甲酯化脂氧素A_4对血管性痴呆大鼠认知功能的影响及对ERK/Nrf2和PI3K/Akt信号转导通路的作用
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摘要
血管性痴呆(vascular dementia，VD)是指各种脑血管疾病引起的获得性智能损害综合征。目前，VD已成为世界范围内继阿尔茨海默病（Alzheimer’s disease，AD)后的第二大类型痴呆，但迄今为止，VD的发病机制尚不完全明确，因而缺乏有效的治疗药物。近年来研究表明，慢性脑低灌注损伤是VD的主要病因之一，因此探讨慢性脑低灌注致VD的发病机制并寻求有效的防治药物成为现代医学的热点课题之一。
在慢性脑低灌注损伤导致VD的发病机制中，氧化应激损伤和神经元的凋亡占据着重要的地位，因此拮抗氧化应激损伤，减少神经元凋亡对于VD的防治至关重要。研究表明细胞外信号调节激酶/核因子E2相关因子2信号传导通路（extracellular signal-regulatedkinase/nuclear factor E2-related factor2, ERK/Nrf2）在拮抗脑缺血导致的神经元氧化应激损伤中发挥着重要的作用；磷脂酰肌醇-3-激酶/蛋白激酶B（phosphoinositide-3-kinase/protein kinase B，PI3K/Akt）信号转导通路是一条经典的抗凋亡，促进细胞存活的信号传导通路，我们的课题组在前期研究中已发现PI3K/Akt信号转导通路参与了双侧颈总动脉反复缺血再灌注致VD小鼠的发病机制。
脂氧素(lipoxins, Lxs)是一类重要的内源性脂质抗炎性介质，其中脂氧素A4（lipoxin A4，LXA4）是其主要的生理形式。LXA4在炎性相关性疾病中得到了充分的关注，但关于LXA4在脑缺血过程中的神经保护作用的研究只是在近数年中才得到重视。研究发现通过侧脑室注射（intracerebroventricular injection, i.c.v.)甲酯化LXA4（LXA4methyl ester, LXA4ME），一种稳定的LXA4合成类似物，能够有效抑制脑局灶性缺血再灌注（cerebral ischemia/reperfusion, I/R)损伤模型或脑局部永久性缺血模型大鼠脑组织中的炎性反应，减少脑梗死体积，改善神经功能评分。除其经典的抗炎作用外，有关LXA4的抗氧化应激损伤作用和抗凋亡作用也引起了越来越多研究者的兴趣。
但到目前为止，尚无关于在慢性脑低灌注致痴呆模型中，LXA4ME能否抑制海马组织氧化应激损伤，减少海马神经元凋亡，改善痴呆大鼠学习记忆能力方面的文献报道。基于上述研究背景，本研究通过双侧颈动脉永久结扎法（permanent bilateral common carotid arteryocclusion，BCCAO）建立大鼠慢性脑低灌注致VD模型，观察并探讨（1）BCCAO术后连续2周应用LXA4ME(i.c.v.)是否能够改善大鼠的认知功能？该作用是否与激活ERK/Nrf2信号转导通路有关？（2）在大鼠慢性脑低灌注早期应用LXA4ME是否能为VD大鼠提供远期神经保护作用，该作用是否与PI3K/Akt信号转导通路的激活有关？在此基础上，进一步观察VD大鼠海马CA1区突触后致密物（postsynaptic density, PSD）厚度以及PSD-95表达的变化，并观察LXA4ME对其的影响，为探讨LXA4ME发挥神经保护作用，改善认知障碍的作用机制提供更多的理论依据。
第一部分甲酯化脂氧素A4改善血管性痴呆大鼠认识功能以及对ERK/Nrf2信号转导通路的影响
目的：通过BCCAO方法建立慢性脑低灌注致VD模型，观察LXA4ME对BCCAO术后2W大鼠学习记忆能力的影响以及LXA4ME对ERK/Nrf2信号转导通路的作用，同时观察LXA4ME对大鼠海马内4-羟基壬烯醛（4-Hydroxynonenal,4-HNE)以及凋亡相关蛋白表达的影响，探讨LXA4ME改善慢性脑低灌注致VD模型大鼠认知功能的作用机制。
方法：清洁级成年健康雄性Sprague-Dawley(SD)大鼠随机分为四组：假手术组（sham组）；模型组（vehicle组）；低剂量组（LXA4ME10ng组）；高剂量组（LXA4ME100ng组）。在BCCAO术前一周，给予大鼠脑立体定位侧脑室置管。BCCAO术后假手术组和模型组大鼠分别通过微量注射器给予生理盐水5μl/d（i.c.v.）, LXA4ME低剂量组和LXA4ME高剂量组大鼠分别注射10ng/5μl LXA4ME和100ng/5μlLXA4ME（i.c.v.），每日一次，连续14天。最后一次给药的次日，所有大鼠进行为期6天的Morris水迷宫实验，测试大鼠空间学习记忆能力。（1）定位航行实验（Place navigation test）：主要用于测试大鼠的空间学习能力。实验历时5天。每天训练4次。两次训练间隔1分钟。训练时随机选择一个象限池壁圆弧中点作为入水点，将大鼠面向池壁轻轻放入水中。大鼠在4次试验中分别从四个象限入水，将大鼠在平台停留10s作为寻台成功的标志。水迷宫装置自动记录大鼠从入水到寻台成功的时间，即逃避潜伏期（escape latency）。如120s大鼠仍未找到平台，将大鼠人为引上平台休息10s，将潜伏期记录为120s。（2）空间探索实验（Spatial probe test）：用于测试大鼠的空间记忆能力。在定位航行实验结束的第二天，撤除平台，选定与原有平台所在象限相对的象限中点为大鼠入水点，将大鼠面向池壁轻轻放入水中，给予120s自由游泳的时间，记录大鼠在120s内穿过原来平台所在位置的次数以及在平台象限的游泳时间占总时间的百分比，作为反映大鼠空间记忆能力的指标。
行为学测试结束后，每组随机取6只大鼠，2%戊巴比妥钠（50mg/kg）麻醉后，4%多聚甲醛灌注固定，观察大鼠海马CA1区组织病理学改变和4-HNE表达变化。此外，每组随机取6只大鼠，充分麻醉后，断头取材，分离海马，采用western blot技术观察大鼠海马组织内total ERK(t-ERK)、phospho-ERK (p-ERK)、total Nrf2、nuclear Nrf2、 NAD(P)H苯醌氧化还原酶（NAD(P)H: quinoneoxidoreductase1，NQO1）、Bcl-2、Bax和cleaved caspase-3蛋白表达的变化。
结果：在定位航行实验的第一天，各组间平均潜伏期差异无统计学意义（P>0.05）；第二天，同其他三组相比，假手术组大鼠平均潜伏期明显缩短（P<0.01），而模型组大鼠和LXA4ME10ng组、LXA4ME100ng组大鼠无统计学意义（P>0.05）。在定位航行的第三天，LXA4ME100ng组大鼠平均潜伏期明显低于模型组大鼠（P<0.01），而LXA4ME10ng组大鼠平均潜伏期同模型组大鼠相比，差异无统计学意义（P>0.05）。在随后的两天中，LXA4ME10ng组和LXA4ME100ng组大鼠的平均潜伏期均明显低于模型组大鼠（P<0.01），且同LXA4ME10ng组大鼠相比，LXA4ME100ng组大鼠的平均潜伏期缩短（P<0.01）。
在空间探索实验中，选用目标象限停留时间百分比和穿越平台次数两个指标进行测定。总的统计分析显示各组间差异具有显著统计学意义（P<0.01）。同假手术组大鼠相比，模型组大鼠在目标象限停留时间以及穿越平台次数显著减少（P<0.01）；然而同模型组大鼠相比，经LXA4ME（10ng和100ng）干预后大鼠在目标象限停留时间明显增加（P<0.01），而LXA4ME10ng组和LXA4ME100ng组组间比较无统计学意义（P>0.05）；与之相似的是，经LXA4ME（10ng和100ng）干预后大鼠穿越平台次数较模型组大鼠明显增加（P<0.05，P<0.01），且LXA4ME100ng组大鼠穿越平台次数多于LXA4ME10ng组（P<0.01）。
假手术组大鼠海马CA1区神经元排列紧密，整齐，细胞形态、大小正常，结构完整，胞核形态饱满，核仁清晰可见，没有明显的神经元丢失现象；和对照组相比，模型组大鼠海马CA1神经元缺失较明显，存活神经元数目减少，排列松散，胞体缩小，部分胞体呈多角形或梭形，胞核固缩，部分核仁模糊不清，胞浆浅染；经LXA4ME（尤其是LXA4ME100ng组）处理后大鼠，其海马CA1区神经元丢失减少，细胞排列较为紧密有序，
BCCAO术后20天，模型组大鼠海马CA1区有广泛的4-HNE免疫阳性表达(0.17±0.02)，阳性产物为棕色颗粒，主要定位于神经元的核周体和轴突部分；同模型组相比，经LXA4ME（10ng和100ng）治疗大鼠海马4-HNE的表达显著下降（P<0.01），且LXA4ME100ng组大鼠海马4-HNE的表达(0.08±0.02)低于LXA4ME10ng组(0.11±0.02)（P<0.05）。
Western blot结果显示，各组间t-ERK1/2蛋白表达水平无明显统计学差异（P>0.05）；同假手术组大鼠相比，模型组大鼠海马p-ERK1/2蛋白表达明显降低（P<0.01），而经LXA4ME（10ng和100ng）干预后，这一下降趋势得到了明显的逆转（P<0.05，P<0.01）。同假手术组相比，模型组大鼠海马组织中total Nrf2、 nuclear Nrf2以及NQO1蛋白均明显下调（P<0.01），经LXA4ME (10ng and100ng)治疗后大鼠海马中total Nrf2、nuclear Nrf2以及NQO1蛋白水平较模型组明显升高。LXA4ME10ng组和LXA4ME100ng组大鼠组间total Nrf2蛋白表达水平比较无明显统计学差异（P>0.05），但LXA4ME100ng组大鼠海马p-ERK1/2、nuclear Nrf2以及NQO1蛋白表达水平明显高于LXA4ME10ng组(P<0.01)。
