左旋丁苯酞对血管性痴呆小鼠认知功能的影响及对PI3K/Akt信号转导通路的作用
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摘要
血管性痴呆(vascular dementia，VaD)是指各种脑血管疾病引起的脑功能障碍而产生的获得性智能损害综合征。VaD以学习、记忆功能损害为主要症状，可伴有运动、语言、视空间及人格障碍。随着社会人口日益老龄化和脑卒中后生存率的提高，由脑卒中造成的精神及智能损害日益突出，给社会和家庭带来沉重负担。脑卒中后缺血-再灌注是诱发VaD的主要原因之一。
脑缺血可以诱导脑梗死或选择性脑神经元死亡，当缺血发生在与学习、记忆密切相关的区域时，即可导致VaD的发生。海马是学习记忆的关键部位，同时又是缺血缺氧的敏感部位。但导致海马CA1区神经细胞死亡的调控机制目前不祥。细胞的死亡方式有2种：细胞坏死和程序性细胞死亡，其中程序性细胞死亡有Ⅰ型(细胞凋亡)和Ⅱ型(自噬性细胞死亡)两种。磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt, phosphoinositide3kinase/Akt)信号转导途径是一个经典抗凋亡、促存活的信号转导途径。它的激活在神经细胞，尤其在缺血缺氧神经元损伤中起着重要作用，并逐渐受到广泛重视。近年来发现其与自噬的调节密切相关。PI3K/Akt信号转导通路在脑缺血和神经退行性变中的作用已被广泛研究，已有研究发现PI3K/Akt信号转导通路与学习认知有关。但PI3K/Akt信号转导通路在VaD发病机制中的作用尚不清楚。因此，研究PI3K/Akt信号通路与VaD的关系，对于VaD的基础及临床研究，具有重要的指导意义。
丁苯酞(3-n-butylphthalide，NBP)是中国医学科学院药物研究所研制开发的一类新型抗脑缺血药物，对急性缺血性脑卒中有明显疗效。既往研究发现丁苯酞具有多靶点抗脑缺血作用，能改善缺血区局部脑血流，缩小脑梗死面积，改善线粒体功能，抑制自由基的产生，抑制炎症反应，抑制谷氨酸释放，减轻脑缺血所致脑水肿，抗血栓形成和抗血小板聚集，并且有研究证实能抑制神经细胞凋亡，保护缺血缺氧引起的神经细胞损伤，神经功能缺失及脑缺血记忆障碍。而这些机制均与血管性痴呆的发生有关。近年来，左旋丁苯酞(l-NBP)和右旋丁苯酞(d-NBP)已经被拆分，并且将拆分后的l-NBP和d-NBP进行了对比研究后发现，l-NBP比d-NBP的作用更强，而且还发现在对神经细胞凋亡研究中d-NBP的存在降低了dl-NBP的作用，从而证明d-NBP有拮抗l-NBP作用，因而开发l-NBP的意义更为重。目前该药应用于VaD的实验研究少有报道，更无其与PI3K/Akt信号转导通路之间关系的报道。基于此，本文拟通过观察PI3K/Akt信号转导通路在VaD小鼠海马组织中的变化，以及l-NBP对VaD的影响，以探讨PI3K/Akt信号转导通路在VaD的发病机制中的作用及l-NBP的作用机制。
第一部分：血管性痴呆小鼠模型的建立和左旋丁苯酞的防治作用
目的：观察脑反复缺血再灌注后C57BL/6小鼠行为学的改变和海马病理学特征变化，并观察l-NBP对脑反复缺血再灌注诱导的VaD小鼠空间学习记忆障碍的影响。
方法：
将C57BL/6小鼠随机分为假手术组（sham组）、模型组（IR组，造模后植物油灌胃6ml.kg-1.d-1，连续28天）、左旋丁苯酞预防性治疗组（Pre-l-NBP组，造模前7天左旋-丁苯酞灌胃30mg.kg-1.d-1，连续7天）、左旋丁苯酞低剂量治疗组（Low-l-NBP组，造模后左旋-丁苯酞灌胃30mg.kg-1.d-1，连续28天)和左旋丁苯酞高剂量治疗组（High-l-NBP组，造模后左旋-丁苯酞灌胃60mg.kg-1.d-1，连续28天）；除假手术组外均给予双侧颈总动脉反复三次缺血-再灌注方法建立小鼠VaD模型，假手术组仅暴露颈总动脉不结扎。造模4周后通过Morris水迷宫试验和跳台试验进行学习和记忆成绩测试，观察其行为学改变；应用琉堇染色观察海马病理学特征变化。
结果：
1空间学习记忆能力的改变——Morris水迷宫实验
在定位航行实验中，IR组小鼠的寻台潜伏期明显长于Sham组小鼠[F(1,27)=30.09，p<0.01]。l-NBP各组小鼠寻台潜伏期与IR组小鼠相比明显缩短[F(3,50)=10.315，P<0.01]。在实验的第2-5天，sham组小鼠、Pre-l-NBP组小鼠寻台潜伏期明显短于IR组小鼠（P<0.05, P<0.01）；在实验的第3-5天，Low-l-NBP组和High-l-NBP组小鼠寻台潜伏期明显短于IR组小鼠（P<0.05，P<0.01）。
在空间探索试验中，与IR组小鼠相比，Sham组小鼠对目标象限表现出明显的偏爱（P<0.01)。与IR组小鼠相比，High-l-NBP和Pre-l-NBP组小鼠对目标象限表现出明显的偏爱(P<0.05, P<0.01)。
2被动逃避反应的改变——跳台实验
2.1下台潜伏时间：IR组与Sham组相比小鼠的首次跳下平台的潜伏时间明显缩短(P<0.01)，提示小鼠记忆形成功能受损；Low-l-NBP组、High-l-NBP组和Pre-l-NBP组与IR组相比跳下平台的潜伏时间明显增加(P<0.05)，提示l-NBP可改善小鼠记忆形成功能。
2.2错误次数：IR组与Sham组相比小鼠跳下平台的错误次数明显增多(P<0.05)，Low-l-NBP组、High-l-NBP组和Pre-l-NBP组与IR组相比跳下平台的错误次数明显减少，但无统计学意义(P>0.05)。
3小鼠海马的组织病理学改变
硫堇染色显示，sham组小鼠海马CA1区锥体神经元排列整齐、致密，细胞形态完整，胞核饱满，核仁清晰。IR组小鼠海马CA1区可见片段性的锥体细胞缺失，部分细胞形态发生明显改变，可见胞体缩小，形态不规则，呈梭型或多角形，胞核固缩、浓染，核仁模糊不清或消失。与IR组相比，Low-l-NBP组、High-l-NBP组和Pre-l-NBP组小鼠海马CA1区的锥体细胞缺失明显减轻。
结论：
1本实验通过双侧颈总动脉反复三次缺血-再灌注方法成功的建立了C57BL/6小鼠VaD模型。预防性和治疗性应用l-NBP可以减轻VaD小鼠学习和记忆障碍，尤以预防性应用l-NBP效果显著。
2反复缺血再灌注4周后小鼠海马组织CA1区出现神经细胞片段性缺失、排列紊乱、神经元密度降低等改变。预防性和治疗性应用l-NBP可以减轻VaD小鼠海马组织病理学变化，进而改善其学习和记忆能力。
第二部分：PI3K激动剂及抑制剂对血管性痴呆小鼠认知功能的影响
目的：观察PI3K激动剂及抑制剂对VaD小鼠认知功能的影响。
方法：将C57BL/6小鼠随机分为假手术组（sham组）、激动剂组(IR+IGF-1组，手术前及手术24小时后经侧脑室给予IGF-12ul/mouse)、激动剂溶剂对照组(IR+DW组，手术前及手术24小时后经侧脑室给予去离子水2μl/mouse)、抑制剂组(IR+LY294002组，手术前及手术24小时后经侧脑室给予LY29400210nmol/mouse)和抑制剂溶剂对照组(IR+DMSO组，手术前及手术24小时后经侧脑室给予同等体积(2μl)的溶剂)；除假手术组外均给予双侧颈总动脉反复三次缺血-再灌注方法建立小鼠VaD模型，侧脑室插管给予相应试剂；假手术组仅暴露颈总动脉不结扎。造模4周后通过Morris水迷宫试验进行学习和记忆成绩测试，观察其行为学改变；应用Western Blot方法测定各组小鼠海马组织中Akt及p-Akt Ser473蛋白的变化。
结果：
1空间学习记忆能力的改变——Morris水迷宫实验
在定位航行实验中，Sham组和IR+IGF-1组小鼠的寻台潜伏期明显短于IR+DW组、IR+LY294002组、IR+DMSO组小鼠[F(4,45)=9.702，P<0.01]。
在空间探索试验中，与IR+DW组、IR+LY294002组、IR+DMSO组小鼠相比，sham组和IR+IGF-1组小鼠对目标象限表现出明显的偏爱(P<0.01)。
2Akt、pAkt Ser473在海马组织中表达的变化
