丁苯酞对血管性痴呆小鼠海马神经元谷氨酸受体及钙信号转导机制的作用研究
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摘要
目的:血管性痴呆(vascular dementia, VD)由于各种脑血管病引起的获得性认知功能障碍,以学习、记忆功能缺损为主要症状,可伴有语言、运动、视空间及人格障碍等。随着社会人口的日益老龄化和脑卒中治愈率的不断提高,VD的发病呈迅速增长趋势,成为神经内科的常见病、多发病,给社会和家庭带来沉重的负担。目前VD的发病机制尚未明确,也无特效的防治方法,因此探讨和阐明VD的发病机理,对于临床制定更加合理的治疗方案具有重要的价值和理论意义。
学习和记忆是大脑的高级功能之一。学习是指经验及技能的获得,记忆是经验及技能的保存及再现,它们是两个不同而又密切联系的神经生物过程。目前认为,海马与学习、记忆等高级认知功能密切相关,其神经元形态正常与否对学习、记忆的形成和维持具有重要意义。现代大量资料研究证实海马与长时程增强(long-term potentiation,LTP)和学习、记忆过程密切相关,人们称LTP可能是“记忆的突触模型”、“记忆的神经元机制”。LTP形成后,动物的学习能力增强;而通过受体阻断剂阻抑LTP的产生,则影响动物的学习能力,记忆亦受损。而谷氨酸及其受体参与了LTP的产生与维持。
谷氨酸(Glutamate,Glu)是中枢神经系统中一种最重要的兴奋性神经递质,主要分布于大脑皮质、海马、小脑和纹状体,在学习、记忆、突触可塑性及大脑发育等方面均起重要作用。研究证实,Glu的神经递质作用是通过兴奋性氨基酸受体而实现的,受体活性的变化、受体数目的增减都会对突触效能产生明显的影响。在对LTP的研究中,人们发现谷氨酸受体对LTP的诱导具有重要意义,这些受体的特异性拮抗剂不但可阻断LTP的诱导,而且可破坏多种学习和记忆行为。
神经细胞内钙离子浓度(intracellular concentration of calcium,[Ca2+]i)、钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin dependent protein kinaseⅡ, CaMPKⅡ)及钙调素(CaM)一直是脑组织缺血缺氧损伤机制研究的焦点。多种脑缺血动物模型的研究表明,神经细胞内钙超载(Ca2+ over-loading)及CaMPKⅡ水平升高参与了脑组织缺血损伤;并且应用Ca2+通道拮抗剂抑制钙超载,可以减轻神经细胞缺血性损伤。有些学者对缺血性脑组织损伤做了进一步研究,发现缺血后神经细胞内环磷酸腺苷反应元件结合蛋白(cAMP- responsive element binding protein, CREB)水平升高,神经突触后膜谷氨酸受体表达增多,二者均参与了脑组织缺血性损伤。
目前认为,在生理状态下神经细胞[Ca2+]i是参与学习和记忆机制的细胞内第二信使;CREB作为核内的“第三信使”,具有调节包括学习记忆在内的广泛生物学的功能,是细胞内多种信号通路的一种关键成分,而神经细胞膜Glu受体N-甲基-D-天门冬氨酸(N-methyl-D-aspartate, NMDA)受体、α-氨基-3-羟基-5-甲基-4-异唑丙酸(α-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid, AMPA)受体及细胞内[Ca2+]i、CaM、CaMPKⅡ、CREB水平的变化对学习和记忆的影响,也逐渐成为了多数学者研究的热点。也有学者对老年性痴呆(Alzheimers dementia, AD)动物模型进行了研究,发现AD模型脑组织内钙离子超载、谷氨酸受体水平降低。这些研究结果为我们探讨VD动物模型脑组织内谷氨酸受体、钙信号转导机制及CREB改变提供了线索。
丁苯酞(dl-3n-butylphthalide,NBP)系从天然食用植物芹菜籽中提取并经消旋合成的黄色油状液体,具有芹菜香味。该药为国家一类新药,国外对该药的研究尚未见报道,国内冯亦璞等对丁苯酞治疗脑缺血的相关机制做了大量的实验研究,发现丁苯酞具有多靶点抗脑缺血作用,能改善线粒体功能,提高脑血管内皮NO和PGI2的水平,抑制谷氨酸释放,降低细胞内钙浓度和花生四烯酸含量,抑制自由基和提高抗氧化酶活性,减轻脑缺血所致脑水肿,抗血栓形成和抗血小板聚集,缩小脑梗死面积,改善局部脑血流量和细胞能量代谢、神经功能缺失及脑缺血记忆障碍。但该药对VD的实验研究尚未见相关报道。基于此,本文通过探讨谷氨酸受体及钙信号转导机制,观察丁苯酞治疗VD的效果并探讨其作用机制。
方法:1采用三次双侧颈总动脉结扎,造成脑组织反复缺血/再灌注,建立了小鼠VD模型,建立假手术组排除手术本身对小鼠所致的影响,通过跳台试验和水迷宫试验进行学习和记忆成绩测试,观察其行为学改变;用HE染色观察海马病理学特征变化。2采用免疫组化和反转录-聚合酶链反应(RT-PCR)方法测定各组小鼠海马谷氨酸AMPA-GluR2蛋白及GluR2 mRNA的变化。3采用免疫组化和RT-PCR方法测定各组小鼠海马谷氨酸NMDA-NR2B蛋白及NR2B mRNA的变化。4采用流式细胞分析技术测定各组小鼠海马细胞[Ca2+]i变化;Western-Blot及RT-PCR方法检测CaMPKII蛋白及mRNA表达的变化。5 Western-Blot及RT-PCR方法检测CREB蛋白及mRNA表达的变化。
结果:
1 VD小鼠模型的行为学评价及海马病理学特征
3月龄雄性昆明小鼠196只,分为假手术对照组、VD模型组、丁苯酞预防治疗组、丁苯酞治疗组。采用双侧颈总动脉结扎,连续三次脑缺血(20 min)再灌注(10min),建立VD小鼠模型。术后29d,各组分别进行跳台试验及水迷宫实验,测试学习成绩;术后30d测试记忆成绩,HE染色观察各组小鼠海马组织病理学特征。
1.1学习成绩
术后第29天,分别进行跳台试验和水迷宫试验,测试跳台试验反应时间(sec)和错误次数(number/5 min)作为学习成绩,水迷宫试验游完全程时间(sec)和错误次数(number/3 min)作为学习成绩。
跳台试验结果显示:⑴与假手术对照组的反应时间(48.76±9.24)s比较,VD模型组小鼠学习阶段的反应时间(89.41±12.08)s明显延长(P<0.01);⑵与VD模型组的反应时间比较,丁苯酞治疗组小鼠学习阶段的反应时间(59.68±10.27)s及丁苯酞预防治疗组小鼠学习阶段的的反应时间(57.23±10.05)s均明显缩短(P<0.01);⑶与假手术对照组的错误次数(1.23±0.27)比较,VD模型组小鼠学习阶段的错误次数(3.74±0.42)明显增加(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠学习阶段的错误次数(1.52±0.38)及丁苯酞预防治疗组小鼠学习阶段的的错误次数(1.48±0.31)均明显减少(P<0.01);⑸假手术对照组、丁苯酞治疗组、丁苯酞预防治疗组之间的学习成绩无显著性差异(P>0.05)。
水迷宫试验结果显示:⑴与假手术对照组的游全程时间(87.32±13.46)s比较,VD模型组小鼠学习阶段的游全程时间(145.80±13.29)s明显延长(P<0.01);⑵与VD模型组的游全程时间比较,丁苯酞治疗组小鼠学习阶段的游全程时间(101.89±18.38)s及丁苯酞预防治疗组小鼠学习阶段的游全程时间(99.18±11.25)s明显缩短(P<0.01);⑶与假手术对照组的错误次数(16.88±4.19)比较,模型组小鼠学习阶段的错误次数(34.60±5.92)明显增多(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠学习阶段的错误次数(18.71±4.35)及丁苯酞预防治疗组小鼠学习阶段的错误次数(17.29±4.82)明显减少(P<0.01);⑸假手术对照组、丁苯酞预防治疗组、丁苯酞治疗组的学习成绩之间无显著性差异(P>0.05)。
以上结果提示VD模型小鼠的学习成绩降低,丁苯酞可以改善其学习成绩。
1.2记忆成绩
术后第30天,分别进行跳台试验和水迷宫试验,测试跳台试验潜伏时间(sec)和错误次数(number/5min)作为记忆成绩,水迷宫试验游完全程时间(sec)和错误次数(number / 3 min)作为记忆成绩。
跳台试验结果显示:⑴与假手术对照组的潜伏时间(148.92±20.83)s比较,VD模型组小鼠记忆阶段的潜伏时间(78.67±19.17)s明显缩短(P<0.01);⑵与VD模型组的潜伏时间比较,丁苯酞治疗组小鼠记忆阶段的潜伏时间(131.41±18.82)s及丁苯酞预防治疗组小鼠记忆阶段的的潜伏时间(128.57±18.29)s均明显延长(P<0.01);⑶与假手术对照组的错误次数(0.32±0.05)比较,VD模型组小鼠记忆阶段的错误次数(1.72±0.13)明显增加(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠记忆阶段的错误次数(0.54±0.08)及丁苯酞预防治疗组小鼠记忆阶段的的错误次数(0.50±0.07)均明显减少(P<0.01);⑸假手术对照组、丁苯酞预防治疗组、丁苯酞治疗组之间的记忆成绩无显著性差异(P>0.05)。结果提示VD模型小鼠的记忆成绩降低,丁苯酞可以改善其记忆成绩。
水迷宫试验结果显示:⑴与假手术对照组的游全程时间(67.82±8.79)比较,VD模型组小鼠记忆阶段的游全程时间(137.40±11.86)明显延长(P<0.01);⑵与VD模型组的游全程时间比较,丁苯酞治疗组小鼠记忆阶段的游全程时间(88.73±9.53)及丁苯酞预防治疗组小鼠记忆阶段的游全程时间(83.87±8.62)明显缩短(P<0.01);⑶与假手术对照组的错误次数(9.57±2.91)比较,模型组小鼠记忆阶段的错误次数(20.90±4.25)明显增多(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠记忆阶段的错误次数(11.17±3.04)及丁苯酞预防治疗组小鼠记忆阶段的错误次数(10.27±2.83)明显减少(P<0.01);⑸假手术对照组、丁苯酞治疗组、丁苯酞预防治疗组的记忆成绩之间无显著性差异(P>0.05)。
以上结果提示VD模型小鼠的记忆成绩降低,丁苯酞可以改善其记忆成绩。
1.3 VD小鼠海马病理学改变
在光镜下,⑴假手术对照组小鼠海马CA1区锥体细胞核大而圆,核仁明显,细胞排列较致密整齐。⑵相比之下,VD模型组小鼠海马CA1区锥体细胞松散,层次不清,有细胞缺失现象;炎性细胞浸润;部分细胞旁有空染区,核固缩、胞质浓染、胞体变小,并且有胶质细胞增生形成结节。⑶丁苯酞治疗组和预防治疗组小鼠海马CA1区锥体细胞排列尚整齐,未发现炎性细胞浸润,核固缩现象较少见。
2 VD小鼠海马CA1区AMPA-GluR2受体表达水平及丁苯酞的影响
2.1 AMPA-GluR2免疫组化染色
小鼠10%水合氯醛麻醉,4%多聚甲醛灌注固定,石蜡包埋,冠状切片,采用SP法行免疫组化染色,观察GluR2阳性神经元数目的变化。结果显示:⑴与假手术对照组海马CA1区AMPA-GluR2阳性神经元面密度(124.37±7.48)比较,VD模型组小鼠海马CA1区GluR2阳性神经元面密度值(27.69±5.15)明显减少(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区GluR2阳性神经元面密度值(88.57±10.98)和丁苯酞预防组小鼠海马CA1区GluR2阳性神经元面密度值(94.42±12.37)明显增多(P<0.05);⑶丁苯酞治疗组、丁苯酞预防治疗组、假手术对照组海马CA1区GluR2阳性神经元面密度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区GluR2水平降低参与了VD的发病机制,丁苯酞和丁苯酞预防治疗组可以防止VD小鼠海马CA1区GluR2水平表达下降而促进其学习和记忆成绩的改善。
2.2 AMPA-GluR2 mRNA RT-PCR检测