和假手术组相比，模型组大鼠海马中Bcl-2蛋白表达明显下调而Bax蛋白表达明显增高，因此Bcl-2/Bax比值显著下降（P<0.05, P<0.01,P<0.01）；然而，和模型组大鼠相比， LXA4ME (10ng and100ng)显著逆转了大鼠海马中Bcl-2（P<0.01）和Bax蛋白的表达变化（P<0.05,P<0.01），升高了Bcl-2/Bax比值（P<0.01）。且同LXA4ME10ng组相比，LXA4ME100ng组大鼠海马Bcl-2/Bax比值升高更为明显（P<0.01）。
同假手术组相比，模型组大鼠海马cleaved caspase-3表达水平显著升高(P<0.01)，100ng干预剂量的LXA4ME明显下调了cleavedcaspase-3蛋白的表达水平(P<0.01)，而LXA4ME10ng组大鼠和模型组大鼠相比，二者cleaved caspase-3蛋白的表达水平无显著统计学差异（P>0.05）。
结论：本实验通过BCCAO方法成功建立慢性脑低灌注致VD大鼠模型，LXA4ME干预后可明显改善大鼠学习记忆能力，减少海马组织内4-HNE的生成，抑制海马神经元的凋亡，而且此作用可能是通过激活ERK/Nrf2信号转导通路来实现的。
第二部分甲酯化脂氧素A4对血管性痴呆大鼠的远期神经保护作用及对PI3K/Akt信号转导通路的影响
目的：观察BCCAO术后早期应用LXA4ME对慢性脑低灌注致VD大鼠是否具有远期神经保护作用，并对其分子生物学机制进行探讨。
方法：清洁级成年健康雄性SD大鼠随机分为四组：假手术组（sham组）；模型组（vehicle组）； LXA4ME100ng组；LY294002+LXA4ME100ng组。在BCCAO术前一周，给予大鼠脑立体定位侧脑室置管。BCCAO术后假手术组大鼠和模型组大鼠即给予LY294002溶媒2μl/d（i.c.v.），30min后给予生理盐水5μl/d（i.c.v.）；LXA4ME100ng组大鼠BCCAO术后即给予LY294002溶媒2μl/d（i.c.v.），30min后给予LXA4ME100ng/d （i.c.v.）；LY294002+LXA4ME100ng组大鼠造模后即给予LY294002溶液2μl/d（i.c.v.），30min后给予LXA4ME100ng/d（i.c.v.）；以上给药过程持续两周。
BCCAO术后29天，所有大鼠进行为期6天的Morris水迷宫实验，测试大鼠空间学习记忆能力。
行为学测试结束后，每组随机取6只大鼠，2%戊巴比妥钠（50mg/kg）麻醉后，4%多聚甲醛灌注固定，常规石蜡包埋，冠状切片，焦油紫染色，观察大鼠海马CA1区组织病理学改变。此外，每组随机取6只大鼠，充分麻醉后，断头取材，分离海马，采用westernblot技术观察大鼠海马组织中total Akt(t-Akt)、phospho-Akt（p-Akt）、total CREB(t-CREB)、phospho-CREB(p-CREB)、Bcl-2和Bax的蛋白表达。
结果：在定位航行第一天，各组大鼠平均潜伏期比较无统计学意义（P>0.05）；从第二天开始，同假手术组大鼠相比，模型组大鼠平均潜伏期明显延长（P<0.01）；而LXA4ME100ng组大鼠平均潜伏期明显短于模型组大鼠（day2：P<0.05；day3-5：P<0.01）；和LXA4ME100ng组大鼠相比，LY294002+LXA4ME100ng组大鼠平均潜伏期明显延长（day2：P<0.05；day3-5：P<0.01）。在空间探索实验中，各组大鼠间目标象限停留时间百分比和穿越平台次数的差异具有显著统计学意义（P<0.01）。同假手术组大鼠相比，模型组大鼠在目标象限停留时间以及穿越平台次数明显减少（P<0.01）；然而同模型组大鼠相比，经LXA4ME100ng干预后大鼠在目标象限停留时间和穿越平台次数均明显增加（P<0.01）；但LY294002+LXA4ME100ng组大鼠在目标象限停留时间和穿越平台次数较LXA4ME100ng组大鼠明显减少（P<0.01）。
光学显微镜下观察，可见假手术组大鼠海马CA1区神经元排列紧密有序，没有明显的神经元丢失现象，细胞大小、形态正常，结构完整，胞核形态饱满，核仁清晰，胞浆尼氏体丰富；和对照组相比，模型组大鼠海马CA1神经元缺失较明显，存活神经元数目减少，排列松散，胞体、胞核皱缩，部分核仁模糊不清，胞浆浅染；经LXA4ME100ng治疗后大鼠，以上情况得以较明显改善；而LY294002+LXA4ME100ng组大鼠海马CA1区可见部分神经元丢失，细胞皱缩等现象。
BCCAO术后34天，同对照组相比，模型组大鼠海马Akt蛋白的磷酸化水平明显降低（P<0.01）；而在LXA4ME100ng大鼠，该趋势得到了明显的逆转（P<0.01）；LY294002+LXA4ME100ng大鼠海马Akt蛋白的磷酸化水平较LXA4ME100ng显著下降（P<0.01）。但各组间t-Akt蛋白表达水平比较无显著统计学差异（P>0.05）。p-CREB在各组间的表达变化趋势同p-Akt相似，t-CREB蛋白表达水平组间比较无显著统计学差异（P>0.05）。
和假手术组相比，慢性脑低灌注模型组大鼠海马组织中Bcl-2蛋白表达明显减少（P<0.01）而Bax蛋白表达明显增加（P<0.01），Bcl-2/Bax比值显著降低（P<0.01）；和模型组大鼠相比，LXA4ME100ng组大鼠海马Bcl-2（P<0.01）和Bax蛋白的表达趋势得以明显逆转（P<0.01），Bcl-2/Bax比值显著升高（P<0.01）。且同LXA4ME100ng组相比，大鼠经LY294002和LXA4ME联合处理后，其海马Bcl-2蛋白表达明显下调（P<0.01）而Bax蛋白表达明显上调（P<0.01），Bcl-2/Bax比值明显降低（P<0.01）。
结论：慢性脑低灌注损伤早期应用LXA4ME能够发挥远期神经保护作用，减少神经元的凋亡，改善VD大鼠认知功能障碍，该作用可能是通过激活PI3K/Akt信号转导通路来实现的。
第三部分：甲酯化脂氧素A4对血管性痴呆大鼠突触后致密物的影响
目的：观察LXA4ME对慢性脑低灌注致VD大鼠海马CA1区神经元PSD厚度及PSD-95表达变化的影响，为LXA4ME的神经保护作用提供更多的理论支持。
方法：清洁级成年健康雄性SD大鼠随机分为三组：假手术组（sham组）；模型组（vehicle组）；LXA4ME100ng组。在BCCAO术前一周，给予大鼠脑立体定位侧脑室置管。BCCAO术后，假手术组大鼠和模型组大鼠即给予生理盐水5μl/d（i.c.v.）；LXA4ME100ng组大鼠BCCAO术后即给予LXA4ME100ng/d（i.c.v.）。以上给药过程持续两周。
BCCAO术后34天，每组大鼠各取3只，4%多聚甲醛（含2.5%戊二醛）灌注固定，利用透射电子显微镜观察各组大鼠海马CA1区PSD厚度的变化。此外，每组随机取6只大鼠，采用免疫组化技术观察大鼠海马CA1区PSD-95的表达变化。
结果：假手术组大鼠海马CA1区突触后致密物厚度为49.65±4.84nm，BCCAO术后34天，模型组大鼠突触后致密物厚度为34.61±5.66nm，较假手术组明显降低（P<0.01）；LXA4ME（100ng/d）干预组大鼠海马CA1区突触后致密物厚度为47.53±6.10nm，较模型组显著增加（P<0.01）。
免疫组化结果显示，假手术组大鼠海马CA1区有广泛的PSD-95免疫阳性表达，阳性细胞密集且规则，免疫染色深，平均光密度值为0.21±0.03；BCCAO术后34天，模型组大鼠海马CA1区PSD-95免疫染色阳性细胞分布稀疏，免疫染色浅，平均光密度值为0.11±0.02，明显低于假手术组（P<0.01）；而经LXA4ME（100ng/d）治疗后大鼠其海马CA1区PSD-95的平均光密度值（0.17±0.02）较模型组明显增加（P<0.01）。
结论：LXA4ME能够促进慢性脑低灌注致VD大鼠海马CA1区的PSD-95的表达，增加PSD厚度，改善突触可塑性，从而改善VD大鼠的认知功能。
Vascular dementia (VD) is an acquired syndrome of cognitiveimpairment which is caused by various kinds of cerebral vascular diseases. Itis incontrovertible that VD is the second most common cause of dementiaafter Alzheimer’s disease (AD) in the world. Up to now, the pathogenesis ofVD is not yet entirely clear and there are no especially effective drugs.Considerable studies characterize chronic cerebral hypoperfusion as acommon pathophysiological status contributing to VD. Thus, exploring themechanism by which chronic cerebral hypoperfusion leads to VD and seekingthe effective drugs for VD have become one of the focuses of currentmedicine.
