Effects and Mechanisms of Xueshuantong on the Cognitive Function and Abnormal Neural Excitability in Mice with Alzheimer’ s Disease
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摘要:
目的 探究血栓通[主要有效成分为三七皂苷（panax notoginseng，PNS）]对阿尔茨海默症（Alzheimer’s disease，AD）模型小鼠认知功能及神经兴奋性的影响，并探讨其潜在分子机制。 方法 用APP/PS1小鼠作为AD研究动物模型，在小鼠淀粉样蛋白尚未检测到阶段（2月龄）开始每日以60 mg/kg对血栓通组（APP/PS1+PNS）行灌胃给药，每日1次，连续给药6个月（给药至8月龄）；对照组小鼠予同等体积的0.9%氯化钠（APP/PS1+vehicle）灌胃处理，同月龄野生型小鼠予0.9%氯化钠灌胃处理作为正常对照组（WT+ vehicle），每组各15只。6个月后，新物体识别实验、Morris水迷宫实验检测小鼠的认知功能；EEG脑电检测、Western blot、细胞表面生物素化试验以检测各组小鼠皮质与海马中BACE1的活性、Nav1.1α的分布、表达以及Navβ2的表达与酶解情况（Navβ2的酶解片段Navβ2 full length及Navβ2-CTF表达检测）。 结果 新物体识别实验显示，与对照组APP/PS1小鼠相比，血栓通用药后APP/PS1小鼠的辨别指数（discrimination index，DI）上升（P < 0.05）；Morris水迷宫检测结果发现，血栓通灌胃6个月后小鼠在探索实验中逃避潜伏期缩短（P < 0.05），撤除平台后在目标象限停留时间增加（P < 0.05）、穿梭平台次数增加（P < 0.05）；EEG脑电检测结果发现，血栓通给药后减少了APP/PS1小鼠棘波放电出现的频率（P < 0.05）。血栓通给药后显著降低了BACE1蛋白水平的表达（P < 0.05），而全长片段Navβ2的蛋白水平显著上升（P < 0.05），并纠正了Nav1.1α在神经元内外的异常分布（P < 0.05）。 结论 血栓通可以改善AD模型小鼠的学习记忆能力、纠正大脑异常兴奋性，其作用机制可能与抑制BACE1的活性从而减少Navβ2由 APP/PS1诱导的过度酶解，纠正皮质、海马神经元Nav1.1α的异常表达与分布，调节神经元的兴奋性有关。 Abstract:Objective To explore the possible effects and the underlying molecular mechanisms of xueshuantong [The main active component is panax notoginseng （PNS）] on the cognitive function and neural excitability of mice with Alzheimer’ s disease （AD）. Methods The APP/PS1 mice were used as an animal model for AD research, at the stage when amyloid protein was not detected in mice （2 months of age）. Mice in the xueshuantong group （APP/PS1+PNS） were administered by gavage once a day at a dose of 60 mg/kg for six months （for 8 months of age）. The mice of the control group were given 0.9% sodium chloride （APP/PS1+Vehicle） intragastric treatment of the same volume, while the wild-type mice of the same age were given 0.9% sodium chloride intragastric treatment as the normal control group （WT+Vehicle） （15 mice in each group, n=15）. After six months, the cognitive function of the mice was evaluated by the Novel Object Recognition （NOR） task and Morris Water Maze （MWM） test. The activity of BACE1, the distribution and expression of Nav1.1α, as well as the expression and enzymatic