冰上迅速分离小鼠海马,Trizol (Invitrogen公司)提取海马总RNA,测定其浓度及纯度。每个样品取2μg RNA反转录合成cDNA,再各取1μL cDNA进行PCR扩增。取RT-PCR产物5μL在2 %琼脂糖凝胶上电泳,用UVP凝胶成像系统扫描定量电泳条带的灰度,以GluR2 /β-actin表示产物的相对含量,凝胶图像分析系统分析结果。结果显示:⑴与假手术对照组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.96±0.21)比较,VD模型组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.28±0.07)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.71±0.15)和丁苯酞预防治疗组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.75±0.18)显著升高(P<0.05);⑶丁苯酞组、丁苯酞预防治疗组、假手术对照组海马CA1区GluR2 mRNA表达的灰度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区GluR2水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区GluR2 mRNA水平表达下降而促进其学习和记忆成绩的改善。
3 VD小鼠海马CA1区NMDA-NR2B受体表达水平及丁苯酞的影响
免疫组化观察NMDA-NR2B受体的变化,RT-PCR观察NMDA-NR2B mRNA表达的变化。方法同2。
3.1 NMDA-NR2B受体免疫组化染色
结果显示:⑴与假手术对照组海马CA1区NMDA-NR2B阳性神经元面密度值为:(149.27±8.67)比较,VD模型组小鼠海马CA1区NR2B阳性神经元面密度值(29.70±2.09)明显减少(P<0.05);⑵与VD模型组比较,丁苯酞预防治疗组小鼠海马CA1区NMDA-NR2B阳性神经元面密度值(108.51±7.28)和丁苯酞组小鼠海马CA1区NMDA-NR2B阳性神经元面密度值(100.53±10.00)明显增多(P<0.05);⑶丁苯酞组、丁苯酞预防治疗组、假手术对照组海马CA1区NMDA-NR2B阳性神经元面密度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区NMDA受体水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区NMDA受体水平表达下降而促进其学习和记忆成绩的改善。
3.2 NMDA受体mRNA RT-PCR检测
结果显示:⑴与假手术对照组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.71±0.18)比较,VD模型组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.22±0.04)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.54±0.11)和丁苯酞预防组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.59±0.20)显著升高(P<0.05);⑶丁苯酞组、丁苯酞预防治疗组、假手术对照组海马CA1区NMDA受体mRNA阳性神经元密度之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区NMDA受体水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区NMDA受体mRNA水平表达下降而促进其学习和记忆成绩的改善。
4 VD小鼠海马神经元钙信号转导机制及丁苯酞的影响
4.1 VD小鼠海马神经元静息态[Ca2+]i的变化及丁苯酞的影响
小鼠经10%水合氯醛麻醉后处死,采用机械法在低温修块台上快速分离双侧海马组织,并制备海马细胞悬液,负载Fluo-3/AM,采用流式细胞分析技术观察了VD海马细胞静息态[Ca2+]i变化特征及丁苯酞干预效果。结果显示:⑴与假手术对照组小鼠海马细胞静息态[Ca2+]i浓度(1.29±0.32)比较,VD模型组小鼠海马细胞静息态[Ca2+]i浓度(1.92±0.35)显著升高(P<0.01);⑵与VD模型组比较,丁苯酞治疗组小鼠海马细胞静息态[Ca2+]i浓度(1.34±0.28)和丁苯酞预防组小鼠海马细胞静息态[Ca2+]i浓度(1.33±0.41)显著降低(P<0.05);⑶丁苯酞治疗组、丁苯酞预防治疗组、假手术对照组小鼠海马细胞静息态[Ca2+]i浓度之间无明显差异(P>0.05)。此可以提示VD小鼠海马细胞钙超载参与了其发病机制,丁苯酞可以抑制VD小鼠海马细胞钙超载而改善其学习和记忆成绩。
4.2 VD小鼠海马神经元CaMPKⅡ表达水平的变化特征及丁苯酞的影响
小鼠断头处死,快速分离海马组织,提取总蛋白。考马斯亮蓝法进行蛋白浓度定量。应用Western-Blot方法对VD小鼠海马组织CaMPKⅡ的蛋白水平进行了检测。结果显示:⑴与假手术对照组小鼠海马组织CaMPKⅡ蛋白表达水平(1.06±0.14)比较,VD模型组小鼠海马组织CaMPKⅡ蛋白表达水平(0.30±0.07)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞预防治疗组小鼠海马组织CaMPKⅡ蛋白表达水平(0.98±0.27)和丁苯酞治疗组小鼠海马组织CaMPKⅡ蛋白表达水平(0.94±0.13)显著升高(P<0.01);⑶假手术对照组、丁苯酞治疗组、丁苯酞预防治疗组小鼠海马组织CaMPKⅡ蛋白表达水平之间无明显差异(P>0.05)。此可以提示VD小鼠海马组织CaMPKⅡ蛋白表达水平的降低参与了VD的发病机制,丁苯酞可以防止并改善VD小鼠海马神经元CaMPKⅡ蛋白表达水平下降而促进其学习和记忆成绩改善。
4.3 VD小鼠海马神经元CaMPKⅡmRNA RT-PCR结果
标本取材同2。结果显示:⑴与假手术对照组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.92±0.15)比较,VD模型组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.31±0.06)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.78±0.11)和丁苯酞预防组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.82±0.14)明显增多(P<0.05);⑶假手术对照组、丁苯酞组、丁苯酞预防治疗组海马CA1区CaMPKⅡmRNA表达的灰度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区CaMPKⅡ水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区CaMPKⅡmRNA水平表达下降而促进其学习和记忆成绩的改善。
5 VD小鼠海马神经元CREB水平变化及丁苯酞的影响
5.1 VD小鼠海马神经元CREB表达水平的变化特征及丁苯酞的影响
小鼠断头处死,快速分离海马组织,提取核蛋白。考马斯亮蓝法进行蛋白浓度定量。应用Western-Blot方法对VD小鼠海马组织CREB的蛋白表达水平进行了检测。结果显示:⑴与假手术对照组小鼠海马组织CREB蛋白表达水平(1.37±0.21)比较,VD模型组小鼠海马组织CREB蛋白表达水平(0.43±0.06)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马组织CREB蛋白表达水平(1.06±0.12)和丁苯酞预防治疗组小鼠海马组织CREB蛋白表达水平(1.12±0.14)显著升高(P<0.01);⑶丁苯酞治疗组、丁苯酞预防治疗组、假手术对照组小鼠海马组织CREB蛋白表达水平之间无明显差异(P>0.05)。此可以提示VD小鼠海马组织CREB蛋白表达水平的降低参与了VD的发病机制,丁苯酞可以防止并改善VD小鼠海马组织CREB蛋白表达水平下降而促进其学习和记忆成绩改善。
5.2 VD小鼠海马神经元CREB mRNA RT-PCR结果
取材方法同2。结果显示:⑴与假手术对照组小鼠海马CA1区CREBmRNA表达的灰度值(0.98±0.17)比较,VD模型组小鼠海马CA1区CREB mRNA表达的灰度值(0.29±0.04)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区CREB mRNA表达的灰度值(0.82±0.09)和丁苯酞预防治疗组小鼠海马CA1区CREB mRNA表达的灰度值(0.84±0.11)明显增多(P<0.05);⑶假手术对照组、丁苯酞组、丁苯酞预防治疗组海马CA1区CREB mRNA表达的灰度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区CREB水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区CREB mRNA水平表达下降而促进其学习和记忆成绩的改善。
结论:
⑴本实验建立的VD小鼠模型可模拟人类VD以学习和记忆能力缺损为主的认知功能障碍,是研究VD比较理想的动物模型,对深入研究VD是可行的。VD模型小鼠海马CA1区出现病理性变化。
⑵VD小鼠海AMPA-GluR2蛋白和mRNA表达水平降低,与其学习和记忆成绩下降相一致,提示GluR2表达水平降低可能参与了VD的发病。
⑶VD小鼠海马NMDA-NR2B蛋白和mRNA表达水平降低,与其学习和记忆成绩下降相一致,提示NMDA-NR2B表达水平降低可能参与了VD的发病。
⑷VD小鼠海马细胞静息态[Ca2+]i升高,CaMPKⅡ蛋白表达及CaMPKⅡmRNA水平降低,导致钙信号转导系统功能紊乱,可能参与了VD的分子生物学发病机制。
⑸VD小鼠海马神经元CREB蛋白及mRNA表达水平降低,与其学习和记忆成绩下降相一致,提示CREB蛋白表达水平降低可能参与了VD的分子生物学发病机制。
丁苯酞在改善VD小鼠学习和记忆成绩的同时,可显著减轻VD小鼠海马细胞内[Ca2+]i、CaMPKⅡ、NR2B、GluR2和CREB水平等方面的异常,从而发挥对VD的治疗作用,并且改善了海马组织CA1区病变,此与其学习和记忆成绩改善相一致。提示此药可能具有改善VD的药理学作用。
Objectives:
As an acquired syndrome of intelligent impairment, vascular dementia (VD), which is a kind of cerebral dysfunction caused by various kinds cerebral vascular diseases, demonstrats mainly as learning and memory dysfunction, accompanied with the possible disorder of tongue, motion, direction and personality. With the increase of the proportion of the elderly in the population and that of the curing rate of cerebral vascular disease, morbidity of VD mounts continuously and hence brings about heavy loads to the society and family. However, both the pathogenesis of VD and its specific treatment remain unknown up to now. It is therefore significant to study the pathophysiological mechanism of VD and to find some effective treatments of VD.