Oxidative injury and the apoptosis of neuron play an important role in thepathogenesis of VD induced by chronic cerebral hypoperfusion. So, inhabitingthe oxidative injury induced by chronic cerebral hypoperfusion and reduingthe apoptosis of neuron may be crucial for managing VD. It has beendemonstrated that the activation of extracellular signal-regulatedkinase/nuclear factor E2-related factor2(ERK/Nrf2) signaling pathway isvery important for neurons to resist the oxidative injury induced by cerebralischemia; the phosphatidylinositol-3kinase/Akt (PI3K/Akt) cell signalingpathway is a classic anti-apoptosis pathway, promoting the survival signaltransduction. In previous studies, our research team have explored that theactivation of PI3K/Akt cell signaling pathway can improve cognitive functiondeficits of VD mice induced by cerebral repetitive ischemia/reperfusion.
Lipoxins (LXs), a class of endogenous anti-inflammatory lipid-basedautacoids, are generated from arachidonic acid via sequential actions oflipoxygenases during the onset of the inflammatory. Lipoxin A4(LXA4) is oneof the main physiologic forms of LXs. LXA4has been widely studied in various diseases related to inflammation. Until recent years, the novelneuroprotection of LXA4for neurons against injury induced by cerebralischemia is being recognized by researchers. several recent studies revealedthat LXA4methyl ester (LXA4ME), a stable synthetic analog of LXA4, whichwas administrated through intracerebroventricular injection (i.c.v.), couldsignificantly reduce infarct volumes by inhibiting inflammatory responses inrat models of cerebral ischemia/reperfusion (I/R) and permanent focalcerebral ischemia. Intriguingly, besides its classic anti-inflammatory effect,considerable attention has been attracted to the novel antioxidant effect ofLXA4in recent years.
However, no information is available with regard to the possibleprotective effect of LXA4on neuronal injury and cognitive deficits induced bychronic cerebral hypoperfusion. So, in the present study, a widely acceptedmodel of chronic cerebral hypoperfusion induced by permanent bilateralcommon carotid artery occlusion (BCCAO) in rats was used to explore:(1)whether long-term administration of LXA4ME can protect neurons inhippocampus against oxidative injury, reduce neuronal apoptosis andameliorate cognitive deficits induced by chronic cerebral hypoperfusionthrough activating the ERK-Nrf2signaling pathway?(2) whetheradministration of LXA4ME during early phase of chronic cerebralhypoperfusion can provide long-term neuroprotection and improve cognitiveimpairments? And if so, whether the effects of LXA4ME is are associatedwith the activation of PI3K/Akt signaling pathway? Based on the abovestudies, we will further observe the effects of LXA4ME on the thickness ofpostsynaptic density (PSD）and the expression of PSD-95in CA1area ofhippocampus of rats underwent chronic cerebral hypoperfusion, and providemore insights into the neuroprotective mechanisms of LXA4ME.
Part Ⅰ LXA4ME ameliorates cognitive deficits induced by chroniccerebral hypoperfusion through activating ERK/Nrf2signalingpathway in rats
Objective: To establish a VD rat model induced by chronic cerebral hypoperfusion through BCCAO and observe the effects of LXA4ME on thecognitive deficits of VD rats. At the same time, we will evaluate the effects ofLXA4ME on the expression of ERK/Nrf2signaling pathway,4-Hydroxynonenal (4-HNE) and apoptosis-associated proteins, and explorethe neuoprotective mechanisms of LXA4ME.
Methods: Adult male Sprague–Dawley rats weighing250-300g wereprovided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into four groups: Sham(sham-operated) group, vehicle (BCCAO+normal saline) group, LXA4ME10group and LXA4ME100group.10ng or100ng LXA4ME (purchased fromCayman Chemical, Ann Arbor, MI, USA) were dissolved in5μl normalsaline, respectively. Rats in LXA4ME10group and LXA4ME100groupreceived10ng or100ng LXA4ME (i.c.v.) immediately after BCCAO,respectively. Rats in sham group and vehicle group received5μl normal saline(i.c.v.). All rats received LXA4ME dissolved in normal saline or normalsaline according to the aforementioned experimental plan every24h up to14days afterwards.
The next day after the final injection，all rats were evaluated spatiallearning and memory abilities by the Morris water maze test. Each rat receivedfour trails per day for five consecutive days，with an intertrial interval of60s.In every trail, each rat was gently placed the water at one starting position,facing the wall of water maze. The time to reach the platform (escape latency)was recorded in each trial. The rat was allowed to find the platform within120s. If the rats failed to find the hidden platform within120s, they were gentlyguided to find the platform by the experimenter and allowed to remain on theplatform for10s and their escape latencies were recorded as120s. Theprocedure was repeated for all the four start locations. On day6, the rats weretested on a spatial probe trial in which the platform was removed, and the ratswere placed in a quadrant which is opposite to the target quadrant and allowedto swim freely for120s. The percentage of rats spent in the target quadrant where the platform had been located and the times of rats crossing theplatform were recorded.
Shortly after the behavioral tests, six rats chosen randomly from eachgroup were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with normal saline rapidly through the left cardiac ventricle andascending aorta, then followed by4%paraformaldehyde. The brains were thenembedded in paraffin. Coronal brain sections were cut and underwent Nisslstaining with0.5%cresyl fast violet and immunohistochemically stained for4-Hydroxynonenal (4-HNE), respectively. IOD/area value was used toindicate the mean optical density for each slice. Furthermore, six rats fromeach group were randomly chosen to be decapitated under anesthesia, Thehippocampi were quickly dissected and homogenated to detect the proteinexpression of p-ERK、Nrf2、NQO1、Bcl-2、Bax and caspase-3in hippocampusby western blot.
Results: In the Morris water maze, beginning on day2, rats in vehiclegroup took significantly longer time to find the platform (P<0.01) comparedwith sham group. Rats in LXA4ME100ng group showed shorter meanlatencies compared with vehicle group (P<0.01) on day3. Over the next twodays, rats treated with LXA4ME (10ng and100ng) showed significantshorter escape latencies compared with vehicle group (both P<0.01),meanwhile, administration of LXA4ME100ng resulted in shorter escapelatencies than LXA4ME10ng group (P<0.01). In the probe trial, rats invehicle group stayed in the target quadrant for significantly less time andpossessed fewer times of crossing the platform than sham group (both P<0.01).Compared with vehicle group, rats received LXA4ME (10ng and100ng)evidently increased the ratio of time spent in the target quadrant,(bothP<0.01). Similarly, administration of LXA4ME (10ng and100ng)significantly increased the times of rats crossed the platform relative to thevehicle group (P<0.05，P<0.01，respectively). In detail, rats in LXA4ME100ng group crossed the platform for more times than LXA4ME10ng group(P<0.01).
The pyramidal neurons in the CA1region of hippocampus in sham groupwere tightly ranked in order, and the neurons were clear and moderate in sizewith normal microstructure. In vehicle group, obvious pathological changeswere exhibited with loosely arranged neurons, neuronal shrinkage, loss andlight color staining. Administration of LXA4ME，especially100ng per day,evidently reversed the morphologic changes.