术后4周Sham组、IR+DW组、IR+IGF-1组、IR+DMSO组和IR+LY294002组之间Akt总蛋白表达无明显差异(P>0.05)；IR+DW组、IR+IGF-1组、IR+DMSO组和IR+LY294002组的p-Akt Ser473的蛋白表达明显高于sham组（P<0.01, P<0.05）。IR+IGF-1组p-Akt的蛋白表达明显高于IR+DW组、IR+DMSO组和IR+LY294002组（P<0.05）。IR+LY294002组p-Akt的蛋白表达低于IR+DMSO组（P<0.05）。
结论：
PI3K激动剂IGF-1可以减轻VaD小鼠学习记忆功能障碍，促进海马组织中p-Akt Ser473表达。其机制可能为IGF-1激活PI3K/Akt信号转导通路，通过调节神经细胞的凋亡和自噬，从而改善VaD小鼠的认知功能。第三部分：血管性痴呆小鼠海马组织中Akt、p-Akt、mTOR、p-mTOR表达水平变化及左旋丁苯酞的影响
目的：观察血管性痴呆小鼠海马组织中Akt、p-Akt、mTOR、p-mTOR表达水平的变化及l-NBP的干预作用。
方法：造模4周后采用Western-Blot方法测定各组小鼠海马组织中Akt、p-Akt Ser473、mTOR和p-mTOR Ser2448蛋白的变化；采用免疫组化方法测定各组小鼠海马组织中p-Akt Ser473蛋白的变化。
结果：
1左旋丁苯酞对Akt、p-Akt Ser473蛋白表达的影响
脑反复缺血再灌注术后4周各组之间Akt总蛋白表达无明显差异(P>0.05)；与sham组相比，IR组、Pre-l-NBP组、Low-l-NBP组和High-l-NBP组p-Akt Ser473蛋白表达明显增强（P<0.01）；与IR组相比，Pre-l-NBP组、Low-l-NBP组和High-l-NBP组三组p-Akt Ser473的表达明显增强（P<0.01）。
2左旋丁苯酞对mTOR、p-mTOR Ser2448蛋白的表达的影响：
脑反复缺血再灌注术后4周各组之间mTOR总蛋白表达无明显差异(P>0.05)；IR组海马组织p-mTOR Ser2448的蛋白明显高于sham组（P<0.01）。Pre-l-NBP组、Low-l-NBP组和High-l-NBP组三组海马组织p-mTOR Ser2448的蛋白表达明显高于IR组（P<0.01, P<0.05）。
结论：
1脑反复缺血再灌注4周后，VaD小鼠海马组织中p-Akt Ser473、p-mTOR Ser2448的表达明显增多。提示脑反复缺血再灌注使PI3K/Akt通路活化，Akt和mTOR活化可抵抗缺血引起的损伤，在神经元的存活中发挥着重要的作用。
2左旋丁苯酞可促进VaD小鼠海马组织中p-Akt Ser473和p-mTORSer2448的表达。提示该药可能通过激活PI3K/Akt通路，促进p-Akt Ser473和p-mTOR Ser2448的表达，从而发挥神经保护作用，改善小鼠认知功能障碍和神经损害。
第四部分：血管性痴呆小鼠海马组织中自噬因子LC3B和Beclin1表达水平变化及左旋丁苯酞的影响
目的：观察血管性痴呆小鼠海马组织中LC3B和Beclin1蛋白及mRNA表达的变化及l-NBP的干预作用。
方法：造模4周后应用Western Blot方法测定各组小鼠海马组织中LC3B蛋白表达的变化，应用FQ RT-PCR方法测定各组小鼠海马LC3B和Beclin1mRNA表达的变化
结果：
1LC3B蛋白的表达情况：
术后4周IR组海马组织LC3B蛋白表达明显高于sham组、Pre-l-NBP组和High-l-NBP组（P<0.01）。
2LC3B、Beclin1的mRNA表达水平
IR组小鼠海马LC3B mRNA和Beclin1mRNA的表达比Sham组明显增加（P<0.05）；Pre-l-NBP组、Low-l-NBP组和High-l-NBP组三组海马组织中LC3B mRNA和Beclin1mRNA的表达无组间差异（P>0.05），与Sham组相比无统计学差异（P>0.05），但较IR组明显下降（P<0.01,P<0.05）。
结论：
1脑反复缺血再灌注4周后，小鼠海马组织LC3B蛋白、LC3B mRNA和Beclin1mRNA表达均显著升高，与小鼠行为学改变和神经元损伤基本一致。
2左旋丁苯酞降低小鼠海马组织LC3B蛋白、LC3B mRNA和Beclin1mRNA表达（与其上游抑制物p-mTOR的表达相反）。提示l-NBP可能通过激活PI3K/Akt通路抑制自噬，从而减少VaD小鼠海马神经元损伤，减轻认知功能障碍。
第五部分：血管性痴呆小鼠侧脑室室管膜扫描电镜的变化特征及左旋丁苯酞的影响
目的：观察VaD小鼠侧脑室脉络丛上皮细胞和室管膜细胞的超微结构变化，以及l-NBP的影响。
方法：通过扫描电镜观察造模4周后sham组、IR组和High-l-NBP组小鼠侧脑室脉络丛上皮细胞和室管膜细胞超微结构的变化。
结果：
1小鼠侧脑室脉络丛上皮细胞超微结构特点
Sham组侧脑室脉络丛上皮细胞细胞胞体形状规则，细胞之间界限清晰，微绒毛密布上皮细胞游离面，较粗短，细胞表面可见大小不一的分泌泡。IR组小鼠侧脑室脉络丛上皮细胞形状变得极不规则，有的细胞有突起，有的呈沟回样皱缩，有的细胞表面光滑，并可见大量的分泌泡，分泌物增多，细胞间界限不清，细胞表面呈泥沙样堆积，脉络丛上皮细胞上微绒毛明显减少甚至消失。High-l-NBP组小鼠侧脑室脉络丛上皮细胞排列尚规律，细胞间界限较清晰。
2小鼠侧脑室室管膜表面超微结构特点
sham组小鼠可见室管膜上皮细胞表面带有大量的纤毛和微绒毛，纤毛，粗细较为均匀，在纤毛的空隙处，分布着众多的微绒毛。IR组小鼠室管膜上皮细胞表面可见纤毛稀疏、倒伏，部分粘附呈缕。部分纤毛表面粘附絮状物，可见纤毛和微绒毛被“淤泥状”分泌物呈片状覆盖。High-l-NBP组小鼠室管膜上皮细胞表面的纤毛和微绒毛较IR组明显增多。
结论：
1VaD小鼠侧脑室脉络丛上皮细胞及室管膜细胞出现超微结构异常，提示脉络丛上皮细胞和室管膜的异常可能参与了VaD的发病机制。
2左旋丁苯酞可以减轻VaD小鼠侧脑室脉络丛上皮细胞室管膜的超微结构异常，从而改善认知功能障碍。
As an acquired syndrome of cognitive impairment, vascular dementia(VaD), which is caused by various kinds of cerebral vascular disease,demonstrates mainly as learning and memory dysfunction, accompanied withthe possible disorder of motion, tongue, direction and personality. VaD, as thesecond most common form of dementia after Alzheimer's disease in the elderpeople, has attracted much attention in recent years. Recently, there isemerging epidemiological evidence that VaD is likely the leading cause ofdementia in Asian population. At present, the pathogenesis of VaD remainsunclear, but increasing evidence suggests that VaD is associated with a groupof diverse pathologies affecting the cerebrovascular circulation. One ofimportant mechanisms in VaD is the pathophysiology of ischemia/reperfusion(I/R) including excitotoxicity, oxidative stress and inflammatory reactions.
