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  新医学  2016, Vol. 47 Issue (12): 803-808  DOI: 10.3969/j.issn.0253.9802.2016.12.003
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江滢, 刘莹莹, 马丽丽, 陈晓红. 多奈哌齐抑制实验性自身免疫性脑脊髓炎小鼠模型血脑屏障渗透性的研究[J]. 新医学, 2016, 47(12): 803-808.
Jiang Ying, Liu Yingying, Ma Lili, Chen Xiaohong. Effect of donepezil on inhibiting blood-brain barrier permeability in mouse models with experimental autoimmune encephalomyelitis[J]. Journal of New Medicine, 2016, 47(12): 803-808.

基金项目

广东省医学科研基金(A2014233)

通讯作者

陈晓红,E-mail:xiaohongchenzssy@aliyun.com

文章历史

收稿日期:2016-08-23
多奈哌齐抑制实验性自身免疫性脑脊髓炎小鼠模型血脑屏障渗透性的研究
江滢, 刘莹莹, 马丽丽, 陈晓红     
510630 广州,中山大学附属第三医院神经病学科
摘要: 目的 探讨多奈哌齐对实验性自身免疫性脑脊髓炎(EAE)小鼠模型血脑屏障渗透性的影响。 方法 将48只6~8周龄雌性C57BL/6小鼠分为多奈哌齐组(19只)、PBS组(19只)与空白对照组(10只)。将多奈哌齐组及PBS组制备为EAE小鼠模型,在接种免疫后第13日,将多奈哌齐2 mg/(kg·d)溶解于PBS中对多奈哌齐组进行灌胃;而PBS组只予PBS进行灌胃。对EAE小鼠进行临床症状评分和组织学评估(炎症评分及脱髓鞘评分),利用伊文思蓝对EAE小鼠脑组织的蓝染程度评估血脑屏障渗透性,用蛋白免疫印迹法检测EAE小鼠脑组织中基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的表达情况。 结果 多奈哌齐组的临床症状评分低于PBS组的临床症状评分(t=4.351, P < 0.001)。多奈哌齐组的炎症评分低于PBS组(t=6.325, P < 0.001);PBS组的腰髓有大片的髓鞘脱失,但多奈哌齐组脱髓鞘病变较其少,2组评分比较差异有统计学意义(t=3.308, P=0.008)。与空白对照组相比,PBS组(t=8.621, P < 0.001)和多奈哌齐组(t=9.974, P < 0.001)脑内伊文思蓝含量均较高;与PBS组相比,多奈哌齐组脑内伊文思蓝含量较低(t=2.286, P=0.045)。与PBS组相比,多奈哌齐可抑制MMP-2(t=4.679, P=0.010)和MMP-9 (t=5.822, P=0.001)的表达。 结论 多奈哌齐可降低EAE小鼠血脑屏障的渗透性。
关键词: 多奈哌齐    实验性自身免疫性脑脊髓炎    血脑屏障    基质金属蛋白酶    
Effect of donepezil on inhibiting blood-brain barrier permeability in mouse models with experimental autoimmune encephalomyelitis
Jiang Ying, Liu Yingying, Ma Lili, Chen Xiaohong     
Department of Neurology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
Corresponding author: Chen Xiaohong, E-mail:xiaohongchenzssy@aliyun.com
Abstract: Objective To evaluate the effect of donepezil upon inhibiting the blood-brain barrier permeability in mouse models with experimental autoimmune encephalomyelitis (EAE). Methods Forty eight female C57BL/6 mice, aged 6-8 weeks, were divided into the donepezil (n=19), phosphate buffer saline (PBS, n=19) and control groups (n=10). EAE mouse models were established in the donepezil and PBS groups. In the donepezil group, intragastric gavage of PBS containing 2 mg/(kg·d) of donepezil was delivered at 13 d after innoculation, whereas PBS alone was administered in the PBS group. The grading of clinical symptoms and histological evaluation were performed in EAE mice (inflammation and demyelinating scores). The blood-brain barrier permeability was assessed by Evans blue staining. The expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 were measured by western blot. Results The score of clinical symptoms in the donepezil group was significantly lower compared with that in the PBS group (t=4.351, P < 0.001). The inflammation score in the donepezil group was considerably lower than that in the PBS group (t=6.325, P < 0.001). In the PBS group, a large mass of myelinoclasis was observed, whereas demyelinating lesion was seldom seen in the donepezil group with statistical significance (t=3.308, P=0.008). Compared with the control group, intracerebral content of Evans blue in the PBS (t=8.621, P < 0.001) and donepezil groups (t=9.974, P < 0.001) was significantly higher. However, intracerebral content of Evans blue in the donepezil group was significantly less compared with that in the PBS group (t=2.286, P=0.045). Compared with the PBS group, donepezil could significantly down-regulate the expression levels of MMP-2 (t=4.679, P=0.010) and MMP-9 (t=5.822, P=0.001). Conclusion Donepezil can reduce the blood-brain barrier permeability in EAE mouse models.
Key words: Donepezil    Experimental autoimmune encephalomyelitis    Blood-brain barrier    Matrix metalloproteinase    