Extensively positive staining for4-HNE was seen in the neuronalperikarya and axons of neurons in hippocampus of rats in vehicle group(0.17±0.02). However, compared with vehicle group, the IOD/area value of4-HNE significantly decreased in group treated with LXA4ME (both P<0.01),which was lower in LXA4ME100ng group (0.08±0.02) than LXA4ME10nggroup(0.11±0.02)(P<0.05).
There was no significant difference in the expression of t-ERK1/2amongall groups (P＞0.05). The expression of p-ERK1/2in vehicle group obviouslydecreased compared with sham group (P<0.01). However, this decrease wasremarkably reversed by LXA4ME at doses of10ng and100ng (P<0.05，P<0.01, respectively). Similarly, the expression of total Nrf2, nuclear Nrf2andNQO1in vehicle group were also significantly downregulaed (all P<0.01).Interestingly, LXA4ME (10ng and100ng) significantly elevated theexpression of total Nrf2, nuclear Nrf2and NQO1（P<0.01，P<0.01，P<0.05,respectively）. Furthermore, except that no significant difference was found inthe expression of total Nrf2between LXA4ME100ng group and LXA4ME10ng group(P＞0.05), there were higher expression of p-ERK1/2, nuclearNrf2and NQO1in LXA4ME100ng group than LXA4ME10ng group(P<0.01).
Compared with sham group, chronic cerebral hypoperfusion significantlydecreased the expression of Bcl-2and increased the expression of Bax, thusmarkedly reduced the ratio of Bcl-2/Bax (P<0.05, P<0.01, P<0.01,respectively). However, administration of LXA4ME (10ng and100ng)significantly reversed the downregulation of Bcl-2(both P<0.01) and theupregulation of Bax (P<0.05, P<0.01, respectively) and elevated the expression ratio of Bcl-2/Bax (both P<0.01). Moreover, compared with LXA4ME10ng group, treatment with LXA4ME100ng more effectivelyupregulated the ratio of Bcl-2/Bax (P<0.01).
Cleaved caspase-3protein was significantly upregulated in vehicle groupcompared with sham group (P<0.01). Adminstration of LXA4ME100ng perday significantly downregulated cleaved caspase-3protein expression(P<0.01).
Conclusion: The present study indicates protective effects of LXA4MEon cognitive impairment induced by chronic cerebral hypoperfusion, andprovides further insight into the antioxidant and anti-apoptotic mechanisms ofLXA4ME. The activation of ERK/Nrf2signaling pathway contributes to theneuroprotection of LXA4ME against chronic cerebral hypoperfusion injury.
PartⅡLXA4ME provides long-term neuroprotection for VD rats inducedby chronic cerebral hypoperfusion through activating PI3K/Aktsignaling pathway
Objective: To evaluate the possible long-term neuroprotection of LXA4ME administrated during early phase of chronic cerebral hypoperfusion andfurther explore the underlying mechanisms.
Methods: Adult male Sprague–Dawley rats weighing250-300g wereprovided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into four groups: Sham group,vehicle group, LXA4ME100group and LXA4ME100+LY294002group. Ratsin Sham group and vehicle group received2μl vehicle of LY294002（i.c.v.）shortly after BCCAO, and which was followed by administration of5μlnormal saline (i.c.v.)30min later; Rats in LXA4ME100group received2μlvehicle of LY294002（i.c.v.）shortly after BCCAO, and which was followedby administration of LXA4ME (100ng in5μl)(i.c.v.)30min later; Rats inLXA4ME100+LY294002group received2μl LY294002（i.c.v.） shortlyafter BCCAO, and which was followed by administration of LXA4ME (100ng in5μl)(i.c.v.)30min later. The aforementioned experimental plan was performed every24h up to14days afterwards.
At day29following BCCAO, all rats received Morris water maze test toevaluate patial learning and memory abilities.
Shortly after the Morris water maze test, six rats chosen randomly fromeach group were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with4%paraformaldehyde. The brains were then embedded inparaffin. Coronal brain sections were cut and underwent Nissl staining with0.5%cresyl fast violet to observe the pathology changes in CA1region ofhippocampus. Moreover, six rats from each group were randomly chosen to bedecapitated under anesthesia. The hippocampi were quickly dissected andhomogenated to detect the protein expression of total Akt (t-Akt),phospho-Akt (p-Akt), total CREB (t-CREB), phospho-CREB (p-CREB),Bcl-2and Bax in hippocampus by western blot.
Results: In the Morris water maze, all animals showed a progressivedecline in the escape latency with training. Beginning on day2, rats in vehiclegroup took significantly longer time to find the platform (P<0.01) comparedwith sham group. Rats in LXA4ME100ng group showed shorter meanlatencies compared with vehicle group （day2：P<0.05；day3-5：P<0.01）.Compared with LXA4ME100ng group, the escape latencies of rats in LXA4ME100+LY294002group significantly increased（day2：P<0.05；day3-5：P<0.01）. In the probe trial, rats in vehicle group stayed in the target quadrantfor significantly less time and possessed fewer times of crossing the platformthan sham group (both P<0.01). Compared with vehicle group, rats receivedLXA4ME (100ng/d) evidently increased the ratio of time spent in the targetquadrant and the times of crossing the platform (both P<0.01). However, ratsin LXA4ME100+LY294002group showed significantly decreased ratio oftime and the fewer times of crossing the platform (both P<0.01).
In sham group, the neurons in the CA1region of hippocampus weretightly ranked in order, and the neurons were clear and moderate in size withnormal microstructure. In vehicle group, obvious pathological changes wereexhibited with loosely arranged neurons, neuronal shrinkage, loss and light color staining. Administration of LXA4ME (100ng/d), evidently reversed themorphologic changes. However, there are obvious phenomena of neuron lossand shrinkage of neurons.
At day34following BCCAO, the expression of p-Akt in vehicle groupobviously decreased compared with sham group (P<0.01); The decrease wasremarkably reversed by LXA4ME (100ng/d)(P<0.01); However, theexpression of p-Akt in LXA4ME100+LY294002group obviously decreased(P<0.01). There was no significant difference in the expression of t-Aktamong all groups (P>0.05). The expression of p-CREB showed a tendencysimilar to p-Akt. There was no significant difference in the expression of t-CREB among all groups (P>0.05).
Compared with sham group, at day34following BCCAO, chroniccerebral hypoperfusion significantly decreased the expression of Bcl-2andincreased the expression of Bax, thus markedly reduced the ratio of Bcl-2/Bax(all P<0.01). Administration of LXA4ME (100ng/d) significantly reversedthe downregulation of Bcl-2(P<0.01) and the upregulation of Bax (P<0.01)and increased the expression ratio of Bcl-2/Bax (P<0.01). Interestingly, thecombined use of LY294002and LXA4ME (100ng/d) blocked the increase ofBcl-2(P<0.01) and the decrease of Bax (P<0.01) induced by LXA4ME only,and decreased the ratio of Bcl-2/Bax (P<0.01).
Conclusion: Administration of LXA4ME during early phase of chroniccerebral hypoperfusion can provide long-term neuroprotection, inhabit theapoptosis of neurons, and improve cognitive impairments induced by chroniccerebral hypoperfusion. The activation of PI3K/Akt signaling pathway may beinvolved in the long-term neuroprotection of LXA4ME.
PartⅢ Effects of LXA4ME on postsynaptic density of rats with VD
Objective: To observe the effects of LXA4ME on the thickness of PSDand the expression of PSD-95in CA1region of rats underwent chroniccerebral hypoperfusion, and provide more insights into the neuroprotectivemechanisms of LXA4ME.
Methods: Adult male Sprague–Dawley rats weighing250-300g were provided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into three groups: Sham group,vehicle group, LXA4ME100group.100ng LXA4ME was dissolved in5μlnormal saline. Rats in LXA4ME100group received100ng LXA4ME (i.c.v.)immediately after BCCAO. Rats in sham group and vehicle group received5μl normal saline (i.c.v.). All rats received LXA4ME dissolved in normalsaline or normal saline according to the aforementioned experimental planevery24h up to14days afterwards.
At day34following BCCAO, three rats chosen randomly from eachgroup were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with4%paraformaldehyde. The thickness of PSD in CA1area ofhippocampus was observed through the electron microscopy. Furthermore,another six rats chosen randomly from each group were fully anesthetized andperfused with4%paraformaldehyde through the left cardiac ventricle. Thebrains were then embedded in paraffin. Coronal brain sections were cut andimmunohistochemically stained for PSD-95. IOD/area value was used toindicate the mean optical density for each slice.
Results: At day34following BCCAO, the thickness of PSD in CA1areaof hippocampus in vehicle group was34.61±5.66nm，which was significantlyless than that of sham group (49.65±4.84nm)(P<0.01); Meanwhile, thethickness of PSD of LXA4ME100group was47.53±6.10nm，which wasevidently more than that of vehicle group (P<0.01).
Immunohistochemical results showed that extensively positive stainingfor PSD-95was seen in the cytoplasm of neurons in hippocampus CA1regionof rats in sham group. The IOD/area value of PSD-95markedly decreased invehicle group compared with sham group (P<0.01). However, compared withsham group, administration of LXA4ME (100ng/d) significantly increased thepositive expression of PSD-95(P<0.01).