Cerebral ischemia can induce the death of cerebral infarction or selectiveneurons in the brain, when ischemia occurred in associated with learning,memory areas, can lead to the occurrence of VaD. Post-IR neurons exhibit twoobvious forms of cell death: necrosis and programmed cell death whichinclude apoptosis and autophagy. Previous researches showed that there mightbe a continuum between autophagy and apoptosis. Hence the extent of thesalvage of apoptosis and autophagy might contribute to the outcome of thepathological process. Recently, accumulating evidence has shown that thephosphatidylinositol-3kinase/Akt (PI3K/Akt) cell signaling pathway takespart in the apoptosis after IR. The PI3K/Akt signal transduction pathway is aclassic anti-apoptosis pathway, promoting the survival signal transduction. Itsactivation in the nerve cells, especially in hypoxic ischemic neuronal injurycells, plays an important role, and has received extensive attention. In recentyears, it found to be closely related with the regulation of autophagy. The neuroprotective role of PI3K/Akt in cerebral ischemia has been widely studied.PI3K/Akt survival signaling pathway regulate cell survival and are importantin the pathogenesis of degenerative diseases, activation of the Akt/proteinkinase B (PKB) kinase pathway can be neuroprotective after stroke throughblocking neuronal death after stroke. Akt/mTOR signal pathway inducedNF-κB (nuclear factor kappa B) activation pathway and Bcl-2and Baxbalance adjustment play an important role in nerve cells inflammatoryresponses and apoptosis of ischemic cerebrovascular disease. Akt may rescuecells from apoptosis by inhibiting the Bax-dependent apoptosis pathwaythrough a forkhead box transcription factor.
3-n-butylphthalide is successfully synthesized to treating ischemic strokewith independent intellectual property rights of a class of chemical drugs inour country. The first is the L-isomer extracted originally from celery seed byYANG Jun-shan, after the artificial synthesis of racemes, also known asbutylphthalide. As the yellow oily liquid, butylphthalide has a celery flavor.3-n-butylphthalide (NBP) has three different stereo isomers known as l-, dl-,and d-NBP. A large number of studies have demonstrated that dl-NBP is ableto be a neuroprotective drug for the treatment of ischemic stroke throughmultiple mechanisms such as improving energy metabolism, reducingoxidative damage, improving microcirculation in arterioles, decreasingneuronal apoptosis, improving mitochondrial function, and inhibitinginflammation. Although the positive effects of NBP on cerebral infarct andAlzheimer’s disease have been verified in clinic trials and animal researches,few studies has investigated whether NBP could be beneficial as a treatmentfor VaD. A previous study discovered that only l-NBP ameliorated thecognitive impairment induced by chronic cerebral hypoperfusion while d-anddl-NBP did not show significant beneficial effects. Furthermore, l-NBPreduces β-amyloid-induced cell death in neuronal cell cultures, and improvescognitive performance in an animal model of Alzheimer’s disease. However,the effects of l-NBP in this area are still not clear.
Therefore, this study set out to examine whether treating with l-NBP and pretreating with l-NBP have the ability to improve cognitive impairment inVaD mice induced by cerebral repetitive IR. Furthermore this study explorewhether activating PI3K/Akt cell signaling pathway is one of the mechanismsof l-NBP providing the protective effect on VaD mice.
Part1: The establishment of the vascular dementia mice model and theeffects of l-3-n-butylphthalide
Objectives: To observe the behavioral changes and hippocampalpathology changes4weeks after cerebral repetitive ischemia/reperfusion inC57BL/6mice, and to observe if l-NBP can improve learning and memoryimpairment induced by cerebral repetitive ischemia/reperfusion.
Methods: C57BL/6mice were randomly divided into five groups: thesham group, IR group(vegetable oil was given daily for28consecutive daysafter IR), Pre-l-NBP group (30mg/kg l-NBP was administered daily for7daysprior to IR), Low-l-NBP group (30mg/kg l-NBP was given daily for28consecutive days after IR) and High-l-NBP group (60mg/kg l-NBP was givendaily for28consecutive days after IR). Mice, except the sham group, weresubjected for continuously three repeated times ischemia-reperfusion throughthe ligation of the bilateral common carotid arteries. In the sham group,protocols were the same as the model group but bilateral common carotidswere only exposed. The capability of learning and memory of mice wereinvestigated by the Morris water maze test and the step-down test, thepathological change of hippocampus in each group was observed by thioninestaining.
Results:
1Learning and memory ability of Morris water maze test:
1.1L-NBP significantly ameliorated learning impairment induced by cerebralrepetitive IR.
For the purpose of evaluating whether the mice cognitive function wasaffected after the oral administration of l-NBP, we used the MWM test toevaluate the mice abilities of spatial learning acquisition and memoryretention. There was a marked treatment effect [F(3,50)=10.315，P<0.01] on escape latency, suggesting that l-NBP was protective against impaired spatiallearning in the cerebral repetitive IR mice. The escape latency of IR group wasevidently longer than that of the sham group (P<0.01), suggesting that cerebralrepetitive IR successfully induced learning deficit. Compared with IR group,after treated with30mg/kg l-NBP or60mg/kg l-NBP, the escape latency wasmarkedly decreased from day3to day5(P<0.01, P<0.05). And comparedwith IR group, after pretreated with30mg/kg l-NBP, the escape latency wasmarkedly decreased from day2to day5(P<0.01). These results indicated thatoral administration of l-NBP, especially pretreatment with l-NBP significantlyrescues learning impairment caused by cerebral repetitive IR
1.2L-NBP significantly rescued memory deficits induced by cerebralrepetitive IR.
To investigate the effects of l-NBP on memory deficits caused bycerebral repetitive IR, we conducted a spatial probe test after the5days oftraining in the water maze. In the test, we discovered that the sham group, thePre-l-NBP group and the High-l-NBP mice were able to show significantpreference for the target quadrant compared with IR group mice (P<0.05,P<0.01).
2Learning ability of step-down test:
2.1Latency time
The latency time of IR group are significant shorter than those of shamgroup (P<0.01), that suggest there are impairment of memory formation in IRgroup mice. The latency time of Low-l-NBP, High-l-NBP and Pre-l-NBPgroup are marked longer than those of IR group (P<0.05), that suggest l-NBPcan improve impairment of memory formation.
2.2The number of error
The number of error of IR group were much more than those ofsham-operated group (P<0.05). The number of error of l-NBP-treated groupwere lower than those of model group, but without statistical significance (P>0.05).
3Neuropathological evaluation
In sham mice, the pyramidal neurons in the CA1hippocampus werearranged in sequence with4-5cell layers, intact lineament, full nuclei andclear nucleoli. In the IR group, part neurons died4weeks after IR, which wererepresented by increases of HG (P<0.05) and by significant decreases of ND(P<0.01) compared with sham group. And, there was significant improvementin l-NBP groups as compared to those in the IR group (P<0.05).