As two advanced functions of human brains, learning and memory are two closely associated neurobiology procedures despite their differences. Learning is a acquirement of experiences and skills. Memory is conservation and playbacks of experience and skills. It is now well known that the hippocampus is correlated with learning and memory. The pathological changes of neurons in the hippocampus have great effects upon the forming and maintance of learning and memory. Many results have confirmed that hippocampus long-term potentiation(LTP) is closely correlated with the process of learning and memory. It has been moreove proved that LTP as a synapse model of memory is produced and maintained with the participation of Glu and its receptor.
Glutamic acid, an important excitabily neurotransmitter in central nervous system, is mainly distributed in the area of cerebral cortex, hippocampus etc, and has important effect upoin learning, memory and synaptic plasticity. It participates in many physiological functions of cerebral cortex and hippocampus, such as learning, memory, motion and sensus. In addition, it helps to nourish trophics nerve, promote growth of neuron and axon. The receptor of glutamate mainly includes the receptor ofα-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA) and that of N-methyl-D-aspartate (NMDA), both are found in postsynapse-dense area. AMPA receptor mainly induces fast synaptic transmission which is necessary for normal information transfer of nervous system. NMDA receptor, once activated, mainly triggers different types of synaptic plasticity, including induction of LTP.
The intracellular ion concentration of calcium ([Ca2+]i) in neurons has been accepted as the second signal which participates in the mechanism of learning and memory. The intracellular ion concentration of calcium ([Ca2+]i) and calcium/calmodulin dependent protein kinaseⅡ(CaMPKⅡ) has been the focus of the researchers on the mechanism of ischemia-hypoxia damage. Many studies on cerebral ischemia animal model show that calcium over- loading and elevation of CaMPKⅡin neurons might participate in the development of ischemic damage and the Ca2+ channel antagons might hold back calcium over-loading and alleviate the ischemic damage in the neurons.
Cyclic AMP(cAMP)-responsive element binding protein(CREB) in neurons, the third signal in intranuclear and a key ingredient of many intracellular signal circuit, has extensive biological functions, including adjustment of the learning and momory by the signal system of Ca2+-CaM- CaMPKⅡand further forming of the transmission circuit Ca2+-CaM- CaMPKⅡ- CREB. According to some related researchers, both the CREB and the expression of glutamate acid receptors in postsynapse membrane of neurons increas after the ischemic damage for their possible participatation.
dl-3n-butylphthalide(NBP), a yellow compound isolated from the seed of celery, is a new drug of Class I of P.R.China and never reported abroad up to now. In China, FengYipu has made lots of related researches which proves it as an anticerebral- ischemic drug which has multitarget effects upon, the prevention of the cerebral ischemic damage, the improvement of both chondriosome function and the level of NO and PGI2 of brain blood vessel endothelium, the restraints of glumatic acid release, the degrades of the intracellular concentration of calcium and the level of arachidonic acid, the restraints of free radicle and improves the activity of antioxidase, the lightenments of cerebral edema and the reduction of area of cerebral infarction. In a word, it is proved that NBP can increase cerebral blood flow and improve brain cell energy metabolism, afunction and dysmnesia. However there is no scientific report the drug in VD at present.
Based on what has been mentioned above, we attempted to study the therapeutic effect of NBP on VD and therefore explore the mechanism of action by observing GluR circuit and pathogenetic mechanism of VD. In this study, the change of AMPA receptor, NMDA receptor on neuron cellular membrane, and the influence of [Ca2+]i, CaMPKⅡ, CREB in neurons and the the contribution on learning and memory were observed as well. The study results provide us with some clues of further exploration of glutamate receptor circuit and calcium signal transduction mechanism in VD animal model. In the mean while, it is expected that our study may offer some evidence of treating VD with NBP.
Methods:
1 The VD model of mice were established for ischemia (20 min)- reperfusion (10 min) thrice by ligating the bilateral common carotid arteries. The changes of behavior were observed through the step-down avoidance test and water maze test. The hippocampal pathologic changes in the mice with VD was observed through HE staining.
2 The changes of AMPA-GluR2 protein of hippocampus and that of the level of mRNA expression of each group of mice were measured with immunohistochemistry and reverse transcription PCR (RT-PCR).
3 The expressions of NMDA receptors NR2B in neurons of hippocampus of every group mice were observed by immunohistochemistry technique. RT-PCR technique was used to measure mRNA expression of NMDA receptor in hippocampal neurons.
4 The changes of [Ca2+]i of hippocampal neurons of the mice in each group were measured with flow cytometry. Western blot was applied to the measurement of the expression of CaMKII in hippocampal neurons of each mouse in the experiment. RT-PCR technique was used to measure the mRNA expression of CaMKII in hippocampal neurons of all mice in the experiment.
5 The expression levels of CREB in the hippocampal of the mice in each group were analyzed with Western-blot. And RT-PCR technique was used to measure mRNA expression of CREB in hippocampal neurons of the mice in each group.
Results:
1 The evaluation of behavior of VD model and its pathologic features of the hippocampus
The mice were divided into the control group, sham-operated group, VD model group, NBP treating group and NBP precaution treating group. The mice were subjected for ischemia (20 min)-reperfusion (10 min) thrice by ligating the bilateral common carotid arteries to establish the VD model. Beginning on day 29 and 30 day after operation, the functions of learning and memory of each mouse in each group were tested through step-down test and water maze test.
1.1 The test of learning of mice
On day 29 after operation, the learning of every group were tested by step-down test and water maze test respectively. Response time (sec), error times (number/5min) in step-down test, and swimming time (sec) and error times (number/3min) in water maze test were used to evaluate the achivements of learning.
The results of step-down test revealed that:⑴The response time in learning phase of VD model group (89.41±12.08)s prolonged distinctly (P<0.01) compared with the response time of sham-operated group(48.76±9.24)s.⑵The response time of both the NBP treating group (59.68±10.27)s and the NBP precaution treating group (57.23±10.05)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of VD control group (3.74±0.42) increased notably (P<0.01), compared with the error time of sham-operated group (1.23±0.27).⑷The error times of both the NBP treating group (1.52±038) and the NBP precaution treating group (1.48±0.31) decreased notably (P<0.01), compared with VD model group.⑸The learning of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).
The results of water maze test revealed that:⑴The swimming time in learning phase of the VD model group (145.80±13.29)s prolonged significantly(P<0.01), compared with sham-operated group (87.32±13.46)s.⑵The swimming time of both the NBP treating group (101.89±18.38)s and the NBP precaution treating group (99.18±11.25)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of the VD model group (34.60±5.92) increased notably (P<0.01), compared with and sham-operated group (16.88±4.19).⑷The error times of both the NBP treating group (18.71±4.35) and the NBP precaution treating group (17.29±4.82) decreased remarkably (P<0.01), compared with VD model group.⑸The learning of the sham-operated group, the NBP treating group and the NBP precaution treating group had no distinct difference (P>0.05).
The results above suggest that the learning ability of VD mice reduced and NBP may improve their learning. 1.2 The test of memory of mice
On day 30 after the operations, the memory of the mice in every group was tested by step-down test and water maze test. The results of latency time (sec) and error times (number / 5 min) in step-down test and that of swimming time (sec) and error times (number / 3 min) are the memory.
