.29.537-542.
49.韩恩吉,胡洪涛,何秀全,等.海风藤对β-APP基因农达的影响.山东大学学报(医学版),2003,41：87-89
50.Takashima A. GSK-3 is essential in the pathogenesis of Alzheimer's disease. J Alzheimers Dis, 2006;9:Suppl:309-17.
51.Akiyama H, Barger S, Barnum S, et al. Inflammation and Alzheimer's disease.Neurobiol Aging, 2000;21:383-421..
52.Cook DG, L 107. everenz JB, McMillan PJ,et al. Reduced hippocampal insulin-degrading enzyme in late-onset Alzheimer's disease is associated with the apolipoprotein E-epsilon4 allele[J]. Am J Pathol,2003; 162:313-9.
53.Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease --a double-edged sword. Neuron,2002;35:419-32.
54.邢华医,郑梅梅，杜怡峰,等.海风藤对活化的新生大鼠小胶质细胞的影响作用.中华行为医学与脑科学杂志,2011，20(9)：778-780.
1. Cummings B J, Cotman C W. Image analysis of beta-amyloid load in Alzheimer's disease and relation to dementia severity. Lancet,1995,346:1524-1528
2. Dickstein D L, Biron K E, Ujiie M, et al. Abeta peptide immunization restores blood-brain barrier integrity in Alzheimer disease. Faseb J,2006,20:426-433
3.韩桂秋。中草药中血小板活化因子受体拮抗活性成分的研究.自然科学进展。1995,5(2)：161-163
4.郑行,马从容。海风藤酮对兔胚胎发育及PAF效应的影响.中国农业大学学报,1999,4(1)：15-18
5.曾武华,姜远英。海风藤醇及对PAF诱导的兔血小板钙内流和胞内游离钙浓度升高的抑制作用,第二军医大学学报。1996,16(5)：437-440
6. 王伟,刘洋,周清明,等。海风藤醇提取物对血小板活化因子诱导血的小板聚集作用的的初步研究.卒中与神经疾病。2000,7(4)：193-197
7.张雄,王伟,阮旭忠,等。海风藤新木脂索类成分对缺血脑区血小板活化因子及花生四烯酸代谢的的影响,中华老年心脑血管病杂志,2002,4(4)：270-275
8.何英,王东武,邓志宽,等.海风藤对犬脑干缺血后BAEP (?)的影响.临床电脑学杂志,1997,6(3)：165-167
9.邓志宽,王东武,何英，锋.海风藤对犬脑干缺血兴奋性氨基酸含量的影响及对其缺血损伤的的保护作用.中国药学杂志,1997,32(5)：276-279
10.王伟,董为伟.海风藤酮对缺血鼠脑磷脂酶A2、三磷酸肌醇及自由基形成的影响.中华神经科杂志，1996,29(6)：325-328
11. 王伟，董为伟.PAF受体拮抗剂海风藤酮保护作用的实验究.卒中与神经疾病,1996,3(1)：8-12.
12.王伟,王雪松,阮旭中.海风藤新木脂素成分对缺血鼠脑细胞间粘附分子-1及其mRNA表达的影响.中华物理医学与康复杂志,2002,24(3)：133-135.
13.王雪松,王伟,阮旭中.海风藤提取物对脑缺血保护作用的实验研究.中国临床神经科学，2003，11(1)：1-5.
14.王雪松，薛峥,刘买利，等.对海风藤酮治疗实验性缺血再灌注脑损伤的磁共振 波谱分析.中华物理医学与康复杂志,2004,26(6)：333-336.
15.王雪松,王伟,阮旭中.海风藤新木脂素类成分对缺血再灌注鼠脑损伤的保护作用.中国药理学通报,2002,18(6)：622-626.
16.郭瑞友,于义英,方思羽.海风藤对局灶性脑缺血治疗作用的实验研究.中西医结合心脑血管病杂志,2003,1(8)：461-464.
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