Conclusions:
1This study has successfully established C57BL/6VaD mouse model bythree repeated times ischemia-reperfusion through the ligation of the bilateralcommon carotid arteries. Mice, treated with l-NBP, especially pre-treated withl-NBP significantly attenuated learning and memory impairment induced bycerebral repetitive IR.
2L-NBP might improve learning and memory ability of mice byattenuating the injury of hippocampus neurons.
Part2: The effects of PI3K agonists and inhibitors on cognitive functionin mice with vascular dementia
Objectives：To observe the effects of PI3K agonists and inhibitors oncognitive function in VaD mice.
Methods: C57BL/6mice were equally and randomly divided into fivegroups: the sham group, IR+IGF-1group, IR+DW group, IR+LY294002group and IR+DMSO group. Mice, except the sham group, were subjected forcontinuously three repeated times ischemia-reperfusion through the ligation ofthe bilateral common carotid arteries. In the sham group, protocols were thesame as the model group but bilateral common carotids were only exposed.The capability of learning and memory of mice were investigated by theMorris water maze test at4weeks after operation. The expression of Akt andp-Akt in hippocampus were studied by immunohistochemistry and WesternBlot technique.
Results:
1Learning and memory ability of Morris water maze test
The escape latency of IR+DW group, IR+LY294002group and IR+DMSO group was evidently longer than that of the sham group andIR+IGF-1group[F(4,45)=9.702，P<0.01].
Compared with IR+DW, IR+LY294002and IR+DMSO group mice, thesham and IR+IGF-1group mice were able to show significant preference forthe target quadrant (P<0.01).
2Protein expression of LC3B in hippocampus by Western blot
There is no marked change in total Akt among five groups (P>0.05) at4weeks after operation. However, compared with the sham group, p-Akt weresignificantly increased in the IR+DW、 IR+IGF-1、 IR+DMSO andIR+LY294002group(P<0.01). Moreover, IR+IGF-1group mice showed astrong expression of p-Akt compared with the IR+DW、IR+DMSO andIR+LY294002group mice (P<0.05). P-Akt were significantly decreased in theIR+LY294002group compared with those of IR+DMSO group (P<0.05)
Conclusions:
PI3K agonist IGF-1could alleviate the learning and memory impairmentand promote the p-Akt protein expression in hippocampus in VaD mice, andits possible mechanism of improving cognitive function may be activating ofPI3K/Akt signal transduction pathway and regulating apoptosis and autophagyof nerve cells.
Part3: The expression level of Akt, p-Akt, mTOR, p-mTOR protein inhippocampus of VaD mice and the effects of l-NBP.
Objectives： To observe the Akt, p-Akt, mTOR, p-mTOR proteinexpression in hippocampus of VaD mice and the effects of l-NBP.
Methods: The expressions of Akt, p-Akt, mTOR and p-mTOR inhippocampus were studied by measure of Western Blot and the expression ofp-Akt were studied by immunohistochemistry at4weeks after operation.
Results:
1L-NBP induce expression of p-Akt Ser473in the hippocampus of VaD mice.
There is no marked change in total Akt among five groups (P>0.05).However, p-Akt Ser473were significantly increased in the IR group micecompared with those in the sham group (P<0.01). Moreover, Low-l-NBP, High-l-NBP and Pre-l-NBP group mice showed a strong expression of p-Aktcompared with the IR group mice (P<0.01).2L-NBP induce expression of p-mTOR Ser2448in the hippocampus ofcerebral repetitive IR mice.
There is no marked change in total mTOR among five groups (P>0.05).However, p-mTOR Ser2448were significantly increased in the IR group micecompared with those in the sham group (P<0.01). Moreover, Low-l-NBP,High-l-NBP and Pre-l-NBP group mice showed a strong expression ofp-mTOR compared with the IR group mice (P<0.01, P<0.05).
Conclusions:
1There is significant increase in the expression of p-Akt Ser473andp-mTOR Ser2448protein in hippocampal tissue of VaD mice at4weeks postsurgery. It is suggest that activation of the PI3K/Akt signal transductionpathway might play a protect role in resisting neuronal damage after repeatedcerebral ischemia and reperfusion.
2L-NBP can promote the expression of p-Akt Ser473and p-mTORSer2448in hippocampal tissue of VaD mice. The result suggest that l-NBPmay play a neuroprotective role by promoting the expression of p-Akt Ser473and p-mTOR Ser2448, thereby improving cognitive dysfunction and neuronaldamage in mice.
Part4: The expression level of LC3B和Beclin1in hippocampus of VaDmice and the effects of l-NBP
Objectives：To observe the LC3B和Beclin1expression in hippocampusof VaD mice and the effects of l-NBP.
Methods: The expression of LC3B和Beclin1in hippocampus werestudied by Western-Blot technique or FQ RT-PCR technique at4weeks afteroperation.
Results:
1The expression level of LC3B protein in hippocampus by Western blot.
The expression level of LC3B protein were significantly increased in theIR group mice compared with those in the sham group (P<0.01). Howover, in High-l-NBP and Pre-l-NBP group, the protein expression of LC3B were lowerthan those of IR group (P<0.05).
2LC3B and Beclin1mRNA expression in hippocampus by FQ RT-PCR
LC3B and Beclin1mRNA expression were significantly increased in theIR group mice compared with those in the sham group (P<0.05). There are nosignificant difference among Low-l-NBP, High-l-NBP, Pre-l-NBP group andsham group. Howover, LC3B and Beclin1mRNA expression of l-NBPgroups were marked lower than that of IR group (P<0.05).
Conclusions:
1There are significant increase in the expression of LC3B protein, LC3BmRNA and Beclin1mRNA in hippocampal tissue of VaD mice at4weekspost surgery. It suggests that increase in the expressions of LC3B and Beclin1was related to cognitive damage and neuronal damage in VaD mice.
2L-NBP can reduce the expression level of LC3B and Beclin1inhippocampal tissue of VaD mice, thereby improving cognitive dysfunction andneuronal damage in VaD mice.
Part5: Features of ependyma of the lateral ventricle in VaD mice byscanning electron microscopy (SEM) and the effect of l-NBP
Objectives：To observe the change of ependyma cells of the lateralventricle in the VaD mice and the effect of l-NBP.
Methods: To observe the change of ependyma cell and choroid plexusepithelial cell of the lateral ventricle in the VaD mice by scanning electronmicroscopy (SEM) and the effect of l-NBP.
Results:
1Structure features of choroid plexus epithelial cell of the lateral ventricle inmice
The choroid plexus epithelial cell of the lateral ventricle of sham groupare of regular shape, clear boundary between cells. On cell surface, there arefull of bushy and shorter microvilli, and kind of secretory vesicles withdifferent sizes. In IR group, the shape of choroids plexus epithelial cellbecame irregular, and some cells have raised, some are of gyrus shrinkage,
some cell surface are smooth. There were a large number of secretory vesiclesand increased secretions. Sediment is accumulated on the cell surface andintercellular boundaries are not clear. Microvillus decreased significantly evendisappear. The arrangement of choroid plexus epithelial cell in High-l-NBPgroup was in order, with clear boundaries between cells.
2Structure features of ependyma cell of the lateral ventricle in mice
In sham group, there were abundant cilia and microvillus on its unevensurface. The quantities of cilia and microvillus on the uneven surface of in IRgroup reduced obviously. Both of them became twisty and stiff or bent and fellon the surface of cell in lesion regions and ‘mud-like’ secretions flaky out onthe ependymal surface. In High-l-NBP group, the quantities of cilia andmicrovillus on the uneven surface increased obviously compared with IRgroup.