The results of step-down test revealed that:⑴The latency time in memory phase of VD model group (78.67±19.17)s shortened remarkably (P<0.01), compared with the latency time of sham-operated group (148.92±20.83)s.⑵The latency time of the NBP treating group (131.41±18.82)s and the NBP precaution treating group (128.57±18.29)s prolonged significantly (P<0.01), compared with VD model group.⑶The error times in memory phase of VD model group (1.72±0.13) increased notably (P<0.01), compared with the error time of sham-operated group (0.32±0.05).⑷The error times of both the NBP treating group (0.54±0.08) and the NBP precaution treating group (0.50±0.07) decreased notably (P<0.01), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).
The results of water maze test revealed that:⑴The swimming time in memory phase of the VD model group (137.40±11.76)s prolonged remarkably (P<0.01), compared with sham-operated group (67.82±8.79)s.⑵The swimming time of both the NBP treating group (88.73±9.53)s and the NBP precaution treating group (83.87±8.62)s shortened significantly (P<0.05), compared with VD model group.⑶The error times in memory phase of the VD model group (20.90±4.25) increased remarkably (P<0.05), compared with sham-operated group (9.57±2.91).⑷The error times of both the NBP treating group (11.17±3.04) and the NBP precaution treating group (10.27±2.83) decreased significantly (P<0.05), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).
These results suggested that the memory of VD mice decreases and NBP may improve their memory.
1.3 The pathologic changes of the hippocampus of the VD mice
The observations under the light microscope show:⑴The arrangement of the pyramydal neurons in the hippocampal CA1 area in sham-operated group were tight and in order with the nucleus being large and round and its neucleolus being evident.⑵In the VD model group, the pyramydal neurons in the hippocampal CA1 decreased and had no clear arrangement. There was empty dye area surrounding the neurons which had condensed nucleus dense cytoplast and small body. In addition, there was infiltration of inflammatory cells.⑶In the NBP treating group and the NBP precaution treating group, the condensed nucleus dense cytoplast and small body decreased apparently and there was no infiltration of inflammatory cells.
2 The level of AMPA-GluR2 receptor in the hippocampal CA1 area of VD mice and the effect of NBP .
2.1 Staining of immunohistochemistry of AMPA-GluR2.
The mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made and AMPA-GluR2 was stained through immunohistochemistry. And then the statistic study of each group (surface density Sv showing their numbers) was conducted. The result revealed that:⑴The AMPA-GluR2 positive neurons of hippocampus CA1 area in VD model group (27.69±5.15) reduced distinctly (P<0.05), compared with sham-operated group (124.37±7.48).⑵The AMPA-GluR2 positive neurons of hippocampus CA1 area of the mice in both the NBP treating group (88.57±10.98) and the NBP precaution treating group (94.42±12.37) increased (P<0.05), compared with VD model group.⑶The NBP treating group, the NBP precaution treating group and the sham-operated group had no distinct difference (P>0.05).
These suggested that the lower level AMPA-GluR2 of hippocampus CA1 area might participate in the pathogenesis of VD and NBP could prevent the level AMPA-GluR2 of hippocampus CA1 area of VD from decreasing and hence improve their learning and memory ability.
2.2 RT-PCR of AMPA-GluR2 mRNA
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of GluR2 mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of AMPA-GluR2 mRNA expression in VD model (0.28±0.07) reduced notably (P<0.01), compared with the magnitude value of GluR2 mRNA expression in sham-operated group (0.96±0.21).⑵The level of AMPA-GluR2 mRNA expression in NBP treating group (0.71±0.15) and NBP precaution treating group (0.75±0.18) elevated remarkably (P<0.01), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of AMPA-GluR2 mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of AMPA-GluR2 mRNA to reduce and improve their learning ability and memory.
3 The level of NMDA-2B receptor in the hippocampal CA1 area of VD mice and the effect of NBP.
3.1 Immunohistochemistry of NMDA-2B receptor
The mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made and NMDA-2B receptor was dyed through immunohistochemistry; and made the statistics of group (surface density Sv showing their numbers).The result revealed that:⑴NMDA-2B receptor positive neurons of hippocampus CA1 area in VD model group (29.70±2.09) reduced distinctly (P<0.05), compared with sham-operated group (149.27±8.67).⑵NMDA-2B receptor positive neurons of hippocampus CA1 area in NBP treating group (100.53±10.00) and NBP precaution treating group (108.51±7.28) increased (P<0.05), compared with VD model group.⑶NBP treating group, NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower level NMDA-2B receptor of hippocampus CA1 area might participate in the pathogenesis of VD; but NBP could prevent the level NMDA-2B receptor of hippocampus CA1 area of VD to reduce and improve their learning ability and memory.
3.2 RT-PCR of NMDA-NR2B receptor mRNA
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of NMDA-2B receptor mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of NMDA-2B receptor mRNA expression in VD model (0.22±0.04) reduced notably (P<0.05), compared with the magnitude value of NMDA-2B receptor mRNA expression in sham-operated group (0.71±0.18).⑵The level of NMDA-2B receptor mRNA expression in NBP treating group (0.54±0.11) and NBP precaution treating group (0.59±0.20) elevated notably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of NMDA-2B receptor mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of NMDA-2B receptor mRNA to reduce and improve their learning ability and memory.
4 Calcium signal transduction pathway in hippocampal neuron of mice with VD and the effect of NBP
4.1 The resting [Ca2+]i in the hippocampus of VD mice and the effect of NBP
The mice were executed quickly after anaesthetized by 10% chloral hydrate solution and their hippocampuses were extracted in low temperature operation. They were separated through mechanical method. Thus the single cell suspension sample of hippocampus was prepared. We observed the change characteristic of the resting [Ca2+]i by flow cytometryanalytical technique and intervention of NBP. The result indicated that:⑴The concentration of intracellular [Ca2+]i in hippocampus of VD model group (1.92±0.35) increased notably (P<0.01), compared with the concentration of intracellular [Ca2+]i in sham-operated group (1.29±0.32);⑵the intracellular [Ca2+]i in hippocampus of NBP treating group (1.34±0.28) and NBP precaution treating group(1.33±0.41) decreased notably (P<0.01),compared with VD model group;⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the calcium over-loading in hippocampus cells of VD might participate in its pathogenesis; but NBP could restrain the change and improve the learning ability and memory.
4.2 Western blot of CaMPKⅡin hippocampus of VD
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. The level of CaMPKⅡexpression in hippocampus was examined through Western blot method. The result indicated that:⑴The level of CaMPKⅡexpression in VD model group (0.30±0.07) reduced notably (P<0.01), compared with the level of CaMPKⅡexpression in sham-operated group (1.06±0.14).⑵The level of CaMPKⅡexpression in NBP treating group (0.94±0.13) and NBP precaution treating group (0.98±0.27) elevated notably (P<0.01), compared with the level of CaMPKⅡexpression in VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CaMPKⅡmight participate in the pathogenesis of VD; but NBP could prevent the expression of CaMPKⅡto reduce and improve their learning ability and memory.
4.3 RT-PCR of CaMPKⅡmRNA
The mice were executed quickly through decollation and their hippocampus were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of CaMPKⅡmRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of CaMPKⅡmRNA expression in VD model (0.31±0.06) reduced notably (P<0.05), compared with the magnitude value of CaMPKⅡmRNA expression in sham-operated group (0.92±0.15).⑵The level of CaMPKⅡmRNA expression in NBP treating group (0.78±0.11) and NBP precaution treating group (0.82±0.14) elevated notably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CaMPKⅡmRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of CaMPKⅡmRNA to reduce and improve their learning ability and memory.
5 The level of CREB in hippocampus of VD and the effect of NBP
5.1 Western blot of CREB in hippocampus of VD
The mice were executed quickly through decollation and their hippocampus were extracted in low temperature operation. The level of CREB expression in hippocampus was examined through Western blot method. The result indicated that:⑴The level of CREB expression in VD model group (0.43±0.06) reduced remarkably (P<0.05), compared with the level of CREB expression in sham-operated group (1.37±0.21).⑵The level of CREB expression in NBP treating group (1.06±0.12) and NBP precaution treating group (0.12±0.14) elevated remarkably (P<0.05), compared with the level of CREB expression in VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CREB might participate in the pathogenesis of VD; but NBP could prevent the expression of CREB to reduce and improve their learning and memory.
5.2 RT-PCR of CREB mRNA
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of CREB mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of CREB mRNA expression in VD model (0.29±0.04) reduced remarkably (P<0.05), compared with the magnitude value of CREB mRNA expression in sham-operated group (0.98±0.17).⑵The level of CREB mRNA expression in NBP treating group (0.82±0.09) and NBP precaution treating group (0.84±0.11) elevated remarkably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CREB mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of CREB mRNA to reduce and improve their learning and memory.
Conclusions:
1 The VD mice model which had the cognization impairment in primary of damage of learning and memory was successfully established. The VD model is the perfect animal model and feasible to the further study of VD. The hippocampus CA1 area in the VD mice model had the pathologic changes.
2 The level of AMPA-GluR2 and its mRNA in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the reduction of the expression of AMPA receptor might participate in the pathogenesis of VD.
3 The level of NMDAR-2B and its mRNA in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the lower expression of NMDA-NR2B might participate in the pathogenesis of VD.
4 The imbalance of calcium signal transduction pathway resulted from the increase of the resting [Ca2+]i in VD mice hippocampus and the reduction of CaMPKⅡin VD mice hippocampus, which might be one part of the molecular pathogenesis of VD.
5 The reduction of the protein level of CREB in VD mice hippocampus consistent with the decline of learning and memory suggested that the reduction of the protein level of CREB might participate in the moleculear pathogenesis of VD.
6 NBP could resist the over-loading of calcium in hippocampus of VD and prevent the expression of CaMPKⅡand AMPA receptor and NMDA receptor and the level of CREB from reducing. These further improve the calcium signal transduction pathway and ameliorate the pathologic changes in hippocampual CA1 area in the mice with VD. This was in accord to their better learnig and memory. All mentioned above suggest that the medicine may improve the pathogenesis of VD.