hydrolysis of Navβ2 （Navβ2 full-length and Navβ2-CTF fragments） in cortex and hippocampus were detected by EEG, Western blot and cell surface biotinylation assay, respectively. Results The NOR task showed that compared with the mice in the APP/PS1+Vehicle group, the Discrimination index （DI） of mice in the APP/PS1 group was significantly increased after xueshuantong administration （P < 0.05）. The MWM test found that, the escape latency of the mice in the xueshuantong group was shortened followed six months in gastric administration （P < 0.05）, while the stay time in the target quadrant and the number of platforms significantly increased （P < 0.05） after the removal of the platform. The results of EEG recording showed that xueshuantong reduced the frequency of spike-wave discharges in APP/PS1 mice （P < 0.05）. Furthermore, xueshuantong significantly reduced the expression of BACE1 （P < 0.05）. In the APP+PNS group, the expression of Navβ2 full-length was increased （P < 0.05）, as well as corrected the abnormal distribution of Nav1.1α inside and outside of neurons （P<0.05）. Conclusion Treatment with xueshuantong can significantly improve the learning and memory ability and correct the abnormal excitability of the brain in AD model mice. The mechanism may be related to the inhibition of BACE1 activity, the reduction of APP/PS1-induced excessive enzyme digestion of Navβ2, the correction of the abnormal expression and distribution of Nav1.1α in cortical and hippocampal neurons, as well as the subsequent regulation of neuronal excitability. -
Key words:
- Xueshuantong /
- Alzheimer’ s disease /
- Cognition /
- BACE1 /
- The enzymolysis of Navβ2 /
- The distribution of Nav1.1α
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九十年代初期的研究表明，在动脉粥样硬化（atherosclerosis，AS）形成过程中，动脉内膜和外膜层聚集着肥大细胞（mast cells，MCs），首次提出MCs参与AS进程[1]。
血管的微环境一旦发生变化，炎性细胞便会从血管外膜的滋养血管中渗出，参与疾病的变化[2-3]。MCs激活时释放出来的趋化因子、细胞因子、组胺、白三烯等炎症因子[4]使血管的通透性增加，导致单核巨噬细胞和淋巴细胞聚集到斑块处[5]，释放出大量的中性蛋白酶，可以加速低密度脂蛋白（low-density lipoprotein，LDL）进入到血管内膜局部并聚集[6]，促进AS斑块形成。
在骨髓细胞向MCs分化及活化过程中白细胞介素3（interleukin-3，IL-3）和干细胞因子（stem cell factor，SCF）起到重要作用[7-8]。本研究旨在通过提取乳兔骨髓细胞，利用IL-3、SCF、β-巯基乙醇联合作用诱导培养骨髓来源肥大细胞并鉴定其纯度与活性，为进一步研究肥大细胞与AS的关系提供依据。