Conclusions:
1The ependyma cells of the lateral ventricle of VaD mice were abnormal,that suggests abnormal ependyma cells may be involved in the pathogenesis ofVaD.
2L-NBP can improve the abnormal ependyma cells of the lateralventricle, thereby improving cognitive dysfunction.
脑缺血可以诱导脑梗死或选择性脑神经元死亡，当缺血发生在与学习、记忆密切相关的区域时，即可导致VaD的发生。海马是学习记忆的关键部位，同时又是缺血缺氧的敏感部位。但导致海马CA1区神经细胞死亡的调控机制目前不祥。细胞的死亡方式有2种：细胞坏死和程序性细胞死亡，其中程序性细胞死亡有Ⅰ型(细胞凋亡)和Ⅱ型(自噬性细胞死亡)两种。磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt, phosphoinositide3kinase/Akt)信号转导途径是一个经典抗凋亡、促存活的信号转导途径。它的激活在神经细胞，尤其在缺血缺氧神经元损伤中起着重要作用，并逐渐受到广泛重视。近年来发现其与自噬的调节密切相关。PI3K/Akt信号转导通路在脑缺血和神经退行性变中的作用已被广泛研究，已有研究发现PI3K/Akt信号转导通路与学习认知有关。但PI3K/Akt信号转导通路在VaD发病机制中的作用尚不清楚。因此，研究PI3K/Akt信号通路与VaD的关系，对于VaD的基础及临床研究，具有重要的指导意义。
丁苯酞(3-n-butylphthalide，NBP)是中国医学科学院药物研究所研制开发的一类新型抗脑缺血药物，对急性缺血性脑卒中有明显疗效。既往研究发现丁苯酞具有多靶点抗脑缺血作用，能改善缺血区局部脑血流，缩小脑梗死面积，改善线粒体功能，抑制自由基的产生，抑制炎症反应，抑制谷氨酸释放，减轻脑缺血所致脑水肿，抗血栓形成和抗血小板聚集，并且有研究证实能抑制神经细胞凋亡，保护缺血缺氧引起的神经细胞损伤，神经功能缺失及脑缺血记忆障碍。而这些机制均与血管性痴呆的发生有关。近年来，左旋丁苯酞(l-NBP)和右旋丁苯酞(d-NBP)已经被拆分，并且将拆分后的l-NBP和d-NBP进行了对比研究后发现，l-NBP比d-NBP的作用更强，而且还发现在对神经细胞凋亡研究中d-NBP的存在降低了dl-NBP的作用，从而证明d-NBP有拮抗l-NBP作用，因而开发l-NBP的意义更为重。目前该药应用于VaD的实验研究少有报道，更无其与PI3K/Akt信号转导通路之间关系的报道。基于此，本文拟通过观察PI3K/Akt信号转导通路在VaD小鼠海马组织中的变化，以及l-NBP对VaD的影响，以探讨PI3K/Akt信号转导通路在VaD的发病机制中的作用及l-NBP的作用机制。
第一部分：血管性痴呆小鼠模型的建立和左旋丁苯酞的防治作用
目的：观察脑反复缺血再灌注后C57BL/6小鼠行为学的改变和海马病理学特征变化，并观察l-NBP对脑反复缺血再灌注诱导的VaD小鼠空间学习记忆障碍的影响。
方法：
将C57BL/6小鼠随机分为假手术组（sham组）、模型组（IR组，造模后植物油灌胃6ml.kg-1.d-1，连续28天）、左旋丁苯酞预防性治疗组（Pre-l-NBP组，造模前7天左旋-丁苯酞灌胃30mg.kg-1.d-1，连续7天）、左旋丁苯酞低剂量治疗组（Low-l-NBP组，造模后左旋-丁苯酞灌胃30mg.kg-1.d-1，连续28天)和左旋丁苯酞高剂量治疗组（High-l-NBP组，造模后左旋-丁苯酞灌胃60mg.kg-1.d-1，连续28天）；除假手术组外均给予双侧颈总动脉反复三次缺血-再灌注方法建立小鼠VaD模型，假手术组仅暴露颈总动脉不结扎。造模4周后通过Morris水迷宫试验和跳台试验进行学习和记忆成绩测试，观察其行为学改变；应用琉堇染色观察海马病理学特征变化。
结果：
1空间学习记忆能力的改变——Morris水迷宫实验
在定位航行实验中，IR组小鼠的寻台潜伏期明显长于Sham组小鼠[F(1,27)=30.09，p<0.01]。l-NBP各组小鼠寻台潜伏期与IR组小鼠相比明显缩短[F(3,50)=10.315，P<0.01]。在实验的第2-5天，sham组小鼠、Pre-l-NBP组小鼠寻台潜伏期明显短于IR组小鼠（P<0.05, P<0.01）；在实验的第3-5天，Low-l-NBP组和High-l-NBP组小鼠寻台潜伏期明显短于IR组小鼠（P<0.05，P<0.01）。
在空间探索试验中，与IR组小鼠相比，Sham组小鼠对目标象限表现出明显的偏爱（P<0.01)。与IR组小鼠相比，High-l-NBP和Pre-l-NBP组小鼠对目标象限表现出明显的偏爱(P<0.05, P<0.01)。
2被动逃避反应的改变——跳台实验
2.1下台潜伏时间：IR组与Sham组相比小鼠的首次跳下平台的潜伏时间明显缩短(P<0.01)，提示小鼠记忆形成功能受损；Low-l-NBP组、High-l-NBP组和Pre-l-NBP组与IR组相比跳下平台的潜伏时间明显增加(P<0.05)，提示l-NBP可改善小鼠记忆形成功能。
2.2错误次数：IR组与Sham组相比小鼠跳下平台的错误次数明显增多(P<0.05)，Low-l-NBP组、High-l-NBP组和Pre-l-NBP组与IR组相比跳下平台的错误次数明显减少，但无统计学意义(P>0.05)。
3小鼠海马的组织病理学改变
硫堇染色显示，sham组小鼠海马CA1区锥体神经元排列整齐、致密，细胞形态完整，胞核饱满，核仁清晰。IR组小鼠海马CA1区可见片段性的锥体细胞缺失，部分细胞形态发生明显改变，可见胞体缩小，形态不规则，呈梭型或多角形，胞核固缩、浓染，核仁模糊不清或消失。与IR组相比，Low-l-NBP组、High-l-NBP组和Pre-l-NBP组小鼠海马CA1区的锥体细胞缺失明显减轻。
结论：
1本实验通过双侧颈总动脉反复三次缺血-再灌注方法成功的建立了C57BL/6小鼠VaD模型。预防性和治疗性应用l-NBP可以减轻VaD小鼠学习和记忆障碍，尤以预防性应用l-NBP效果显著。
2反复缺血再灌注4周后小鼠海马组织CA1区出现神经细胞片段性缺失、排列紊乱、神经元密度降低等改变。预防性和治疗性应用l-NBP可以减轻VaD小鼠海马组织病理学变化，进而改善其学习和记忆能力。
第二部分：PI3K激动剂及抑制剂对血管性痴呆小鼠认知功能的影响
目的：观察PI3K激动剂及抑制剂对VaD小鼠认知功能的影响。
方法：将C57BL/6小鼠随机分为假手术组（sham组）、激动剂组(IR+IGF-1组，手术前及手术24小时后经侧脑室给予IGF-12ul/mouse)、激动剂溶剂对照组(IR+DW组，手术前及手术24小时后经侧脑室给予去离子水2μl/mouse)、抑制剂组(IR+LY294002组，手术前及手术24小时后经侧脑室给予LY29400210nmol/mouse)和抑制剂溶剂对照组(IR+DMSO组，手术前及手术24小时后经侧脑室给予同等体积(2μl)的溶剂)；除假手术组外均给予双侧颈总动脉反复三次缺血-再灌注方法建立小鼠VaD模型，侧脑室插管给予相应试剂；假手术组仅暴露颈总动脉不结扎。造模4周后通过Morris水迷宫试验进行学习和记忆成绩测试，观察其行为学改变；应用Western Blot方法测定各组小鼠海马组织中Akt及p-Akt Ser473蛋白的变化。
结果：
1空间学习记忆能力的改变——Morris水迷宫实验
在定位航行实验中，Sham组和IR+IGF-1组小鼠的寻台潜伏期明显短于IR+DW组、IR+LY294002组、IR+DMSO组小鼠[F(4,45)=9.702，P<0.01]。
在空间探索试验中，与IR+DW组、IR+LY294002组、IR+DMSO组小鼠相比，sham组和IR+IGF-1组小鼠对目标象限表现出明显的偏爱(P<0.01)。
2Akt、pAkt Ser473在海马组织中表达的变化