学习和记忆是大脑的高级功能之一。学习是指经验及技能的获得,记忆是经验及技能的保存及再现,它们是两个不同而又密切联系的神经生物过程。目前认为,海马与学习、记忆等高级认知功能密切相关,其神经元形态正常与否对学习、记忆的形成和维持具有重要意义。现代大量资料研究证实海马与长时程增强(long-term potentiation,LTP)和学习、记忆过程密切相关,人们称LTP可能是“记忆的突触模型”、“记忆的神经元机制”。LTP形成后,动物的学习能力增强;而通过受体阻断剂阻抑LTP的产生,则影响动物的学习能力,记忆亦受损。而谷氨酸及其受体参与了LTP的产生与维持。
谷氨酸(Glutamate,Glu)是中枢神经系统中一种最重要的兴奋性神经递质,主要分布于大脑皮质、海马、小脑和纹状体,在学习、记忆、突触可塑性及大脑发育等方面均起重要作用。研究证实,Glu的神经递质作用是通过兴奋性氨基酸受体而实现的,受体活性的变化、受体数目的增减都会对突触效能产生明显的影响。在对LTP的研究中,人们发现谷氨酸受体对LTP的诱导具有重要意义,这些受体的特异性拮抗剂不但可阻断LTP的诱导,而且可破坏多种学习和记忆行为。
神经细胞内钙离子浓度(intracellular concentration of calcium,[Ca2+]i)、钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin dependent protein kinaseⅡ, CaMPKⅡ)及钙调素(CaM)一直是脑组织缺血缺氧损伤机制研究的焦点。多种脑缺血动物模型的研究表明,神经细胞内钙超载(Ca2+ over-loading)及CaMPKⅡ水平升高参与了脑组织缺血损伤;并且应用Ca2+通道拮抗剂抑制钙超载,可以减轻神经细胞缺血性损伤。有些学者对缺血性脑组织损伤做了进一步研究,发现缺血后神经细胞内环磷酸腺苷反应元件结合蛋白(cAMP- responsive element binding protein, CREB)水平升高,神经突触后膜谷氨酸受体表达增多,二者均参与了脑组织缺血性损伤。
目前认为,在生理状态下神经细胞[Ca2+]i是参与学习和记忆机制的细胞内第二信使;CREB作为核内的“第三信使”,具有调节包括学习记忆在内的广泛生物学的功能,是细胞内多种信号通路的一种关键成分,而神经细胞膜Glu受体N-甲基-D-天门冬氨酸(N-methyl-D-aspartate, NMDA)受体、α-氨基-3-羟基-5-甲基-4-异唑丙酸(α-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid, AMPA)受体及细胞内[Ca2+]i、CaM、CaMPKⅡ、CREB水平的变化对学习和记忆的影响,也逐渐成为了多数学者研究的热点。也有学者对老年性痴呆(Alzheimers dementia, AD)动物模型进行了研究,发现AD模型脑组织内钙离子超载、谷氨酸受体水平降低。这些研究结果为我们探讨VD动物模型脑组织内谷氨酸受体、钙信号转导机制及CREB改变提供了线索。
丁苯酞(dl-3n-butylphthalide,NBP)系从天然食用植物芹菜籽中提取并经消旋合成的黄色油状液体,具有芹菜香味。该药为国家一类新药,国外对该药的研究尚未见报道,国内冯亦璞等对丁苯酞治疗脑缺血的相关机制做了大量的实验研究,发现丁苯酞具有多靶点抗脑缺血作用,能改善线粒体功能,提高脑血管内皮NO和PGI2的水平,抑制谷氨酸释放,降低细胞内钙浓度和花生四烯酸含量,抑制自由基和提高抗氧化酶活性,减轻脑缺血所致脑水肿,抗血栓形成和抗血小板聚集,缩小脑梗死面积,改善局部脑血流量和细胞能量代谢、神经功能缺失及脑缺血记忆障碍。但该药对VD的实验研究尚未见相关报道。基于此,本文通过探讨谷氨酸受体及钙信号转导机制,观察丁苯酞治疗VD的效果并探讨其作用机制。
方法:1采用三次双侧颈总动脉结扎,造成脑组织反复缺血/再灌注,建立了小鼠VD模型,建立假手术组排除手术本身对小鼠所致的影响,通过跳台试验和水迷宫试验进行学习和记忆成绩测试,观察其行为学改变;用HE染色观察海马病理学特征变化。2采用免疫组化和反转录-聚合酶链反应(RT-PCR)方法测定各组小鼠海马谷氨酸AMPA-GluR2蛋白及GluR2 mRNA的变化。3采用免疫组化和RT-PCR方法测定各组小鼠海马谷氨酸NMDA-NR2B蛋白及NR2B mRNA的变化。4采用流式细胞分析技术测定各组小鼠海马细胞[Ca2+]i变化;Western-Blot及RT-PCR方法检测CaMPKII蛋白及mRNA表达的变化。5 Western-Blot及RT-PCR方法检测CREB蛋白及mRNA表达的变化。
结果:
1 VD小鼠模型的行为学评价及海马病理学特征
3月龄雄性昆明小鼠196只,分为假手术对照组、VD模型组、丁苯酞预防治疗组、丁苯酞治疗组。采用双侧颈总动脉结扎,连续三次脑缺血(20 min)再灌注(10min),建立VD小鼠模型。术后29d,各组分别进行跳台试验及水迷宫实验,测试学习成绩;术后30d测试记忆成绩,HE染色观察各组小鼠海马组织病理学特征。
1.1学习成绩
术后第29天,分别进行跳台试验和水迷宫试验,测试跳台试验反应时间(sec)和错误次数(number/5 min)作为学习成绩,水迷宫试验游完全程时间(sec)和错误次数(number/3 min)作为学习成绩。
跳台试验结果显示:⑴与假手术对照组的反应时间(48.76±9.24)s比较,VD模型组小鼠学习阶段的反应时间(89.41±12.08)s明显延长(P<0.01);⑵与VD模型组的反应时间比较,丁苯酞治疗组小鼠学习阶段的反应时间(59.68±10.27)s及丁苯酞预防治疗组小鼠学习阶段的的反应时间(57.23±10.05)s均明显缩短(P<0.01);⑶与假手术对照组的错误次数(1.23±0.27)比较,VD模型组小鼠学习阶段的错误次数(3.74±0.42)明显增加(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠学习阶段的错误次数(1.52±0.38)及丁苯酞预防治疗组小鼠学习阶段的的错误次数(1.48±0.31)均明显减少(P<0.01);⑸假手术对照组、丁苯酞治疗组、丁苯酞预防治疗组之间的学习成绩无显著性差异(P>0.05)。
水迷宫试验结果显示:⑴与假手术对照组的游全程时间(87.32±13.46)s比较,VD模型组小鼠学习阶段的游全程时间(145.80±13.29)s明显延长(P<0.01);⑵与VD模型组的游全程时间比较,丁苯酞治疗组小鼠学习阶段的游全程时间(101.89±18.38)s及丁苯酞预防治疗组小鼠学习阶段的游全程时间(99.18±11.25)s明显缩短(P<0.01);⑶与假手术对照组的错误次数(16.88±4.19)比较,模型组小鼠学习阶段的错误次数(34.60±5.92)明显增多(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠学习阶段的错误次数(18.71±4.35)及丁苯酞预防治疗组小鼠学习阶段的错误次数(17.29±4.82)明显减少(P<0.01);⑸假手术对照组、丁苯酞预防治疗组、丁苯酞治疗组的学习成绩之间无显著性差异(P>0.05)。
以上结果提示VD模型小鼠的学习成绩降低,丁苯酞可以改善其学习成绩。
1.2记忆成绩
术后第30天,分别进行跳台试验和水迷宫试验,测试跳台试验潜伏时间(sec)和错误次数(number/5min)作为记忆成绩,水迷宫试验游完全程时间(sec)和错误次数(number / 3 min)作为记忆成绩。
跳台试验结果显示:⑴与假手术对照组的潜伏时间(148.92±20.83)s比较,VD模型组小鼠记忆阶段的潜伏时间(78.67±19.17)s明显缩短(P<0.01);⑵与VD模型组的潜伏时间比较,丁苯酞治疗组小鼠记忆阶段的潜伏时间(131.41±18.82)s及丁苯酞预防治疗组小鼠记忆阶段的的潜伏时间(128.57±18.29)s均明显延长(P<0.01);⑶与假手术对照组的错误次数(0.32±0.05)比较,VD模型组小鼠记忆阶段的错误次数(1.72±0.13)明显增加(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠记忆阶段的错误次数(0.54±0.08)及丁苯酞预防治疗组小鼠记忆阶段的的错误次数(0.50±0.07)均明显减少(P<0.01);⑸假手术对照组、丁苯酞预防治疗组、丁苯酞治疗组之间的记忆成绩无显著性差异(P>0.05)。结果提示VD模型小鼠的记忆成绩降低,丁苯酞可以改善其记忆成绩。
水迷宫试验结果显示:⑴与假手术对照组的游全程时间(67.82±8.79)比较,VD模型组小鼠记忆阶段的游全程时间(137.40±11.86)明显延长(P<0.01);⑵与VD模型组的游全程时间比较,丁苯酞治疗组小鼠记忆阶段的游全程时间(88.73±9.53)及丁苯酞预防治疗组小鼠记忆阶段的游全程时间(83.87±8.62)明显缩短(P<0.01);⑶与假手术对照组的错误次数(9.57±2.91)比较,模型组小鼠记忆阶段的错误次数(20.90±4.25)明显增多(P<0.01);⑷与VD模型组的错误次数比较,丁苯酞治疗组小鼠记忆阶段的错误次数(11.17±3.04)及丁苯酞预防治疗组小鼠记忆阶段的错误次数(10.27±2.83)明显减少(P<0.01);⑸假手术对照组、丁苯酞治疗组、丁苯酞预防治疗组的记忆成绩之间无显著性差异(P>0.05)。
以上结果提示VD模型小鼠的记忆成绩降低,丁苯酞可以改善其记忆成绩。
1.3 VD小鼠海马病理学改变
在光镜下,⑴假手术对照组小鼠海马CA1区锥体细胞核大而圆,核仁明显,细胞排列较致密整齐。⑵相比之下,VD模型组小鼠海马CA1区锥体细胞松散,层次不清,有细胞缺失现象;炎性细胞浸润;部分细胞旁有空染区,核固缩、胞质浓染、胞体变小,并且有胶质细胞增生形成结节。⑶丁苯酞治疗组和预防治疗组小鼠海马CA1区锥体细胞排列尚整齐,未发现炎性细胞浸润,核固缩现象较少见。
2 VD小鼠海马CA1区AMPA-GluR2受体表达水平及丁苯酞的影响
2.1 AMPA-GluR2免疫组化染色
小鼠10%水合氯醛麻醉,4%多聚甲醛灌注固定,石蜡包埋,冠状切片,采用SP法行免疫组化染色,观察GluR2阳性神经元数目的变化。结果显示:⑴与假手术对照组海马CA1区AMPA-GluR2阳性神经元面密度(124.37±7.48)比较,VD模型组小鼠海马CA1区GluR2阳性神经元面密度值(27.69±5.15)明显减少(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区GluR2阳性神经元面密度值(88.57±10.98)和丁苯酞预防组小鼠海马CA1区GluR2阳性神经元面密度值(94.42±12.37)明显增多(P<0.05);⑶丁苯酞治疗组、丁苯酞预防治疗组、假手术对照组海马CA1区GluR2阳性神经元面密度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区GluR2水平降低参与了VD的发病机制,丁苯酞和丁苯酞预防治疗组可以防止VD小鼠海马CA1区GluR2水平表达下降而促进其学习和记忆成绩的改善。
2.2 AMPA-GluR2 mRNA RT-PCR检测