1. 材料与方法
1.1 材料
IL-3、SCF（美国Peprotech公司）；FBS、DMEM、DMEM/F12、RPMI-1640（美国Gibco公司）；实验动物：2周龄乳兔，昆明医科大学实验动物学部，环境条件符合国家实验动物环境及设施标准要求，室内保持安静、清洁、干燥和通风。自由饮水。实验动物使用许可证号：SCXK（滇）2015-0002。
1.2 方法
1.2.1 兔骨髓细胞的获取
麻醉：（经2周龄乳兔耳缘静脉注射3%戊巴比妥钠，1 mL/kg）→浸泡（75%C2H5OH，10 min）→剥离（后肢皮肤切开，逐层分离，剥离肌肉）→暴露股骨→取出→浸泡（75% C2H5OH的无菌培养皿中浸泡3～5 min）→股骨两端剪去→吹打骨髓（吸取DMEM高糖培养基后吹打骨髓，股骨变白）→获取细胞（1000 r/min，5 min。如果在获取的细胞中有较多红细胞，则应用红细胞裂解液进行处理）→培养体系（含84 mL DMEM高糖培养基、15 mL FBS、1 mL双抗）。
1.2.2 细胞的培养
接种到T25瓶中（含DMEM高糖培养基）→隔天换液→细胞铺满→消化细胞→新培养瓶→贴壁→改用F12诱导培养基（含84 mL DMEM/F12、15 mL FBS、1 mL双抗、10 ng/mL SCF、10 ng/mL IL-3及0、10×10-5 mol/L的β-巯基乙醇）→放置37 ℃、5% CO2 饱和湿度环境→隔天换液→镜下观察细胞有变圆→更换为1640促成熟培养基（含84 mL RPMI 1640、15 mFBS、1 mL双抗、10 ng/mL SCF、10 ng/mL IL-3及0、10×10-5 mol/L的β-巯基乙醇）→7 d换液→直至细胞诱导成熟。
1.2.3 肥大细胞功能学鉴定
诱导成熟的重悬细胞液→载玻片→干燥→甲苯胺蓝染色→95% C2H5OH脱色→清洗→镜下观察。
1.2.4 肥大细胞形态学鉴定
通过光镜下观察不同培养时间的细胞形态，直至诱导成熟。
消化细胞→洗2次（PBS）→细胞密度1×10 7个/mL→每管100 μL→单染管各加入5.0 μL的 CD117-PE抗体及FcεR1α-FITC抗体→双染管加两种抗体→空白对照管（无抗体）→避光孵育30 min→每管加1 mL PBS→离心10 min（400 g）→去上清→上法洗涤1次→去上清→每管加100 μL PBS→检测。
2. 结果
2.1 光镜下观察肥大细胞生长形态
2 d后开始出现以形态为长梭形的贴壁细胞；7 d时：细胞基本将培养瓶铺满；2周时：细胞逐渐变形；4周时：细胞基本变为圆形，悬浮细胞变多；6周时：细胞基本变为类圆形，且形态均匀具有折光性（图1）。
图 1 肥大细胞变形过程（100×）A：7 d时长梭形贴壁细胞；B：2周时细胞逐渐变形；C：4周时细胞逐渐变圆；D：6周时变为类圆形细胞。Figure 1. Changes in mast cells（100×）2.2 肥大细胞的甲苯胺蓝染色
培养6周细胞成熟后利用甲苯胺蓝染色：可以观察到细胞质为紫红色，细胞核为蓝色。异染颗粒的细胞加入β-巯基乙醇比不加入β-巯基乙醇的能得到更多（图2）。
图 2 甲苯胺蓝染色（100×）A：β-巯基乙醇浓度0 mol/L；B：β-巯基乙醇浓度10×10-5 mol/L。Figure 2. Toluene blue staining（100×）2.3 流式细胞术检测肥大细胞
培养6周收集细胞，利用流式细胞术检测细胞表面CD117及FcεR1α的表达情况，加入β-巯基乙醇双阳性细胞的比例达96.2%，大于不加入β-巯基乙醇的（图3），且与不加β-巯基乙醇相比有统计学意义（P < 0.05），见表1。
图 3 流式细胞仪检测细胞的双阳性率A：β-巯基乙醇为0 mol/L；B：β-巯基乙醇为10×10-5 mol/L。Figure 3. Double positive rate of cells detected by flow cytometry表 1 不同浓度β-巯基乙醇诱导后CD117+ FcεRIα+肥大细胞的比例Table 1. The proportion of CD117+ FcεRIα+ mast cells induced by different concentrations of β-mercaptoethanolβ-巯基乙醇
（mol/L）实验次数 CD117+ FcεRIα+
肥大细胞的比例（%）P 0 5 48.97 ± 3.12 0.7069 10×10−5 5 95.64 ± 4.66# 与β-巯基乙醇浓度为0 mol/L比较，#P < 0.05。 3. 讨论
在正常人的心脏、主动脉及脂肪组织中有少量的MCs存在，当人类发生疾病时MCs的数量就会增多，比如AS。近年来，研究表明MCs在AS的发生、发展及斑块稳定性中有着重要作用[9]。
当下有研究认为MCs是利用其表面的趋化因子受体-3与AS斑块中表达的嗜酸细胞活化CC趋化因子亚族中趋化因子配体-11结合而向病变部位聚集[10]。MCs起源于骨髓造血干细胞[11]，促进MCs成熟的分子主要有SCF和神经生长因子（nerve growthfactor，NGF）[17]，SCF可能在MCs向受累区域聚集发挥作用，而MCs侵入受累区域可能会进一步导致更多的炎性细胞浸润，促进AS斑块形成。