术后4周Sham组、IR+DW组、IR+IGF-1组、IR+DMSO组和IR+LY294002组之间Akt总蛋白表达无明显差异(P>0.05)；IR+DW组、IR+IGF-1组、IR+DMSO组和IR+LY294002组的p-Akt Ser473的蛋白表达明显高于sham组（P<0.01, P<0.05）。IR+IGF-1组p-Akt的蛋白表达明显高于IR+DW组、IR+DMSO组和IR+LY294002组（P<0.05）。IR+LY294002组p-Akt的蛋白表达低于IR+DMSO组（P<0.05）。
结论：
PI3K激动剂IGF-1可以减轻VaD小鼠学习记忆功能障碍，促进海马组织中p-Akt Ser473表达。其机制可能为IGF-1激活PI3K/Akt信号转导通路，通过调节神经细胞的凋亡和自噬，从而改善VaD小鼠的认知功能。第三部分：血管性痴呆小鼠海马组织中Akt、p-Akt、mTOR、p-mTOR表达水平变化及左旋丁苯酞的影响
目的：观察血管性痴呆小鼠海马组织中Akt、p-Akt、mTOR、p-mTOR表达水平的变化及l-NBP的干预作用。
方法：造模4周后采用Western-Blot方法测定各组小鼠海马组织中Akt、p-Akt Ser473、mTOR和p-mTOR Ser2448蛋白的变化；采用免疫组化方法测定各组小鼠海马组织中p-Akt Ser473蛋白的变化。
结果：
1左旋丁苯酞对Akt、p-Akt Ser473蛋白表达的影响
脑反复缺血再灌注术后4周各组之间Akt总蛋白表达无明显差异(P>0.05)；与sham组相比，IR组、Pre-l-NBP组、Low-l-NBP组和High-l-NBP组p-Akt Ser473蛋白表达明显增强（P<0.01）；与IR组相比，Pre-l-NBP组、Low-l-NBP组和High-l-NBP组三组p-Akt Ser473的表达明显增强（P<0.01）。
2左旋丁苯酞对mTOR、p-mTOR Ser2448蛋白的表达的影响：
脑反复缺血再灌注术后4周各组之间mTOR总蛋白表达无明显差异(P>0.05)；IR组海马组织p-mTOR Ser2448的蛋白明显高于sham组（P<0.01）。Pre-l-NBP组、Low-l-NBP组和High-l-NBP组三组海马组织p-mTOR Ser2448的蛋白表达明显高于IR组（P<0.01, P<0.05）。
结论：
1脑反复缺血再灌注4周后，VaD小鼠海马组织中p-Akt Ser473、p-mTOR Ser2448的表达明显增多。提示脑反复缺血再灌注使PI3K/Akt通路活化，Akt和mTOR活化可抵抗缺血引起的损伤，在神经元的存活中发挥着重要的作用。
2左旋丁苯酞可促进VaD小鼠海马组织中p-Akt Ser473和p-mTORSer2448的表达。提示该药可能通过激活PI3K/Akt通路，促进p-Akt Ser473和p-mTOR Ser2448的表达，从而发挥神经保护作用，改善小鼠认知功能障碍和神经损害。
第四部分：血管性痴呆小鼠海马组织中自噬因子LC3B和Beclin1表达水平变化及左旋丁苯酞的影响
目的：观察血管性痴呆小鼠海马组织中LC3B和Beclin1蛋白及mRNA表达的变化及l-NBP的干预作用。
方法：造模4周后应用Western Blot方法测定各组小鼠海马组织中LC3B蛋白表达的变化，应用FQ RT-PCR方法测定各组小鼠海马LC3B和Beclin1mRNA表达的变化
结果：
1LC3B蛋白的表达情况：
术后4周IR组海马组织LC3B蛋白表达明显高于sham组、Pre-l-NBP组和High-l-NBP组（P<0.01）。
2LC3B、Beclin1的mRNA表达水平
IR组小鼠海马LC3B mRNA和Beclin1mRNA的表达比Sham组明显增加（P<0.05）；Pre-l-NBP组、Low-l-NBP组和High-l-NBP组三组海马组织中LC3B mRNA和Beclin1mRNA的表达无组间差异（P>0.05），与Sham组相比无统计学差异（P>0.05），但较IR组明显下降（P<0.01,P<0.05）。
结论：
1脑反复缺血再灌注4周后，小鼠海马组织LC3B蛋白、LC3B mRNA和Beclin1mRNA表达均显著升高，与小鼠行为学改变和神经元损伤基本一致。
2左旋丁苯酞降低小鼠海马组织LC3B蛋白、LC3B mRNA和Beclin1mRNA表达（与其上游抑制物p-mTOR的表达相反）。提示l-NBP可能通过激活PI3K/Akt通路抑制自噬，从而减少VaD小鼠海马神经元损伤，减轻认知功能障碍。
第五部分：血管性痴呆小鼠侧脑室室管膜扫描电镜的变化特征及左旋丁苯酞的影响
目的：观察VaD小鼠侧脑室脉络丛上皮细胞和室管膜细胞的超微结构变化，以及l-NBP的影响。
方法：通过扫描电镜观察造模4周后sham组、IR组和High-l-NBP组小鼠侧脑室脉络丛上皮细胞和室管膜细胞超微结构的变化。
结果：
1小鼠侧脑室脉络丛上皮细胞超微结构特点
Sham组侧脑室脉络丛上皮细胞细胞胞体形状规则，细胞之间界限清晰，微绒毛密布上皮细胞游离面，较粗短，细胞表面可见大小不一的分泌泡。IR组小鼠侧脑室脉络丛上皮细胞形状变得极不规则，有的细胞有突起，有的呈沟回样皱缩，有的细胞表面光滑，并可见大量的分泌泡，分泌物增多，细胞间界限不清，细胞表面呈泥沙样堆积，脉络丛上皮细胞上微绒毛明显减少甚至消失。High-l-NBP组小鼠侧脑室脉络丛上皮细胞排列尚规律，细胞间界限较清晰。
2小鼠侧脑室室管膜表面超微结构特点
sham组小鼠可见室管膜上皮细胞表面带有大量的纤毛和微绒毛，纤毛，粗细较为均匀，在纤毛的空隙处，分布着众多的微绒毛。IR组小鼠室管膜上皮细胞表面可见纤毛稀疏、倒伏，部分粘附呈缕。部分纤毛表面粘附絮状物，可见纤毛和微绒毛被“淤泥状”分泌物呈片状覆盖。High-l-NBP组小鼠室管膜上皮细胞表面的纤毛和微绒毛较IR组明显增多。
结论：
1VaD小鼠侧脑室脉络丛上皮细胞及室管膜细胞出现超微结构异常，提示脉络丛上皮细胞和室管膜的异常可能参与了VaD的发病机制。
2左旋丁苯酞可以减轻VaD小鼠侧脑室脉络丛上皮细胞室管膜的超微结构异常，从而改善认知功能障碍。
As an acquired syndrome of cognitive impairment, vascular dementia(VaD), which is caused by various kinds of cerebral vascular disease,demonstrates mainly as learning and memory dysfunction, accompanied withthe possible disorder of motion, tongue, direction and personality. VaD, as thesecond most common form of dementia after Alzheimer's disease in the elderpeople, has attracted much attention in recent years. Recently, there isemerging epidemiological evidence that VaD is likely the leading cause ofdementia in Asian population. At present, the pathogenesis of VaD remainsunclear, but increasing evidence suggests that VaD is associated with a groupof diverse pathologies affecting the cerebrovascular circulation. One ofimportant mechanisms in VaD is the pathophysiology of ischemia/reperfusion(I/R) including excitotoxicity, oxidative stress and inflammatory reactions.