多发性硬化(MS)是最常见的发生于青年人的中枢神经慢性炎性脱髓鞘疾病[1]。实验性自身免疫性脑脊髓炎(EAE)小鼠是研究MS免疫病理学机制及其实验治疗的最佳动物模型[2]。研究显示,EAE的特征性病理变化是血脑屏障破坏和T细胞进入中枢神经系统,导致脑内细胞因子、基质金属蛋白酶(MMP)和自由基的含量升高,最终导致髓鞘脱失和神经传导受阻[1, 3]。多奈哌齐是一种具有高选择性的胆碱酯酶抑制剂,可用于阿尔茨海默病的治疗,且目前发现其具有抗炎效应及神经保护作用[4-7]。虽然多奈哌齐已经被用于治疗MS患者和EAE小鼠的认知功能障碍,但其在EAE/MS中的抗炎作用目前尚未清楚[8-10]。在本研究中,我们探讨了多奈哌齐对EAE小鼠模型的血脑屏障渗透性及MMP-2/9的影响,现将结果报告如下。

材料与方法 一、动物和试剂

6~8周龄雌性C57BL/6小鼠(体质量16~18 g)48只,购自中山大学动物实验中心。将小鼠分为多奈哌齐组19只(其中7只用于临床症状评估、6只用于组织学评估及MMP-2/9蛋白免疫印迹测定、6只用于血脑屏障受损的评价),PBS组19只(只数分配与多奈哌齐组相同)与空白对照组10只(其中6只作为血脑屏障受损的评价对照、4只作为MMP-2/9蛋白免疫印迹测定对照)。多奈哌齐和完全弗氏佐剂(CFA)购自Sigma公司。MOG35-55(MEVGWYRSP FSRVVHLYRNGK)肽段由CL.BioScientific公司合成(中国西安)。纯度超过95%的结核分支杆菌H37Ra购自Difco公司,百日咳毒素(PTX)购自Alexis公司。单克隆MMP-2抗体和多克隆MMP-9抗体分别购自Thermo Fisher Scientific公司和Abcam公司。