Conclusion: Administration of LXA4ME can promote the expression ofPSD-95and increase the thickness of PSD, which contribute to the neuroprotective effects of LXA4ME.
在慢性脑低灌注损伤导致VD的发病机制中，氧化应激损伤和神经元的凋亡占据着重要的地位，因此拮抗氧化应激损伤，减少神经元凋亡对于VD的防治至关重要。研究表明细胞外信号调节激酶/核因子E2相关因子2信号传导通路（extracellular signal-regulatedkinase/nuclear factor E2-related factor2, ERK/Nrf2）在拮抗脑缺血导致的神经元氧化应激损伤中发挥着重要的作用；磷脂酰肌醇-3-激酶/蛋白激酶B（phosphoinositide-3-kinase/protein kinase B，PI3K/Akt）信号转导通路是一条经典的抗凋亡，促进细胞存活的信号传导通路，我们的课题组在前期研究中已发现PI3K/Akt信号转导通路参与了双侧颈总动脉反复缺血再灌注致VD小鼠的发病机制。
脂氧素(lipoxins, Lxs)是一类重要的内源性脂质抗炎性介质，其中脂氧素A4（lipoxin A4，LXA4）是其主要的生理形式。LXA4在炎性相关性疾病中得到了充分的关注，但关于LXA4在脑缺血过程中的神经保护作用的研究只是在近数年中才得到重视。研究发现通过侧脑室注射（intracerebroventricular injection, i.c.v.)甲酯化LXA4（LXA4methyl ester, LXA4ME），一种稳定的LXA4合成类似物，能够有效抑制脑局灶性缺血再灌注（cerebral ischemia/reperfusion, I/R)损伤模型或脑局部永久性缺血模型大鼠脑组织中的炎性反应，减少脑梗死体积，改善神经功能评分。除其经典的抗炎作用外，有关LXA4的抗氧化应激损伤作用和抗凋亡作用也引起了越来越多研究者的兴趣。
但到目前为止，尚无关于在慢性脑低灌注致痴呆模型中，LXA4ME能否抑制海马组织氧化应激损伤，减少海马神经元凋亡，改善痴呆大鼠学习记忆能力方面的文献报道。基于上述研究背景，本研究通过双侧颈动脉永久结扎法（permanent bilateral common carotid arteryocclusion，BCCAO）建立大鼠慢性脑低灌注致VD模型，观察并探讨（1）BCCAO术后连续2周应用LXA4ME(i.c.v.)是否能够改善大鼠的认知功能？该作用是否与激活ERK/Nrf2信号转导通路有关？（2）在大鼠慢性脑低灌注早期应用LXA4ME是否能为VD大鼠提供远期神经保护作用，该作用是否与PI3K/Akt信号转导通路的激活有关？在此基础上，进一步观察VD大鼠海马CA1区突触后致密物（postsynaptic density, PSD）厚度以及PSD-95表达的变化，并观察LXA4ME对其的影响，为探讨LXA4ME发挥神经保护作用，改善认知障碍的作用机制提供更多的理论依据。
第一部分甲酯化脂氧素A4改善血管性痴呆大鼠认识功能以及对ERK/Nrf2信号转导通路的影响
目的：通过BCCAO方法建立慢性脑低灌注致VD模型，观察LXA4ME对BCCAO术后2W大鼠学习记忆能力的影响以及LXA4ME对ERK/Nrf2信号转导通路的作用，同时观察LXA4ME对大鼠海马内4-羟基壬烯醛（4-Hydroxynonenal,4-HNE)以及凋亡相关蛋白表达的影响，探讨LXA4ME改善慢性脑低灌注致VD模型大鼠认知功能的作用机制。
方法：清洁级成年健康雄性Sprague-Dawley(SD)大鼠随机分为四组：假手术组（sham组）；模型组（vehicle组）；低剂量组（LXA4ME10ng组）；高剂量组（LXA4ME100ng组）。在BCCAO术前一周，给予大鼠脑立体定位侧脑室置管。BCCAO术后假手术组和模型组大鼠分别通过微量注射器给予生理盐水5μl/d（i.c.v.）, LXA4ME低剂量组和LXA4ME高剂量组大鼠分别注射10ng/5μl LXA4ME和100ng/5μlLXA4ME（i.c.v.），每日一次，连续14天。最后一次给药的次日，所有大鼠进行为期6天的Morris水迷宫实验，测试大鼠空间学习记忆能力。（1）定位航行实验（Place navigation test）：主要用于测试大鼠的空间学习能力。实验历时5天。每天训练4次。两次训练间隔1分钟。训练时随机选择一个象限池壁圆弧中点作为入水点，将大鼠面向池壁轻轻放入水中。大鼠在4次试验中分别从四个象限入水，将大鼠在平台停留10s作为寻台成功的标志。水迷宫装置自动记录大鼠从入水到寻台成功的时间，即逃避潜伏期（escape latency）。如120s大鼠仍未找到平台，将大鼠人为引上平台休息10s，将潜伏期记录为120s。（2）空间探索实验（Spatial probe test）：用于测试大鼠的空间记忆能力。在定位航行实验结束的第二天，撤除平台，选定与原有平台所在象限相对的象限中点为大鼠入水点，将大鼠面向池壁轻轻放入水中，给予120s自由游泳的时间，记录大鼠在120s内穿过原来平台所在位置的次数以及在平台象限的游泳时间占总时间的百分比，作为反映大鼠空间记忆能力的指标。
行为学测试结束后，每组随机取6只大鼠，2%戊巴比妥钠（50mg/kg）麻醉后，4%多聚甲醛灌注固定，观察大鼠海马CA1区组织病理学改变和4-HNE表达变化。此外，每组随机取6只大鼠，充分麻醉后，断头取材，分离海马，采用western blot技术观察大鼠海马组织内total ERK(t-ERK)、phospho-ERK (p-ERK)、total Nrf2、nuclear Nrf2、 NAD(P)H苯醌氧化还原酶（NAD(P)H: quinoneoxidoreductase1，NQO1）、Bcl-2、Bax和cleaved caspase-3蛋白表达的变化。
结果：在定位航行实验的第一天，各组间平均潜伏期差异无统计学意义（P>0.05）；第二天，同其他三组相比，假手术组大鼠平均潜伏期明显缩短（P<0.01），而模型组大鼠和LXA4ME10ng组、LXA4ME100ng组大鼠无统计学意义（P>0.05）。在定位航行的第三天，LXA4ME100ng组大鼠平均潜伏期明显低于模型组大鼠（P<0.01），而LXA4ME10ng组大鼠平均潜伏期同模型组大鼠相比，差异无统计学意义（P>0.05）。在随后的两天中，LXA4ME10ng组和LXA4ME100ng组大鼠的平均潜伏期均明显低于模型组大鼠（P<0.01），且同LXA4ME10ng组大鼠相比，LXA4ME100ng组大鼠的平均潜伏期缩短（P<0.01）。
在空间探索实验中，选用目标象限停留时间百分比和穿越平台次数两个指标进行测定。总的统计分析显示各组间差异具有显著统计学意义（P<0.01）。同假手术组大鼠相比，模型组大鼠在目标象限停留时间以及穿越平台次数显著减少（P<0.01）；然而同模型组大鼠相比，经LXA4ME（10ng和100ng）干预后大鼠在目标象限停留时间明显增加（P<0.01），而LXA4ME10ng组和LXA4ME100ng组组间比较无统计学意义（P>0.05）；与之相似的是，经LXA4ME（10ng和100ng）干预后大鼠穿越平台次数较模型组大鼠明显增加（P<0.05，P<0.01），且LXA4ME100ng组大鼠穿越平台次数多于LXA4ME10ng组（P<0.01）。
假手术组大鼠海马CA1区神经元排列紧密，整齐，细胞形态、大小正常，结构完整，胞核形态饱满，核仁清晰可见，没有明显的神经元丢失现象；和对照组相比，模型组大鼠海马CA1神经元缺失较明显，存活神经元数目减少，排列松散，胞体缩小，部分胞体呈多角形或梭形，胞核固缩，部分核仁模糊不清，胞浆浅染；经LXA4ME（尤其是LXA4ME100ng组）处理后大鼠，其海马CA1区神经元丢失减少，细胞排列较为紧密有序，
BCCAO术后20天，模型组大鼠海马CA1区有广泛的4-HNE免疫阳性表达(0.17±0.02)，阳性产物为棕色颗粒，主要定位于神经元的核周体和轴突部分；同模型组相比，经LXA4ME（10ng和100ng）治疗大鼠海马4-HNE的表达显著下降（P<0.01），且LXA4ME100ng组大鼠海马4-HNE的表达(0.08±0.02)低于LXA4ME10ng组(0.11±0.02)（P<0.05）。
Western blot结果显示，各组间t-ERK1/2蛋白表达水平无明显统计学差异（P>0.05）；同假手术组大鼠相比，模型组大鼠海马p-ERK1/2蛋白表达明显降低（P<0.01），而经LXA4ME（10ng和100ng）干预后，这一下降趋势得到了明显的逆转（P<0.05，P<0.01）。同假手术组相比，模型组大鼠海马组织中total Nrf2、 nuclear Nrf2以及NQO1蛋白均明显下调（P<0.01），经LXA4ME (10ng and100ng)治疗后大鼠海马中total Nrf2、nuclear Nrf2以及NQO1蛋白水平较模型组明显升高。LXA4ME10ng组和LXA4ME100ng组大鼠组间total Nrf2蛋白表达水平比较无明显统计学差异（P>0.05），但LXA4ME100ng组大鼠海马p-ERK1/2、nuclear Nrf2以及NQO1蛋白表达水平明显高于LXA4ME10ng组(P<0.01)。