Cerebral ischemia can induce the death of cerebral infarction or selectiveneurons in the brain, when ischemia occurred in associated with learning,memory areas, can lead to the occurrence of VaD. Post-IR neurons exhibit twoobvious forms of cell death: necrosis and programmed cell death whichinclude apoptosis and autophagy. Previous researches showed that there mightbe a continuum between autophagy and apoptosis. Hence the extent of thesalvage of apoptosis and autophagy might contribute to the outcome of thepathological process. Recently, accumulating evidence has shown that thephosphatidylinositol-3kinase/Akt (PI3K/Akt) cell signaling pathway takespart in the apoptosis after IR. The PI3K/Akt signal transduction pathway is aclassic anti-apoptosis pathway, promoting the survival signal transduction. Itsactivation in the nerve cells, especially in hypoxic ischemic neuronal injurycells, plays an important role, and has received extensive attention. In recentyears, it found to be closely related with the regulation of autophagy. The neuroprotective role of PI3K/Akt in cerebral ischemia has been widely studied.PI3K/Akt survival signaling pathway regulate cell survival and are importantin the pathogenesis of degenerative diseases, activation of the Akt/proteinkinase B (PKB) kinase pathway can be neuroprotective after stroke throughblocking neuronal death after stroke. Akt/mTOR signal pathway inducedNF-κB (nuclear factor kappa B) activation pathway and Bcl-2and Baxbalance adjustment play an important role in nerve cells inflammatoryresponses and apoptosis of ischemic cerebrovascular disease. Akt may rescuecells from apoptosis by inhibiting the Bax-dependent apoptosis pathwaythrough a forkhead box transcription factor.
3-n-butylphthalide is successfully synthesized to treating ischemic strokewith independent intellectual property rights of a class of chemical drugs inour country. The first is the L-isomer extracted originally from celery seed byYANG Jun-shan, after the artificial synthesis of racemes, also known asbutylphthalide. As the yellow oily liquid, butylphthalide has a celery flavor.3-n-butylphthalide (NBP) has three different stereo isomers known as l-, dl-,and d-NBP. A large number of studies have demonstrated that dl-NBP is ableto be a neuroprotective drug for the treatment of ischemic stroke throughmultiple mechanisms such as improving energy metabolism, reducingoxidative damage, improving microcirculation in arterioles, decreasingneuronal apoptosis, improving mitochondrial function, and inhibitinginflammation. Although the positive effects of NBP on cerebral infarct andAlzheimer’s disease have been verified in clinic trials and animal researches,few studies has investigated whether NBP could be beneficial as a treatmentfor VaD. A previous study discovered that only l-NBP ameliorated thecognitive impairment induced by chronic cerebral hypoperfusion while d-anddl-NBP did not show significant beneficial effects. Furthermore, l-NBPreduces β-amyloid-induced cell death in neuronal cell cultures, and improvescognitive performance in an animal model of Alzheimer’s disease. However,the effects of l-NBP in this area are still not clear.
Therefore, this study set out to examine whether treating with l-NBP and pretreating with l-NBP have the ability to improve cognitive impairment inVaD mice induced by cerebral repetitive IR. Furthermore this study explorewhether activating PI3K/Akt cell signaling pathway is one of the mechanismsof l-NBP providing the protective effect on VaD mice.
Part1: The establishment of the vascular dementia mice model and theeffects of l-3-n-butylphthalide
Objectives: To observe the behavioral changes and hippocampalpathology changes4weeks after cerebral repetitive ischemia/reperfusion inC57BL/6mice, and to observe if l-NBP can improve learning and memoryimpairment induced by cerebral repetitive ischemia/reperfusion.
Methods: C57BL/6mice were randomly divided into five groups: thesham group, IR group(vegetable oil was given daily for28consecutive daysafter IR), Pre-l-NBP group (30mg/kg l-NBP was administered daily for7daysprior to IR), Low-l-NBP group (30mg/kg l-NBP was given daily for28consecutive days after IR) and High-l-NBP group (60mg/kg l-NBP was givendaily for28consecutive days after IR). Mice, except the sham group, weresubjected for continuously three repeated times ischemia-reperfusion throughthe ligation of the bilateral common carotid arteries. In the sham group,protocols were the same as the model group but bilateral common carotidswere only exposed. The capability of learning and memory of mice wereinvestigated by the Morris water maze test and the step-down test, thepathological change of hippocampus in each group was observed by thioninestaining.
Results:
1Learning and memory ability of Morris water maze test:
1.1L-NBP significantly ameliorated learning impairment induced by cerebralrepetitive IR.
For the purpose of evaluating whether the mice cognitive function wasaffected after the oral administration of l-NBP, we used the MWM test toevaluate the mice abilities of spatial learning acquisition and memoryretention. There was a marked treatment effect [F(3,50)=10.315，P<0.01] on escape latency, suggesting that l-NBP was protective against impaired spatiallearning in the cerebral repetitive IR mice. The escape latency of IR group wasevidently longer than that of the sham group (P<0.01), suggesting that cerebralrepetitive IR successfully induced learning deficit. Compared with IR group,after treated with30mg/kg l-NBP or60mg/kg l-NBP, the escape latency wasmarkedly decreased from day3to day5(P<0.01, P<0.05). And comparedwith IR group, after pretreated with30mg/kg l-NBP, the escape latency wasmarkedly decreased from day2to day5(P<0.01). These results indicated thatoral administration of l-NBP, especially pretreatment with l-NBP significantlyrescues learning impairment caused by cerebral repetitive IR
1.2L-NBP significantly rescued memory deficits induced by cerebralrepetitive IR.
To investigate the effects of l-NBP on memory deficits caused bycerebral repetitive IR, we conducted a spatial probe test after the5days oftraining in the water maze. In the test, we discovered that the sham group, thePre-l-NBP group and the High-l-NBP mice were able to show significantpreference for the target quadrant compared with IR group mice (P<0.05,P<0.01).
2Learning ability of step-down test:
2.1Latency time
The latency time of IR group are significant shorter than those of shamgroup (P<0.01), that suggest there are impairment of memory formation in IRgroup mice. The latency time of Low-l-NBP, High-l-NBP and Pre-l-NBPgroup are marked longer than those of IR group (P<0.05), that suggest l-NBPcan improve impairment of memory formation.
2.2The number of error
The number of error of IR group were much more than those ofsham-operated group (P<0.05). The number of error of l-NBP-treated groupwere lower than those of model group, but without statistical significance (P>0.05).
3Neuropathological evaluation
In sham mice, the pyramidal neurons in the CA1hippocampus werearranged in sequence with4-5cell layers, intact lineament, full nuclei andclear nucleoli. In the IR group, part neurons died4weeks after IR, which wererepresented by increases of HG (P<0.05) and by significant decreases of ND(P<0.01) compared with sham group. And, there was significant improvementin l-NBP groups as compared to those in the IR group (P<0.05).