IL-3又被叫做肥大细胞生长因子，在MCs的生长、分化、迁移和效应中具有重要作用。活化T细胞、天然杀伤（NK）细胞和MCs都可产生IL-3，且对于SCF在MCs前体的发生、发展、扩增具有促进意义[12-13]。SCF的配体CD117（c-kit）除在其表面外，各种造血祖细胞中均可存在。MCs发挥重要表面标志的是FcεRIα，但FcεRIα还表达于嗜酸性粒细胞等细胞表面，只有MCs同时表达CD117和FcεRIα[14]。甲苯胺蓝染色是一种常用于识别及判断MCs功能状态的方法，同时也是MCs的特异性染色，可染细胞核为蓝色，胞质内为异染性紫红色颗粒，说明其具有吞噬功能[15]。通过甲苯胺蓝染色和流式细胞仪检测结果对诱导获得的MCs的纯度进行鉴定，就目前来说，利用兔的骨髓间质干细胞来诱导培养MCs的报道是极少的。
本研究选用2周龄的乳兔来获取骨髓间质干细胞，因为当兔龄大于或者小于2周龄时，笔者发现细胞生长速度变得缓慢，可能是2周龄的骨髓间质干细胞的活性更好，更有利于细胞的培养。当IL-3浓度10 ng/mL、SCF浓度为10 ng/mL，加入β-巯基乙醇时：甲苯胺蓝染色及流式检测均较不加入β-巯基乙醇时可以获取更多、双阳性率更高的MSc，原因可能为β-巯基乙醇作为一种还原剂，在降低氧对细胞产生氧化损伤的同时促进干细胞生长。所以该培养体系是一种良好的体外诱导培养体系，其操作简单，且能获得更加成熟、更具有典型特征的MCs，为下一步对兔行进炎性损伤研究提供了优势。综上所述，本研究利用形态学、功能学2个方面对诱导的兔骨髓来源MCs进行鉴定，培养出的细胞不仅具有成熟MCs的生物学特性，还具有其功能，这便为后续的基础研究及临床研究奠定基础。
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图 1 Morris水迷宫任务显示血栓通对APP/PS1小鼠空间学习记忆变化的影响
A：各组小鼠在训练期间从第1天到第6天的逃避潜伏期；B：探针实验测试期间在目标象限所花费的时间；C：探针实验中在目标平台的穿梭次数；D：探针实验中APP/PS1小鼠的游泳路径。（$ \bar x \pm s $，n=15），*P < 0.05 vs. WT+Vehicle，#P<0.05 vs. APP/PS1+PNS。
Figure 1. Morris water maze task shows the effect of xueshuantong on spatial learning and memory changes in APP/PS1 mice
图 2 血栓通对APP/PS1小鼠新物体识别任务的影响
A：APP/PS1小鼠对熟悉物体和新物体的探索时间的比较；B：从不同的组中获得的DI指数。（$ \bar x \pm s $，n=15），*P < 0.05 vs WT+Vehicle，#P < 0.05 vs APP/PS1+PNS，&P < 0.05 vs Novel。
Figure 2. Effect of xueshuantong on novel object recognition task in APP/PS1 mice
图 3 通过EEG记录检测血栓通给药诱导的神经元高兴奋性改善和异常神经活动
A：2月龄各组小鼠的EEG记录情况；B：8月龄各组小鼠的EEG记录情况。
Figure 3. Detection of improved neuronal hyperexcitability and abnormal neural activity induced by xueshuantong administration by EEG recordings
图 4 血栓通改变APP/PS1小鼠中Nav1.1α的分布和Navβ2的裂解
A：各组小鼠额叶皮层和海马中BACE1、Navβ2全长、Navβ2-CTF的蛋白电泳图；B：各组小鼠额叶皮层和海马中BACE1的蛋白表达比较；C：各组小鼠额叶皮层和海马中Navβ2全长的蛋白表达比较；D：各组小鼠额叶皮层和海马中Navβ2-CTF的蛋白表达比较；E：各组小鼠额叶皮层和海马中的Nav1.1α的总量、细胞外Nav1.1α和细胞内Nav1.1α的蛋白电泳图；F：各组小鼠额叶皮层和海马中Nav1.1α总量的蛋白表达比较；G：各组小鼠额叶皮层和海马中细胞外Nav1.1α的蛋白表达比较；H：各组小鼠额叶皮层和海马中细胞外Nav1.1α的蛋白表达比较。（$ \bar x \pm s $，n=15），*P<0.05 vs. WT+ Vehicle，#P<0.05 vs. APP/PS1 + PNS。
Figure 4. Xueshuantong alters Nav1.1α distribution and Navβ2 cleavage in APP/PS1 mice
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