和假手术组相比，模型组大鼠海马中Bcl-2蛋白表达明显下调而Bax蛋白表达明显增高，因此Bcl-2/Bax比值显著下降（P<0.05, P<0.01,P<0.01）；然而，和模型组大鼠相比， LXA4ME (10ng and100ng)显著逆转了大鼠海马中Bcl-2（P<0.01）和Bax蛋白的表达变化（P<0.05,P<0.01），升高了Bcl-2/Bax比值（P<0.01）。且同LXA4ME10ng组相比，LXA4ME100ng组大鼠海马Bcl-2/Bax比值升高更为明显（P<0.01）。
同假手术组相比，模型组大鼠海马cleaved caspase-3表达水平显著升高(P<0.01)，100ng干预剂量的LXA4ME明显下调了cleavedcaspase-3蛋白的表达水平(P<0.01)，而LXA4ME10ng组大鼠和模型组大鼠相比，二者cleaved caspase-3蛋白的表达水平无显著统计学差异（P>0.05）。
结论：本实验通过BCCAO方法成功建立慢性脑低灌注致VD大鼠模型，LXA4ME干预后可明显改善大鼠学习记忆能力，减少海马组织内4-HNE的生成，抑制海马神经元的凋亡，而且此作用可能是通过激活ERK/Nrf2信号转导通路来实现的。
第二部分甲酯化脂氧素A4对血管性痴呆大鼠的远期神经保护作用及对PI3K/Akt信号转导通路的影响
目的：观察BCCAO术后早期应用LXA4ME对慢性脑低灌注致VD大鼠是否具有远期神经保护作用，并对其分子生物学机制进行探讨。
方法：清洁级成年健康雄性SD大鼠随机分为四组：假手术组（sham组）；模型组（vehicle组）； LXA4ME100ng组；LY294002+LXA4ME100ng组。在BCCAO术前一周，给予大鼠脑立体定位侧脑室置管。BCCAO术后假手术组大鼠和模型组大鼠即给予LY294002溶媒2μl/d（i.c.v.），30min后给予生理盐水5μl/d（i.c.v.）；LXA4ME100ng组大鼠BCCAO术后即给予LY294002溶媒2μl/d（i.c.v.），30min后给予LXA4ME100ng/d （i.c.v.）；LY294002+LXA4ME100ng组大鼠造模后即给予LY294002溶液2μl/d（i.c.v.），30min后给予LXA4ME100ng/d（i.c.v.）；以上给药过程持续两周。
BCCAO术后29天，所有大鼠进行为期6天的Morris水迷宫实验，测试大鼠空间学习记忆能力。
行为学测试结束后，每组随机取6只大鼠，2%戊巴比妥钠（50mg/kg）麻醉后，4%多聚甲醛灌注固定，常规石蜡包埋，冠状切片，焦油紫染色，观察大鼠海马CA1区组织病理学改变。此外，每组随机取6只大鼠，充分麻醉后，断头取材，分离海马，采用westernblot技术观察大鼠海马组织中total Akt(t-Akt)、phospho-Akt（p-Akt）、total CREB(t-CREB)、phospho-CREB(p-CREB)、Bcl-2和Bax的蛋白表达。
结果：在定位航行第一天，各组大鼠平均潜伏期比较无统计学意义（P>0.05）；从第二天开始，同假手术组大鼠相比，模型组大鼠平均潜伏期明显延长（P<0.01）；而LXA4ME100ng组大鼠平均潜伏期明显短于模型组大鼠（day2：P<0.05；day3-5：P<0.01）；和LXA4ME100ng组大鼠相比，LY294002+LXA4ME100ng组大鼠平均潜伏期明显延长（day2：P<0.05；day3-5：P<0.01）。在空间探索实验中，各组大鼠间目标象限停留时间百分比和穿越平台次数的差异具有显著统计学意义（P<0.01）。同假手术组大鼠相比，模型组大鼠在目标象限停留时间以及穿越平台次数明显减少（P<0.01）；然而同模型组大鼠相比，经LXA4ME100ng干预后大鼠在目标象限停留时间和穿越平台次数均明显增加（P<0.01）；但LY294002+LXA4ME100ng组大鼠在目标象限停留时间和穿越平台次数较LXA4ME100ng组大鼠明显减少（P<0.01）。
光学显微镜下观察，可见假手术组大鼠海马CA1区神经元排列紧密有序，没有明显的神经元丢失现象，细胞大小、形态正常，结构完整，胞核形态饱满，核仁清晰，胞浆尼氏体丰富；和对照组相比，模型组大鼠海马CA1神经元缺失较明显，存活神经元数目减少，排列松散，胞体、胞核皱缩，部分核仁模糊不清，胞浆浅染；经LXA4ME100ng治疗后大鼠，以上情况得以较明显改善；而LY294002+LXA4ME100ng组大鼠海马CA1区可见部分神经元丢失，细胞皱缩等现象。
BCCAO术后34天，同对照组相比，模型组大鼠海马Akt蛋白的磷酸化水平明显降低（P<0.01）；而在LXA4ME100ng大鼠，该趋势得到了明显的逆转（P<0.01）；LY294002+LXA4ME100ng大鼠海马Akt蛋白的磷酸化水平较LXA4ME100ng显著下降（P<0.01）。但各组间t-Akt蛋白表达水平比较无显著统计学差异（P>0.05）。p-CREB在各组间的表达变化趋势同p-Akt相似，t-CREB蛋白表达水平组间比较无显著统计学差异（P>0.05）。
和假手术组相比，慢性脑低灌注模型组大鼠海马组织中Bcl-2蛋白表达明显减少（P<0.01）而Bax蛋白表达明显增加（P<0.01），Bcl-2/Bax比值显著降低（P<0.01）；和模型组大鼠相比，LXA4ME100ng组大鼠海马Bcl-2（P<0.01）和Bax蛋白的表达趋势得以明显逆转（P<0.01），Bcl-2/Bax比值显著升高（P<0.01）。且同LXA4ME100ng组相比，大鼠经LY294002和LXA4ME联合处理后，其海马Bcl-2蛋白表达明显下调（P<0.01）而Bax蛋白表达明显上调（P<0.01），Bcl-2/Bax比值明显降低（P<0.01）。
结论：慢性脑低灌注损伤早期应用LXA4ME能够发挥远期神经保护作用，减少神经元的凋亡，改善VD大鼠认知功能障碍，该作用可能是通过激活PI3K/Akt信号转导通路来实现的。
第三部分：甲酯化脂氧素A4对血管性痴呆大鼠突触后致密物的影响
目的：观察LXA4ME对慢性脑低灌注致VD大鼠海马CA1区神经元PSD厚度及PSD-95表达变化的影响，为LXA4ME的神经保护作用提供更多的理论支持。
方法：清洁级成年健康雄性SD大鼠随机分为三组：假手术组（sham组）；模型组（vehicle组）；LXA4ME100ng组。在BCCAO术前一周，给予大鼠脑立体定位侧脑室置管。BCCAO术后，假手术组大鼠和模型组大鼠即给予生理盐水5μl/d（i.c.v.）；LXA4ME100ng组大鼠BCCAO术后即给予LXA4ME100ng/d（i.c.v.）。以上给药过程持续两周。
BCCAO术后34天，每组大鼠各取3只，4%多聚甲醛（含2.5%戊二醛）灌注固定，利用透射电子显微镜观察各组大鼠海马CA1区PSD厚度的变化。此外，每组随机取6只大鼠，采用免疫组化技术观察大鼠海马CA1区PSD-95的表达变化。
结果：假手术组大鼠海马CA1区突触后致密物厚度为49.65±4.84nm，BCCAO术后34天，模型组大鼠突触后致密物厚度为34.61±5.66nm，较假手术组明显降低（P<0.01）；LXA4ME（100ng/d）干预组大鼠海马CA1区突触后致密物厚度为47.53±6.10nm，较模型组显著增加（P<0.01）。
免疫组化结果显示，假手术组大鼠海马CA1区有广泛的PSD-95免疫阳性表达，阳性细胞密集且规则，免疫染色深，平均光密度值为0.21±0.03；BCCAO术后34天，模型组大鼠海马CA1区PSD-95免疫染色阳性细胞分布稀疏，免疫染色浅，平均光密度值为0.11±0.02，明显低于假手术组（P<0.01）；而经LXA4ME（100ng/d）治疗后大鼠其海马CA1区PSD-95的平均光密度值（0.17±0.02）较模型组明显增加（P<0.01）。
结论：LXA4ME能够促进慢性脑低灌注致VD大鼠海马CA1区的PSD-95的表达，增加PSD厚度，改善突触可塑性，从而改善VD大鼠的认知功能。
Vascular dementia (VD) is an acquired syndrome of cognitiveimpairment which is caused by various kinds of cerebral vascular diseases. Itis incontrovertible that VD is the second most common cause of dementiaafter Alzheimer’s disease (AD) in the world. Up to now, the pathogenesis ofVD is not yet entirely clear and there are no especially effective drugs.Considerable studies characterize chronic cerebral hypoperfusion as acommon pathophysiological status contributing to VD. Thus, exploring themechanism by which chronic cerebral hypoperfusion leads to VD and seekingthe effective drugs for VD have become one of the focuses of currentmedicine.