二、EAE小鼠模型的诱导和临床症状评估

采用经典的MOG35-55肽段免疫诱导方法将多奈哌齐组及PBS组制备为EAE小鼠模型[11]。首先,将200 μg MOG35-55肽段乳化于含结核分支杆菌H37RA 500 μg的完全弗氏剂中,并对2组小鼠皮下注射混合后的完全弗氏剂,同时腹腔注射含PTX 300 ng的PBS,并在48 h后腹腔注射相同剂量的PTX,在第7日追加皮下注射含MOG35-55肽段的完全弗氏剂。在接种免疫后第13日,将多奈哌齐2 mg/(kg·d)(此剂量的选择来源于已发表文章的数据和寻找最佳剂量的预实验)溶解于PBS中对多奈哌齐组进行灌胃[8];而PBS组只予PBS进行灌胃。每日评估EAE小鼠的残疾程度,临床症状评分标准如下:0分,无标志;1分,尾部张力减退;2分,松弛尾;3分,后肢共济失调和(或)轻瘫;4分,后肢完全瘫痪;5分,垂死或死亡[11]。在我们的实验中,我们定义个体小鼠出现第一种症状的时间为疾病的发病时间,临床症状评分的均值为在实验中每只小鼠的临床症状评分总和的平均值。临床症状评分越高,个体小鼠的病情越重。

三、组织学评估

应用常规苏木素-伊红(HE)染色和依来铬花青浸渍染色对EAE小鼠腰髓组织分别进行炎性浸润和脱髓鞘评估。炎症评分量表如下所示:0分,没有炎性细胞;1分,一些散在的炎性细胞;2分,炎性细胞浸润在血管周围;3分,广泛血管周围袖套带形成并扩展到相邻实质,或实质浸润无明显的袖套[12]。腰髓的脱髓鞘情况据文献[13]报导的评分细则进行评分:1分,软脑膜下脱髓鞘;2分,软脑膜下及血管周围发生明显的脱髓鞘;3分,血管周围或软膜下脱髓鞘发生融合;4分,血管周和软脑膜下脱髓鞘,并累及一个半腰髓,伴随着大量细胞侵入中枢神经系统实质;5分,血管周和软脑膜下脱髓鞘,并累及整个腰髓,伴随着大量细胞侵入中枢神经系统实质。

四、血脑屏障受损的评价

我们用伊文思蓝染色评估空白对照组、PBS组和多奈哌齐组血脑屏障的完整性[14]。2%伊文思蓝染色剂均经过灭菌消毒。在小鼠接种免疫后第20日,每只小鼠静脉接受的伊文思蓝剂量为4 ml/kg。静脉注射伊文思蓝30 min后,伊文思蓝染色液向全身组织扩散,用生理盐水冲洗掉血管内的伊文思蓝染色液。迅速取出小鼠脑组织并称重,用2.5 ml PBS和2.5 ml 60%的三氯乙酸混合液浸泡脑组织,以达到沉淀蛋白质的目的。样品在1 000×g的离心力下离心30 min。取上清液,用分光光度计在610 nm波长下测量EAE小鼠脑内的伊文思蓝含量(EB的吸光度)。根据伊文思蓝的溶液标准曲线计算其含量。

五、MMP-2/9蛋白免疫印迹测定

MMP (尤其是MMP-2/9)可以增加EAE小鼠和MS患者的血脑屏障渗透性,我们用蛋白免疫印迹法检测空白对照组、PBS组和多奈哌齐组脑内的MMP-2和MMP-9的表达。各组小鼠脑组织均用10% SDS-聚丙烯酰胺凝胶(每孔含有20 μg)进行电泳,然后把所分离的蛋白转移至PVDF膜(Bio-Rad),并用5%的脱脂奶粉封闭,接着分别加入多克隆抗MMP-2抗体(1:2 000)和多克隆抗MMP-9抗体(1:2 000)在4℃摇育过夜。在TBST缓冲液洗膜3次后,分别加入辣根过氧化物酶标记的鼠抗体和兔抗体孵育30 min。整个实验重复3次,选择-actin作为内参。最后将所得谱带照相并经Quantity one图像分析软件分析相应谱带密度值,结果以谱带的光密度值和空白地方(没有谱带部位)光密度值的比值表示。

六、统计学处理

采用SPSS 13.0软件处理数据。正态分布数据均采用x±s表示,2组间比较采用t检验(正态分布);多组间比较先行方差分析,差异有统计学意义的,再用LSD-t检验作两两比较。P < 0.05为差异具有统计学意义。