Conclusions:
1This study has successfully established C57BL/6VaD mouse model bythree repeated times ischemia-reperfusion through the ligation of the bilateralcommon carotid arteries. Mice, treated with l-NBP, especially pre-treated withl-NBP significantly attenuated learning and memory impairment induced bycerebral repetitive IR.
2L-NBP might improve learning and memory ability of mice byattenuating the injury of hippocampus neurons.
Part2: The effects of PI3K agonists and inhibitors on cognitive functionin mice with vascular dementia
Objectives：To observe the effects of PI3K agonists and inhibitors oncognitive function in VaD mice.
Methods: C57BL/6mice were equally and randomly divided into fivegroups: the sham group, IR+IGF-1group, IR+DW group, IR+LY294002group and IR+DMSO group. Mice, except the sham group, were subjected forcontinuously three repeated times ischemia-reperfusion through the ligation ofthe bilateral common carotid arteries. In the sham group, protocols were thesame as the model group but bilateral common carotids were only exposed.The capability of learning and memory of mice were investigated by theMorris water maze test at4weeks after operation. The expression of Akt andp-Akt in hippocampus were studied by immunohistochemistry and WesternBlot technique.
Results:
1Learning and memory ability of Morris water maze test
The escape latency of IR+DW group, IR+LY294002group and IR+DMSO group was evidently longer than that of the sham group andIR+IGF-1group[F(4,45)=9.702，P<0.01].
Compared with IR+DW, IR+LY294002and IR+DMSO group mice, thesham and IR+IGF-1group mice were able to show significant preference forthe target quadrant (P<0.01).
2Protein expression of LC3B in hippocampus by Western blot
There is no marked change in total Akt among five groups (P>0.05) at4weeks after operation. However, compared with the sham group, p-Akt weresignificantly increased in the IR+DW、 IR+IGF-1、 IR+DMSO andIR+LY294002group(P<0.01). Moreover, IR+IGF-1group mice showed astrong expression of p-Akt compared with the IR+DW、IR+DMSO andIR+LY294002group mice (P<0.05). P-Akt were significantly decreased in theIR+LY294002group compared with those of IR+DMSO group (P<0.05)
Conclusions:
PI3K agonist IGF-1could alleviate the learning and memory impairmentand promote the p-Akt protein expression in hippocampus in VaD mice, andits possible mechanism of improving cognitive function may be activating ofPI3K/Akt signal transduction pathway and regulating apoptosis and autophagyof nerve cells.
Part3: The expression level of Akt, p-Akt, mTOR, p-mTOR protein inhippocampus of VaD mice and the effects of l-NBP.
Objectives： To observe the Akt, p-Akt, mTOR, p-mTOR proteinexpression in hippocampus of VaD mice and the effects of l-NBP.
Methods: The expressions of Akt, p-Akt, mTOR and p-mTOR inhippocampus were studied by measure of Western Blot and the expression ofp-Akt were studied by immunohistochemistry at4weeks after operation.
Results:
1L-NBP induce expression of p-Akt Ser473in the hippocampus of VaD mice.
There is no marked change in total Akt among five groups (P>0.05).However, p-Akt Ser473were significantly increased in the IR group micecompared with those in the sham group (P<0.01). Moreover, Low-l-NBP, High-l-NBP and Pre-l-NBP group mice showed a strong expression of p-Aktcompared with the IR group mice (P<0.01).2L-NBP induce expression of p-mTOR Ser2448in the hippocampus ofcerebral repetitive IR mice.
There is no marked change in total mTOR among five groups (P>0.05).However, p-mTOR Ser2448were significantly increased in the IR group micecompared with those in the sham group (P<0.01). Moreover, Low-l-NBP,High-l-NBP and Pre-l-NBP group mice showed a strong expression ofp-mTOR compared with the IR group mice (P<0.01, P<0.05).
Conclusions:
1There is significant increase in the expression of p-Akt Ser473andp-mTOR Ser2448protein in hippocampal tissue of VaD mice at4weeks postsurgery. It is suggest that activation of the PI3K/Akt signal transductionpathway might play a protect role in resisting neuronal damage after repeatedcerebral ischemia and reperfusion.
2L-NBP can promote the expression of p-Akt Ser473and p-mTORSer2448in hippocampal tissue of VaD mice. The result suggest that l-NBPmay play a neuroprotective role by promoting the expression of p-Akt Ser473and p-mTOR Ser2448, thereby improving cognitive dysfunction and neuronaldamage in mice.
Part4: The expression level of LC3B和Beclin1in hippocampus of VaDmice and the effects of l-NBP
Objectives：To observe the LC3B和Beclin1expression in hippocampusof VaD mice and the effects of l-NBP.
Methods: The expression of LC3B和Beclin1in hippocampus werestudied by Western-Blot technique or FQ RT-PCR technique at4weeks afteroperation.
Results:
1The expression level of LC3B protein in hippocampus by Western blot.
The expression level of LC3B protein were significantly increased in theIR group mice compared with those in the sham group (P<0.01). Howover, in High-l-NBP and Pre-l-NBP group, the protein expression of LC3B were lowerthan those of IR group (P<0.05).
2LC3B and Beclin1mRNA expression in hippocampus by FQ RT-PCR
LC3B and Beclin1mRNA expression were significantly increased in theIR group mice compared with those in the sham group (P<0.05). There are nosignificant difference among Low-l-NBP, High-l-NBP, Pre-l-NBP group andsham group. Howover, LC3B and Beclin1mRNA expression of l-NBPgroups were marked lower than that of IR group (P<0.05).
Conclusions:
1There are significant increase in the expression of LC3B protein, LC3BmRNA and Beclin1mRNA in hippocampal tissue of VaD mice at4weekspost surgery. It suggests that increase in the expressions of LC3B and Beclin1was related to cognitive damage and neuronal damage in VaD mice.
2L-NBP can reduce the expression level of LC3B and Beclin1inhippocampal tissue of VaD mice, thereby improving cognitive dysfunction andneuronal damage in VaD mice.
Part5: Features of ependyma of the lateral ventricle in VaD mice byscanning electron microscopy (SEM) and the effect of l-NBP
Objectives：To observe the change of ependyma cells of the lateralventricle in the VaD mice and the effect of l-NBP.
Methods: To observe the change of ependyma cell and choroid plexusepithelial cell of the lateral ventricle in the VaD mice by scanning electronmicroscopy (SEM) and the effect of l-NBP.
Results:
1Structure features of choroid plexus epithelial cell of the lateral ventricle inmice
The choroid plexus epithelial cell of the lateral ventricle of sham groupare of regular shape, clear boundary between cells. On cell surface, there arefull of bushy and shorter microvilli, and kind of secretory vesicles withdifferent sizes. In IR group, the shape of choroids plexus epithelial cellbecame irregular, and some cells have raised, some are of gyrus shrinkage,
some cell surface are smooth. There were a large number of secretory vesiclesand increased secretions. Sediment is accumulated on the cell surface andintercellular boundaries are not clear. Microvillus decreased significantly evendisappear. The arrangement of choroid plexus epithelial cell in High-l-NBPgroup was in order, with clear boundaries between cells.
2Structure features of ependyma cell of the lateral ventricle in mice
In sham group, there were abundant cilia and microvillus on its unevensurface. The quantities of cilia and microvillus on the uneven surface of in IRgroup reduced obviously. Both of them became twisty and stiff or bent and fellon the surface of cell in lesion regions and ‘mud-like’ secretions flaky out onthe ependymal surface. In High-l-NBP group, the quantities of cilia andmicrovillus on the uneven surface increased obviously compared with IRgroup.
Conclusions:
1The ependyma cells of the lateral ventricle of VaD mice were abnormal,that suggests abnormal ependyma cells may be involved in the pathogenesis ofVaD.
2L-NBP can improve the abnormal ependyma cells of the lateralventricle, thereby improving cognitive dysfunction.
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