冰上迅速分离小鼠海马,Trizol (Invitrogen公司)提取海马总RNA,测定其浓度及纯度。每个样品取2μg RNA反转录合成cDNA,再各取1μL cDNA进行PCR扩增。取RT-PCR产物5μL在2 %琼脂糖凝胶上电泳,用UVP凝胶成像系统扫描定量电泳条带的灰度,以GluR2 /β-actin表示产物的相对含量,凝胶图像分析系统分析结果。结果显示:⑴与假手术对照组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.96±0.21)比较,VD模型组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.28±0.07)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.71±0.15)和丁苯酞预防治疗组小鼠海马CA1区GluR2 mRNA表达的灰度值(0.75±0.18)显著升高(P<0.05);⑶丁苯酞组、丁苯酞预防治疗组、假手术对照组海马CA1区GluR2 mRNA表达的灰度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区GluR2水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区GluR2 mRNA水平表达下降而促进其学习和记忆成绩的改善。
3 VD小鼠海马CA1区NMDA-NR2B受体表达水平及丁苯酞的影响
免疫组化观察NMDA-NR2B受体的变化,RT-PCR观察NMDA-NR2B mRNA表达的变化。方法同2。
3.1 NMDA-NR2B受体免疫组化染色
结果显示:⑴与假手术对照组海马CA1区NMDA-NR2B阳性神经元面密度值为:(149.27±8.67)比较,VD模型组小鼠海马CA1区NR2B阳性神经元面密度值(29.70±2.09)明显减少(P<0.05);⑵与VD模型组比较,丁苯酞预防治疗组小鼠海马CA1区NMDA-NR2B阳性神经元面密度值(108.51±7.28)和丁苯酞组小鼠海马CA1区NMDA-NR2B阳性神经元面密度值(100.53±10.00)明显增多(P<0.05);⑶丁苯酞组、丁苯酞预防治疗组、假手术对照组海马CA1区NMDA-NR2B阳性神经元面密度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区NMDA受体水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区NMDA受体水平表达下降而促进其学习和记忆成绩的改善。
3.2 NMDA受体mRNA RT-PCR检测
结果显示:⑴与假手术对照组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.71±0.18)比较,VD模型组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.22±0.04)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.54±0.11)和丁苯酞预防组小鼠海马CA1区NMDA受体mRNA表达的灰度值(0.59±0.20)显著升高(P<0.05);⑶丁苯酞组、丁苯酞预防治疗组、假手术对照组海马CA1区NMDA受体mRNA阳性神经元密度之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区NMDA受体水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区NMDA受体mRNA水平表达下降而促进其学习和记忆成绩的改善。
4 VD小鼠海马神经元钙信号转导机制及丁苯酞的影响
4.1 VD小鼠海马神经元静息态[Ca2+]i的变化及丁苯酞的影响
小鼠经10%水合氯醛麻醉后处死,采用机械法在低温修块台上快速分离双侧海马组织,并制备海马细胞悬液,负载Fluo-3/AM,采用流式细胞分析技术观察了VD海马细胞静息态[Ca2+]i变化特征及丁苯酞干预效果。结果显示:⑴与假手术对照组小鼠海马细胞静息态[Ca2+]i浓度(1.29±0.32)比较,VD模型组小鼠海马细胞静息态[Ca2+]i浓度(1.92±0.35)显著升高(P<0.01);⑵与VD模型组比较,丁苯酞治疗组小鼠海马细胞静息态[Ca2+]i浓度(1.34±0.28)和丁苯酞预防组小鼠海马细胞静息态[Ca2+]i浓度(1.33±0.41)显著降低(P<0.05);⑶丁苯酞治疗组、丁苯酞预防治疗组、假手术对照组小鼠海马细胞静息态[Ca2+]i浓度之间无明显差异(P>0.05)。此可以提示VD小鼠海马细胞钙超载参与了其发病机制,丁苯酞可以抑制VD小鼠海马细胞钙超载而改善其学习和记忆成绩。
4.2 VD小鼠海马神经元CaMPKⅡ表达水平的变化特征及丁苯酞的影响
小鼠断头处死,快速分离海马组织,提取总蛋白。考马斯亮蓝法进行蛋白浓度定量。应用Western-Blot方法对VD小鼠海马组织CaMPKⅡ的蛋白水平进行了检测。结果显示:⑴与假手术对照组小鼠海马组织CaMPKⅡ蛋白表达水平(1.06±0.14)比较,VD模型组小鼠海马组织CaMPKⅡ蛋白表达水平(0.30±0.07)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞预防治疗组小鼠海马组织CaMPKⅡ蛋白表达水平(0.98±0.27)和丁苯酞治疗组小鼠海马组织CaMPKⅡ蛋白表达水平(0.94±0.13)显著升高(P<0.01);⑶假手术对照组、丁苯酞治疗组、丁苯酞预防治疗组小鼠海马组织CaMPKⅡ蛋白表达水平之间无明显差异(P>0.05)。此可以提示VD小鼠海马组织CaMPKⅡ蛋白表达水平的降低参与了VD的发病机制,丁苯酞可以防止并改善VD小鼠海马神经元CaMPKⅡ蛋白表达水平下降而促进其学习和记忆成绩改善。
4.3 VD小鼠海马神经元CaMPKⅡmRNA RT-PCR结果
标本取材同2。结果显示:⑴与假手术对照组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.92±0.15)比较,VD模型组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.31±0.06)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.78±0.11)和丁苯酞预防组小鼠海马CA1区CaMPKⅡmRNA表达的灰度值(0.82±0.14)明显增多(P<0.05);⑶假手术对照组、丁苯酞组、丁苯酞预防治疗组海马CA1区CaMPKⅡmRNA表达的灰度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区CaMPKⅡ水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区CaMPKⅡmRNA水平表达下降而促进其学习和记忆成绩的改善。
5 VD小鼠海马神经元CREB水平变化及丁苯酞的影响
5.1 VD小鼠海马神经元CREB表达水平的变化特征及丁苯酞的影响
小鼠断头处死,快速分离海马组织,提取核蛋白。考马斯亮蓝法进行蛋白浓度定量。应用Western-Blot方法对VD小鼠海马组织CREB的蛋白表达水平进行了检测。结果显示:⑴与假手术对照组小鼠海马组织CREB蛋白表达水平(1.37±0.21)比较,VD模型组小鼠海马组织CREB蛋白表达水平(0.43±0.06)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马组织CREB蛋白表达水平(1.06±0.12)和丁苯酞预防治疗组小鼠海马组织CREB蛋白表达水平(1.12±0.14)显著升高(P<0.01);⑶丁苯酞治疗组、丁苯酞预防治疗组、假手术对照组小鼠海马组织CREB蛋白表达水平之间无明显差异(P>0.05)。此可以提示VD小鼠海马组织CREB蛋白表达水平的降低参与了VD的发病机制,丁苯酞可以防止并改善VD小鼠海马组织CREB蛋白表达水平下降而促进其学习和记忆成绩改善。
5.2 VD小鼠海马神经元CREB mRNA RT-PCR结果
取材方法同2。结果显示:⑴与假手术对照组小鼠海马CA1区CREBmRNA表达的灰度值(0.98±0.17)比较,VD模型组小鼠海马CA1区CREB mRNA表达的灰度值(0.29±0.04)显著降低(P<0.05);⑵与VD模型组比较,丁苯酞治疗组小鼠海马CA1区CREB mRNA表达的灰度值(0.82±0.09)和丁苯酞预防治疗组小鼠海马CA1区CREB mRNA表达的灰度值(0.84±0.11)明显增多(P<0.05);⑶假手术对照组、丁苯酞组、丁苯酞预防治疗组海马CA1区CREB mRNA表达的灰度值之间无明显差异(P>0.05)。此可以提示VD小鼠海马CA1区CREB水平降低参与了VD的发病机制,丁苯酞可以防止VD小鼠海马CA1区CREB mRNA水平表达下降而促进其学习和记忆成绩的改善。
结论:
⑴本实验建立的VD小鼠模型可模拟人类VD以学习和记忆能力缺损为主的认知功能障碍,是研究VD比较理想的动物模型,对深入研究VD是可行的。VD模型小鼠海马CA1区出现病理性变化。
⑵VD小鼠海AMPA-GluR2蛋白和mRNA表达水平降低,与其学习和记忆成绩下降相一致,提示GluR2表达水平降低可能参与了VD的发病。
⑶VD小鼠海马NMDA-NR2B蛋白和mRNA表达水平降低,与其学习和记忆成绩下降相一致,提示NMDA-NR2B表达水平降低可能参与了VD的发病。
⑷VD小鼠海马细胞静息态[Ca2+]i升高,CaMPKⅡ蛋白表达及CaMPKⅡmRNA水平降低,导致钙信号转导系统功能紊乱,可能参与了VD的分子生物学发病机制。
⑸VD小鼠海马神经元CREB蛋白及mRNA表达水平降低,与其学习和记忆成绩下降相一致,提示CREB蛋白表达水平降低可能参与了VD的分子生物学发病机制。
丁苯酞在改善VD小鼠学习和记忆成绩的同时,可显著减轻VD小鼠海马细胞内[Ca2+]i、CaMPKⅡ、NR2B、GluR2和CREB水平等方面的异常,从而发挥对VD的治疗作用,并且改善了海马组织CA1区病变,此与其学习和记忆成绩改善相一致。提示此药可能具有改善VD的药理学作用。
Objectives:
As an acquired syndrome of intelligent impairment, vascular dementia (VD), which is a kind of cerebral dysfunction caused by various kinds cerebral vascular diseases, demonstrats mainly as learning and memory dysfunction, accompanied with the possible disorder of tongue, motion, direction and personality. With the increase of the proportion of the elderly in the population and that of the curing rate of cerebral vascular disease, morbidity of VD mounts continuously and hence brings about heavy loads to the society and family. However, both the pathogenesis of VD and its specific treatment remain unknown up to now. It is therefore significant to study the pathophysiological mechanism of VD and to find some effective treatments of VD.