Oxidative injury and the apoptosis of neuron play an important role in thepathogenesis of VD induced by chronic cerebral hypoperfusion. So, inhabitingthe oxidative injury induced by chronic cerebral hypoperfusion and reduingthe apoptosis of neuron may be crucial for managing VD. It has beendemonstrated that the activation of extracellular signal-regulatedkinase/nuclear factor E2-related factor2(ERK/Nrf2) signaling pathway isvery important for neurons to resist the oxidative injury induced by cerebralischemia; the phosphatidylinositol-3kinase/Akt (PI3K/Akt) cell signalingpathway is a classic anti-apoptosis pathway, promoting the survival signaltransduction. In previous studies, our research team have explored that theactivation of PI3K/Akt cell signaling pathway can improve cognitive functiondeficits of VD mice induced by cerebral repetitive ischemia/reperfusion.
Lipoxins (LXs), a class of endogenous anti-inflammatory lipid-basedautacoids, are generated from arachidonic acid via sequential actions oflipoxygenases during the onset of the inflammatory. Lipoxin A4(LXA4) is oneof the main physiologic forms of LXs. LXA4has been widely studied in various diseases related to inflammation. Until recent years, the novelneuroprotection of LXA4for neurons against injury induced by cerebralischemia is being recognized by researchers. several recent studies revealedthat LXA4methyl ester (LXA4ME), a stable synthetic analog of LXA4, whichwas administrated through intracerebroventricular injection (i.c.v.), couldsignificantly reduce infarct volumes by inhibiting inflammatory responses inrat models of cerebral ischemia/reperfusion (I/R) and permanent focalcerebral ischemia. Intriguingly, besides its classic anti-inflammatory effect,considerable attention has been attracted to the novel antioxidant effect ofLXA4in recent years.
However, no information is available with regard to the possibleprotective effect of LXA4on neuronal injury and cognitive deficits induced bychronic cerebral hypoperfusion. So, in the present study, a widely acceptedmodel of chronic cerebral hypoperfusion induced by permanent bilateralcommon carotid artery occlusion (BCCAO) in rats was used to explore:(1)whether long-term administration of LXA4ME can protect neurons inhippocampus against oxidative injury, reduce neuronal apoptosis andameliorate cognitive deficits induced by chronic cerebral hypoperfusionthrough activating the ERK-Nrf2signaling pathway?(2) whetheradministration of LXA4ME during early phase of chronic cerebralhypoperfusion can provide long-term neuroprotection and improve cognitiveimpairments? And if so, whether the effects of LXA4ME is are associatedwith the activation of PI3K/Akt signaling pathway? Based on the abovestudies, we will further observe the effects of LXA4ME on the thickness ofpostsynaptic density (PSD）and the expression of PSD-95in CA1area ofhippocampus of rats underwent chronic cerebral hypoperfusion, and providemore insights into the neuroprotective mechanisms of LXA4ME.
Part Ⅰ LXA4ME ameliorates cognitive deficits induced by chroniccerebral hypoperfusion through activating ERK/Nrf2signalingpathway in rats
Objective: To establish a VD rat model induced by chronic cerebral hypoperfusion through BCCAO and observe the effects of LXA4ME on thecognitive deficits of VD rats. At the same time, we will evaluate the effects ofLXA4ME on the expression of ERK/Nrf2signaling pathway,4-Hydroxynonenal (4-HNE) and apoptosis-associated proteins, and explorethe neuoprotective mechanisms of LXA4ME.
Methods: Adult male Sprague–Dawley rats weighing250-300g wereprovided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into four groups: Sham(sham-operated) group, vehicle (BCCAO+normal saline) group, LXA4ME10group and LXA4ME100group.10ng or100ng LXA4ME (purchased fromCayman Chemical, Ann Arbor, MI, USA) were dissolved in5μl normalsaline, respectively. Rats in LXA4ME10group and LXA4ME100groupreceived10ng or100ng LXA4ME (i.c.v.) immediately after BCCAO,respectively. Rats in sham group and vehicle group received5μl normal saline(i.c.v.). All rats received LXA4ME dissolved in normal saline or normalsaline according to the aforementioned experimental plan every24h up to14days afterwards.
The next day after the final injection，all rats were evaluated spatiallearning and memory abilities by the Morris water maze test. Each rat receivedfour trails per day for five consecutive days，with an intertrial interval of60s.In every trail, each rat was gently placed the water at one starting position,facing the wall of water maze. The time to reach the platform (escape latency)was recorded in each trial. The rat was allowed to find the platform within120s. If the rats failed to find the hidden platform within120s, they were gentlyguided to find the platform by the experimenter and allowed to remain on theplatform for10s and their escape latencies were recorded as120s. Theprocedure was repeated for all the four start locations. On day6, the rats weretested on a spatial probe trial in which the platform was removed, and the ratswere placed in a quadrant which is opposite to the target quadrant and allowedto swim freely for120s. The percentage of rats spent in the target quadrant where the platform had been located and the times of rats crossing theplatform were recorded.
Shortly after the behavioral tests, six rats chosen randomly from eachgroup were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with normal saline rapidly through the left cardiac ventricle andascending aorta, then followed by4%paraformaldehyde. The brains were thenembedded in paraffin. Coronal brain sections were cut and underwent Nisslstaining with0.5%cresyl fast violet and immunohistochemically stained for4-Hydroxynonenal (4-HNE), respectively. IOD/area value was used toindicate the mean optical density for each slice. Furthermore, six rats fromeach group were randomly chosen to be decapitated under anesthesia, Thehippocampi were quickly dissected and homogenated to detect the proteinexpression of p-ERK、Nrf2、NQO1、Bcl-2、Bax and caspase-3in hippocampusby western blot.
Results: In the Morris water maze, beginning on day2, rats in vehiclegroup took significantly longer time to find the platform (P<0.01) comparedwith sham group. Rats in LXA4ME100ng group showed shorter meanlatencies compared with vehicle group (P<0.01) on day3. Over the next twodays, rats treated with LXA4ME (10ng and100ng) showed significantshorter escape latencies compared with vehicle group (both P<0.01),meanwhile, administration of LXA4ME100ng resulted in shorter escapelatencies than LXA4ME10ng group (P<0.01). In the probe trial, rats invehicle group stayed in the target quadrant for significantly less time andpossessed fewer times of crossing the platform than sham group (both P<0.01).Compared with vehicle group, rats received LXA4ME (10ng and100ng)evidently increased the ratio of time spent in the target quadrant,(bothP<0.01). Similarly, administration of LXA4ME (10ng and100ng)significantly increased the times of rats crossed the platform relative to thevehicle group (P<0.05，P<0.01，respectively). In detail, rats in LXA4ME100ng group crossed the platform for more times than LXA4ME10ng group(P<0.01).
The pyramidal neurons in the CA1region of hippocampus in sham groupwere tightly ranked in order, and the neurons were clear and moderate in sizewith normal microstructure. In vehicle group, obvious pathological changeswere exhibited with loosely arranged neurons, neuronal shrinkage, loss andlight color staining. Administration of LXA4ME，especially100ng per day,evidently reversed the morphologic changes.
Extensively positive staining for4-HNE was seen in the neuronalperikarya and axons of neurons in hippocampus of rats in vehicle group(0.17±0.02). However, compared with vehicle group, the IOD/area value of4-HNE significantly decreased in group treated with LXA4ME (both P<0.01),which was lower in LXA4ME100ng group (0.08±0.02) than LXA4ME10nggroup(0.11±0.02)(P<0.05).