As two advanced functions of human brains, learning and memory are two closely associated neurobiology procedures despite their differences. Learning is a acquirement of experiences and skills. Memory is conservation and playbacks of experience and skills. It is now well known that the hippocampus is correlated with learning and memory. The pathological changes of neurons in the hippocampus have great effects upon the forming and maintance of learning and memory. Many results have confirmed that hippocampus long-term potentiation(LTP) is closely correlated with the process of learning and memory. It has been moreove proved that LTP as a synapse model of memory is produced and maintained with the participation of Glu and its receptor.
Glutamic acid, an important excitabily neurotransmitter in central nervous system, is mainly distributed in the area of cerebral cortex, hippocampus etc, and has important effect upoin learning, memory and synaptic plasticity. It participates in many physiological functions of cerebral cortex and hippocampus, such as learning, memory, motion and sensus. In addition, it helps to nourish trophics nerve, promote growth of neuron and axon. The receptor of glutamate mainly includes the receptor ofα-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA) and that of N-methyl-D-aspartate (NMDA), both are found in postsynapse-dense area. AMPA receptor mainly induces fast synaptic transmission which is necessary for normal information transfer of nervous system. NMDA receptor, once activated, mainly triggers different types of synaptic plasticity, including induction of LTP.
The intracellular ion concentration of calcium ([Ca2+]i) in neurons has been accepted as the second signal which participates in the mechanism of learning and memory. The intracellular ion concentration of calcium ([Ca2+]i) and calcium/calmodulin dependent protein kinaseⅡ(CaMPKⅡ) has been the focus of the researchers on the mechanism of ischemia-hypoxia damage. Many studies on cerebral ischemia animal model show that calcium over- loading and elevation of CaMPKⅡin neurons might participate in the development of ischemic damage and the Ca2+ channel antagons might hold back calcium over-loading and alleviate the ischemic damage in the neurons.
Cyclic AMP(cAMP)-responsive element binding protein(CREB) in neurons, the third signal in intranuclear and a key ingredient of many intracellular signal circuit, has extensive biological functions, including adjustment of the learning and momory by the signal system of Ca2+-CaM- CaMPKⅡand further forming of the transmission circuit Ca2+-CaM- CaMPKⅡ- CREB. According to some related researchers, both the CREB and the expression of glutamate acid receptors in postsynapse membrane of neurons increas after the ischemic damage for their possible participatation.
dl-3n-butylphthalide(NBP), a yellow compound isolated from the seed of celery, is a new drug of Class I of P.R.China and never reported abroad up to now. In China, FengYipu has made lots of related researches which proves it as an anticerebral- ischemic drug which has multitarget effects upon, the prevention of the cerebral ischemic damage, the improvement of both chondriosome function and the level of NO and PGI2 of brain blood vessel endothelium, the restraints of glumatic acid release, the degrades of the intracellular concentration of calcium and the level of arachidonic acid, the restraints of free radicle and improves the activity of antioxidase, the lightenments of cerebral edema and the reduction of area of cerebral infarction. In a word, it is proved that NBP can increase cerebral blood flow and improve brain cell energy metabolism, afunction and dysmnesia. However there is no scientific report the drug in VD at present.
Based on what has been mentioned above, we attempted to study the therapeutic effect of NBP on VD and therefore explore the mechanism of action by observing GluR circuit and pathogenetic mechanism of VD. In this study, the change of AMPA receptor, NMDA receptor on neuron cellular membrane, and the influence of [Ca2+]i, CaMPKⅡ, CREB in neurons and the the contribution on learning and memory were observed as well. The study results provide us with some clues of further exploration of glutamate receptor circuit and calcium signal transduction mechanism in VD animal model. In the mean while, it is expected that our study may offer some evidence of treating VD with NBP.
Methods:
1 The VD model of mice were established for ischemia (20 min)- reperfusion (10 min) thrice by ligating the bilateral common carotid arteries. The changes of behavior were observed through the step-down avoidance test and water maze test. The hippocampal pathologic changes in the mice with VD was observed through HE staining.
2 The changes of AMPA-GluR2 protein of hippocampus and that of the level of mRNA expression of each group of mice were measured with immunohistochemistry and reverse transcription PCR (RT-PCR).
3 The expressions of NMDA receptors NR2B in neurons of hippocampus of every group mice were observed by immunohistochemistry technique. RT-PCR technique was used to measure mRNA expression of NMDA receptor in hippocampal neurons.
4 The changes of [Ca2+]i of hippocampal neurons of the mice in each group were measured with flow cytometry. Western blot was applied to the measurement of the expression of CaMKII in hippocampal neurons of each mouse in the experiment. RT-PCR technique was used to measure the mRNA expression of CaMKII in hippocampal neurons of all mice in the experiment.
5 The expression levels of CREB in the hippocampal of the mice in each group were analyzed with Western-blot. And RT-PCR technique was used to measure mRNA expression of CREB in hippocampal neurons of the mice in each group.
Results:
1 The evaluation of behavior of VD model and its pathologic features of the hippocampus
The mice were divided into the control group, sham-operated group, VD model group, NBP treating group and NBP precaution treating group. The mice were subjected for ischemia (20 min)-reperfusion (10 min) thrice by ligating the bilateral common carotid arteries to establish the VD model. Beginning on day 29 and 30 day after operation, the functions of learning and memory of each mouse in each group were tested through step-down test and water maze test.
1.1 The test of learning of mice
On day 29 after operation, the learning of every group were tested by step-down test and water maze test respectively. Response time (sec), error times (number/5min) in step-down test, and swimming time (sec) and error times (number/3min) in water maze test were used to evaluate the achivements of learning.
The results of step-down test revealed that:⑴The response time in learning phase of VD model group (89.41±12.08)s prolonged distinctly (P<0.01) compared with the response time of sham-operated group(48.76±9.24)s.⑵The response time of both the NBP treating group (59.68±10.27)s and the NBP precaution treating group (57.23±10.05)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of VD control group (3.74±0.42) increased notably (P<0.01), compared with the error time of sham-operated group (1.23±0.27).⑷The error times of both the NBP treating group (1.52±038) and the NBP precaution treating group (1.48±0.31) decreased notably (P<0.01), compared with VD model group.⑸The learning of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).
The results of water maze test revealed that:⑴The swimming time in learning phase of the VD model group (145.80±13.29)s prolonged significantly(P<0.01), compared with sham-operated group (87.32±13.46)s.⑵The swimming time of both the NBP treating group (101.89±18.38)s and the NBP precaution treating group (99.18±11.25)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of the VD model group (34.60±5.92) increased notably (P<0.01), compared with and sham-operated group (16.88±4.19).⑷The error times of both the NBP treating group (18.71±4.35) and the NBP precaution treating group (17.29±4.82) decreased remarkably (P<0.01), compared with VD model group.⑸The learning of the sham-operated group, the NBP treating group and the NBP precaution treating group had no distinct difference (P>0.05).
The results above suggest that the learning ability of VD mice reduced and NBP may improve their learning. 1.2 The test of memory of mice
On day 30 after the operations, the memory of the mice in every group was tested by step-down test and water maze test. The results of latency time (sec) and error times (number / 5 min) in step-down test and that of swimming time (sec) and error times (number / 3 min) are the memory.