There was no significant difference in the expression of t-ERK1/2amongall groups (P＞0.05). The expression of p-ERK1/2in vehicle group obviouslydecreased compared with sham group (P<0.01). However, this decrease wasremarkably reversed by LXA4ME at doses of10ng and100ng (P<0.05，P<0.01, respectively). Similarly, the expression of total Nrf2, nuclear Nrf2andNQO1in vehicle group were also significantly downregulaed (all P<0.01).Interestingly, LXA4ME (10ng and100ng) significantly elevated theexpression of total Nrf2, nuclear Nrf2and NQO1（P<0.01，P<0.01，P<0.05,respectively）. Furthermore, except that no significant difference was found inthe expression of total Nrf2between LXA4ME100ng group and LXA4ME10ng group(P＞0.05), there were higher expression of p-ERK1/2, nuclearNrf2and NQO1in LXA4ME100ng group than LXA4ME10ng group(P<0.01).
Compared with sham group, chronic cerebral hypoperfusion significantlydecreased the expression of Bcl-2and increased the expression of Bax, thusmarkedly reduced the ratio of Bcl-2/Bax (P<0.05, P<0.01, P<0.01,respectively). However, administration of LXA4ME (10ng and100ng)significantly reversed the downregulation of Bcl-2(both P<0.01) and theupregulation of Bax (P<0.05, P<0.01, respectively) and elevated the expression ratio of Bcl-2/Bax (both P<0.01). Moreover, compared with LXA4ME10ng group, treatment with LXA4ME100ng more effectivelyupregulated the ratio of Bcl-2/Bax (P<0.01).
Cleaved caspase-3protein was significantly upregulated in vehicle groupcompared with sham group (P<0.01). Adminstration of LXA4ME100ng perday significantly downregulated cleaved caspase-3protein expression(P<0.01).
Conclusion: The present study indicates protective effects of LXA4MEon cognitive impairment induced by chronic cerebral hypoperfusion, andprovides further insight into the antioxidant and anti-apoptotic mechanisms ofLXA4ME. The activation of ERK/Nrf2signaling pathway contributes to theneuroprotection of LXA4ME against chronic cerebral hypoperfusion injury.
PartⅡLXA4ME provides long-term neuroprotection for VD rats inducedby chronic cerebral hypoperfusion through activating PI3K/Aktsignaling pathway
Objective: To evaluate the possible long-term neuroprotection of LXA4ME administrated during early phase of chronic cerebral hypoperfusion andfurther explore the underlying mechanisms.
Methods: Adult male Sprague–Dawley rats weighing250-300g wereprovided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into four groups: Sham group,vehicle group, LXA4ME100group and LXA4ME100+LY294002group. Ratsin Sham group and vehicle group received2μl vehicle of LY294002（i.c.v.）shortly after BCCAO, and which was followed by administration of5μlnormal saline (i.c.v.)30min later; Rats in LXA4ME100group received2μlvehicle of LY294002（i.c.v.）shortly after BCCAO, and which was followedby administration of LXA4ME (100ng in5μl)(i.c.v.)30min later; Rats inLXA4ME100+LY294002group received2μl LY294002（i.c.v.） shortlyafter BCCAO, and which was followed by administration of LXA4ME (100ng in5μl)(i.c.v.)30min later. The aforementioned experimental plan was performed every24h up to14days afterwards.
At day29following BCCAO, all rats received Morris water maze test toevaluate patial learning and memory abilities.
Shortly after the Morris water maze test, six rats chosen randomly fromeach group were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with4%paraformaldehyde. The brains were then embedded inparaffin. Coronal brain sections were cut and underwent Nissl staining with0.5%cresyl fast violet to observe the pathology changes in CA1region ofhippocampus. Moreover, six rats from each group were randomly chosen to bedecapitated under anesthesia. The hippocampi were quickly dissected andhomogenated to detect the protein expression of total Akt (t-Akt),phospho-Akt (p-Akt), total CREB (t-CREB), phospho-CREB (p-CREB),Bcl-2and Bax in hippocampus by western blot.
Results: In the Morris water maze, all animals showed a progressivedecline in the escape latency with training. Beginning on day2, rats in vehiclegroup took significantly longer time to find the platform (P<0.01) comparedwith sham group. Rats in LXA4ME100ng group showed shorter meanlatencies compared with vehicle group （day2：P<0.05；day3-5：P<0.01）.Compared with LXA4ME100ng group, the escape latencies of rats in LXA4ME100+LY294002group significantly increased（day2：P<0.05；day3-5：P<0.01）. In the probe trial, rats in vehicle group stayed in the target quadrantfor significantly less time and possessed fewer times of crossing the platformthan sham group (both P<0.01). Compared with vehicle group, rats receivedLXA4ME (100ng/d) evidently increased the ratio of time spent in the targetquadrant and the times of crossing the platform (both P<0.01). However, ratsin LXA4ME100+LY294002group showed significantly decreased ratio oftime and the fewer times of crossing the platform (both P<0.01).
In sham group, the neurons in the CA1region of hippocampus weretightly ranked in order, and the neurons were clear and moderate in size withnormal microstructure. In vehicle group, obvious pathological changes wereexhibited with loosely arranged neurons, neuronal shrinkage, loss and light color staining. Administration of LXA4ME (100ng/d), evidently reversed themorphologic changes. However, there are obvious phenomena of neuron lossand shrinkage of neurons.
At day34following BCCAO, the expression of p-Akt in vehicle groupobviously decreased compared with sham group (P<0.01); The decrease wasremarkably reversed by LXA4ME (100ng/d)(P<0.01); However, theexpression of p-Akt in LXA4ME100+LY294002group obviously decreased(P<0.01). There was no significant difference in the expression of t-Aktamong all groups (P>0.05). The expression of p-CREB showed a tendencysimilar to p-Akt. There was no significant difference in the expression of t-CREB among all groups (P>0.05).
Compared with sham group, at day34following BCCAO, chroniccerebral hypoperfusion significantly decreased the expression of Bcl-2andincreased the expression of Bax, thus markedly reduced the ratio of Bcl-2/Bax(all P<0.01). Administration of LXA4ME (100ng/d) significantly reversedthe downregulation of Bcl-2(P<0.01) and the upregulation of Bax (P<0.01)and increased the expression ratio of Bcl-2/Bax (P<0.01). Interestingly, thecombined use of LY294002and LXA4ME (100ng/d) blocked the increase ofBcl-2(P<0.01) and the decrease of Bax (P<0.01) induced by LXA4ME only,and decreased the ratio of Bcl-2/Bax (P<0.01).
Conclusion: Administration of LXA4ME during early phase of chroniccerebral hypoperfusion can provide long-term neuroprotection, inhabit theapoptosis of neurons, and improve cognitive impairments induced by chroniccerebral hypoperfusion. The activation of PI3K/Akt signaling pathway may beinvolved in the long-term neuroprotection of LXA4ME.
PartⅢ Effects of LXA4ME on postsynaptic density of rats with VD
Objective: To observe the effects of LXA4ME on the thickness of PSDand the expression of PSD-95in CA1region of rats underwent chroniccerebral hypoperfusion, and provide more insights into the neuroprotectivemechanisms of LXA4ME.
Methods: Adult male Sprague–Dawley rats weighing250-300g were provided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into three groups: Sham group,vehicle group, LXA4ME100group.100ng LXA4ME was dissolved in5μlnormal saline. Rats in LXA4ME100group received100ng LXA4ME (i.c.v.)immediately after BCCAO. Rats in sham group and vehicle group received5μl normal saline (i.c.v.). All rats received LXA4ME dissolved in normalsaline or normal saline according to the aforementioned experimental planevery24h up to14days afterwards.
At day34following BCCAO, three rats chosen randomly from eachgroup were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with4%paraformaldehyde. The thickness of PSD in CA1area ofhippocampus was observed through the electron microscopy. Furthermore,another six rats chosen randomly from each group were fully anesthetized andperfused with4%paraformaldehyde through the left cardiac ventricle. Thebrains were then embedded in paraffin. Coronal brain sections were cut andimmunohistochemically stained for PSD-95. IOD/area value was used toindicate the mean optical density for each slice.
Results: At day34following BCCAO, the thickness of PSD in CA1areaof hippocampus in vehicle group was34.61±5.66nm，which was significantlyless than that of sham group (49.65±4.84nm)(P<0.01); Meanwhile, thethickness of PSD of LXA4ME100group was47.53±6.10nm，which wasevidently more than that of vehicle group (P<0.01).
Immunohistochemical results showed that extensively positive stainingfor PSD-95was seen in the cytoplasm of neurons in hippocampus CA1regionof rats in sham group. The IOD/area value of PSD-95markedly decreased invehicle group compared with sham group (P<0.01). However, compared withsham group, administration of LXA4ME (100ng/d) significantly increased thepositive expression of PSD-95(P<0.01).
Conclusion: Administration of LXA4ME can promote the expression ofPSD-95and increase the thickness of PSD, which contribute to the neuroprotective effects of LXA4ME.
引文
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