The results of step-down test revealed that:⑴The latency time in memory phase of VD model group (78.67±19.17)s shortened remarkably (P<0.01), compared with the latency time of sham-operated group (148.92±20.83)s.⑵The latency time of the NBP treating group (131.41±18.82)s and the NBP precaution treating group (128.57±18.29)s prolonged significantly (P<0.01), compared with VD model group.⑶The error times in memory phase of VD model group (1.72±0.13) increased notably (P<0.01), compared with the error time of sham-operated group (0.32±0.05).⑷The error times of both the NBP treating group (0.54±0.08) and the NBP precaution treating group (0.50±0.07) decreased notably (P<0.01), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).
The results of water maze test revealed that:⑴The swimming time in memory phase of the VD model group (137.40±11.76)s prolonged remarkably (P<0.01), compared with sham-operated group (67.82±8.79)s.⑵The swimming time of both the NBP treating group (88.73±9.53)s and the NBP precaution treating group (83.87±8.62)s shortened significantly (P<0.05), compared with VD model group.⑶The error times in memory phase of the VD model group (20.90±4.25) increased remarkably (P<0.05), compared with sham-operated group (9.57±2.91).⑷The error times of both the NBP treating group (11.17±3.04) and the NBP precaution treating group (10.27±2.83) decreased significantly (P<0.05), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).
These results suggested that the memory of VD mice decreases and NBP may improve their memory.
1.3 The pathologic changes of the hippocampus of the VD mice
The observations under the light microscope show:⑴The arrangement of the pyramydal neurons in the hippocampal CA1 area in sham-operated group were tight and in order with the nucleus being large and round and its neucleolus being evident.⑵In the VD model group, the pyramydal neurons in the hippocampal CA1 decreased and had no clear arrangement. There was empty dye area surrounding the neurons which had condensed nucleus dense cytoplast and small body. In addition, there was infiltration of inflammatory cells.⑶In the NBP treating group and the NBP precaution treating group, the condensed nucleus dense cytoplast and small body decreased apparently and there was no infiltration of inflammatory cells.
2 The level of AMPA-GluR2 receptor in the hippocampal CA1 area of VD mice and the effect of NBP .
2.1 Staining of immunohistochemistry of AMPA-GluR2.
The mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made and AMPA-GluR2 was stained through immunohistochemistry. And then the statistic study of each group (surface density Sv showing their numbers) was conducted. The result revealed that:⑴The AMPA-GluR2 positive neurons of hippocampus CA1 area in VD model group (27.69±5.15) reduced distinctly (P<0.05), compared with sham-operated group (124.37±7.48).⑵The AMPA-GluR2 positive neurons of hippocampus CA1 area of the mice in both the NBP treating group (88.57±10.98) and the NBP precaution treating group (94.42±12.37) increased (P<0.05), compared with VD model group.⑶The NBP treating group, the NBP precaution treating group and the sham-operated group had no distinct difference (P>0.05).
These suggested that the lower level AMPA-GluR2 of hippocampus CA1 area might participate in the pathogenesis of VD and NBP could prevent the level AMPA-GluR2 of hippocampus CA1 area of VD from decreasing and hence improve their learning and memory ability.
2.2 RT-PCR of AMPA-GluR2 mRNA
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of GluR2 mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of AMPA-GluR2 mRNA expression in VD model (0.28±0.07) reduced notably (P<0.01), compared with the magnitude value of GluR2 mRNA expression in sham-operated group (0.96±0.21).⑵The level of AMPA-GluR2 mRNA expression in NBP treating group (0.71±0.15) and NBP precaution treating group (0.75±0.18) elevated remarkably (P<0.01), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of AMPA-GluR2 mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of AMPA-GluR2 mRNA to reduce and improve their learning ability and memory.
3 The level of NMDA-2B receptor in the hippocampal CA1 area of VD mice and the effect of NBP.
3.1 Immunohistochemistry of NMDA-2B receptor
The mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made and NMDA-2B receptor was dyed through immunohistochemistry; and made the statistics of group (surface density Sv showing their numbers).The result revealed that:⑴NMDA-2B receptor positive neurons of hippocampus CA1 area in VD model group (29.70±2.09) reduced distinctly (P<0.05), compared with sham-operated group (149.27±8.67).⑵NMDA-2B receptor positive neurons of hippocampus CA1 area in NBP treating group (100.53±10.00) and NBP precaution treating group (108.51±7.28) increased (P<0.05), compared with VD model group.⑶NBP treating group, NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower level NMDA-2B receptor of hippocampus CA1 area might participate in the pathogenesis of VD; but NBP could prevent the level NMDA-2B receptor of hippocampus CA1 area of VD to reduce and improve their learning ability and memory.
3.2 RT-PCR of NMDA-NR2B receptor mRNA
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of NMDA-2B receptor mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of NMDA-2B receptor mRNA expression in VD model (0.22±0.04) reduced notably (P<0.05), compared with the magnitude value of NMDA-2B receptor mRNA expression in sham-operated group (0.71±0.18).⑵The level of NMDA-2B receptor mRNA expression in NBP treating group (0.54±0.11) and NBP precaution treating group (0.59±0.20) elevated notably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of NMDA-2B receptor mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of NMDA-2B receptor mRNA to reduce and improve their learning ability and memory.
4 Calcium signal transduction pathway in hippocampal neuron of mice with VD and the effect of NBP
4.1 The resting [Ca2+]i in the hippocampus of VD mice and the effect of NBP
The mice were executed quickly after anaesthetized by 10% chloral hydrate solution and their hippocampuses were extracted in low temperature operation. They were separated through mechanical method. Thus the single cell suspension sample of hippocampus was prepared. We observed the change characteristic of the resting [Ca2+]i by flow cytometryanalytical technique and intervention of NBP. The result indicated that:⑴The concentration of intracellular [Ca2+]i in hippocampus of VD model group (1.92±0.35) increased notably (P<0.01), compared with the concentration of intracellular [Ca2+]i in sham-operated group (1.29±0.32);⑵the intracellular [Ca2+]i in hippocampus of NBP treating group (1.34±0.28) and NBP precaution treating group(1.33±0.41) decreased notably (P<0.01),compared with VD model group;⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the calcium over-loading in hippocampus cells of VD might participate in its pathogenesis; but NBP could restrain the change and improve the learning ability and memory.
4.2 Western blot of CaMPKⅡin hippocampus of VD
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. The level of CaMPKⅡexpression in hippocampus was examined through Western blot method. The result indicated that:⑴The level of CaMPKⅡexpression in VD model group (0.30±0.07) reduced notably (P<0.01), compared with the level of CaMPKⅡexpression in sham-operated group (1.06±0.14).⑵The level of CaMPKⅡexpression in NBP treating group (0.94±0.13) and NBP precaution treating group (0.98±0.27) elevated notably (P<0.01), compared with the level of CaMPKⅡexpression in VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CaMPKⅡmight participate in the pathogenesis of VD; but NBP could prevent the expression of CaMPKⅡto reduce and improve their learning ability and memory.
4.3 RT-PCR of CaMPKⅡmRNA
The mice were executed quickly through decollation and their hippocampus were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of CaMPKⅡmRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of CaMPKⅡmRNA expression in VD model (0.31±0.06) reduced notably (P<0.05), compared with the magnitude value of CaMPKⅡmRNA expression in sham-operated group (0.92±0.15).⑵The level of CaMPKⅡmRNA expression in NBP treating group (0.78±0.11) and NBP precaution treating group (0.82±0.14) elevated notably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CaMPKⅡmRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of CaMPKⅡmRNA to reduce and improve their learning ability and memory.
5 The level of CREB in hippocampus of VD and the effect of NBP
5.1 Western blot of CREB in hippocampus of VD
The mice were executed quickly through decollation and their hippocampus were extracted in low temperature operation. The level of CREB expression in hippocampus was examined through Western blot method. The result indicated that:⑴The level of CREB expression in VD model group (0.43±0.06) reduced remarkably (P<0.05), compared with the level of CREB expression in sham-operated group (1.37±0.21).⑵The level of CREB expression in NBP treating group (1.06±0.12) and NBP precaution treating group (0.12±0.14) elevated remarkably (P<0.05), compared with the level of CREB expression in VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CREB might participate in the pathogenesis of VD; but NBP could prevent the expression of CREB to reduce and improve their learning and memory.
5.2 RT-PCR of CREB mRNA
The mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of CREB mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of CREB mRNA expression in VD model (0.29±0.04) reduced remarkably (P<0.05), compared with the magnitude value of CREB mRNA expression in sham-operated group (0.98±0.17).⑵The level of CREB mRNA expression in NBP treating group (0.82±0.09) and NBP precaution treating group (0.84±0.11) elevated remarkably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CREB mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of CREB mRNA to reduce and improve their learning and memory.
Conclusions:
1 The VD mice model which had the cognization impairment in primary of damage of learning and memory was successfully established. The VD model is the perfect animal model and feasible to the further study of VD. The hippocampus CA1 area in the VD mice model had the pathologic changes.
2 The level of AMPA-GluR2 and its mRNA in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the reduction of the expression of AMPA receptor might participate in the pathogenesis of VD.
3 The level of NMDAR-2B and its mRNA in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the lower expression of NMDA-NR2B might participate in the pathogenesis of VD.
4 The imbalance of calcium signal transduction pathway resulted from the increase of the resting [Ca2+]i in VD mice hippocampus and the reduction of CaMPKⅡin VD mice hippocampus, which might be one part of the molecular pathogenesis of VD.
5 The reduction of the protein level of CREB in VD mice hippocampus consistent with the decline of learning and memory suggested that the reduction of the protein level of CREB might participate in the moleculear pathogenesis of VD.
6 NBP could resist the over-loading of calcium in hippocampus of VD and prevent the expression of CaMPKⅡand AMPA receptor and NMDA receptor and the level of CREB from reducing. These further improve the calcium signal transduction pathway and ameliorate the pathologic changes in hippocampual CA1 area in the mice with VD. This was in accord to their better learnig and memory. All mentioned above suggest that the medicine may improve the pathogenesis of VD.
引文
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