结果 一、多奈哌齐对EAE小鼠临床症状严重程度的影响

PBS组与多奈哌齐组的发病时间无明显差异[(14.0±0.3) d vs. (14.7±0.2) d, t=1.987, P=0.070]。多奈哌齐组的临床症状评分为(1.08±0.13)分,而PBS组的临床症状评分为(2.05±0.18)分,比较差异有统计学意义(t=4.351, P < 0.001)。

二、多奈哌齐对EAE小鼠病理结果的影响

多奈哌齐组的炎症评分明显低于PBS组[(1.50±0.18)分 vs. (2.83±0.11)分, t=6.325, P < 0.001]。此外,在接种免疫后第20日,我们发现PBS组的腰髓有大片的髓鞘脱失,但多奈哌齐组脱髓鞘病变较其少,2组评分比较差异有统计学意义[(1.92±0.20)分 vs. (2.67±0.12)分, t=3.308, P=0.008]。2组典型炎症病变和脱髓鞘病变的切片如图 1所示。

图 1 多奈哌齐组和PBS组腰髓组织病理结果 A、a:PBS组HE染色切片,可见普遍存在着炎性浸润与广泛的血管袖套形,A为×40,a为×400;B、b:多奈哌齐组HE染色切片,炎性浸润与血管袖套形成较PBS组少,B为×40,b为×400;C、c:PBS组依来铬花青浸渍染色切片,可见大片的脱髓鞘斑块形成,C为×40,c为×400;D、d:多奈哌齐组依来铬花青浸渍染色切片,脱髓鞘斑块较PBS组少,D为×40,d为×400
三、多奈哌齐对EAE小鼠血脑屏障的影响

空白对照组、PBS组和多奈哌齐组脑内伊文思蓝含量分别为(67.2±3.2) mg/g、(185.8±13.4) mg/g与(150.5±7.7) mg/g。与空白对照组相比,PBS组(t=8.621, P < 0.001)和多奈哌齐组(t=9.974, P < 0.001)小鼠脑内伊文思蓝含量均较高。但与PBS组相比,多奈哌齐组脑内伊文思蓝含量较低(t=2.286, P=0.045)。

四、多奈哌齐对EAE小鼠脑内MMP-2和MMP-9表达的影响

与空白对照组相比,PBS组MMP-2 (5.60±0.65 vs. 0.54±0.25, t=7.286, P=0.002)和MMP-9 (10.93±0.87 vs. 2.61±0.49, t=8.322, P < 0.001)的表达更高,而与PBS组比较,多奈哌齐可以抑制EAE小鼠脑内MMP-2 (1.95±0.44 vs. 5.60±0.65,t=4.679, P=0.010)和MMP-9 (4.45±0.70 vs. 10.93±0.87, t=5.822, P=0.001)的表达,见图 2

图 2 不同组小鼠脑内MMP-2和MMP-9的表达情况 与空白对照(control)组相比,**P < 0.01,与PBS组比较,##P < 0.01
讨论

据文献报道,多奈哌齐可以改善MS患者和EAE小鼠的认知功能[8-10, 15-16]。在本研究中,我们观察到多奈哌齐可以减轻EAE小鼠病情的严重程度及其腰椎细胞浸润和脱髓鞘征象,这恰恰与PBS组截然相反,近期也有研究显示多奈哌齐可以快速增加调节少突胶质细胞分化及髓鞘化的髓鞘相关基因的转录水平[17]。以上结果表明多奈哌齐对于疾病临床期的EAE小鼠是具有治疗作用的。

多奈哌齐发挥抗炎作用的潜在机制是多因素的。De Vries等[18]发现,血脑屏障渗透性升高,导致大量的白细胞进入中枢神经系统,这一病理变化在MS和EAE小鼠疾病的发生和进展中扮演着重要角色。MMP-2和MMP-9可能与T细胞迁移进入中枢神经系统和血脑屏障破坏相关,它们可以特异性降解Ⅳ型胶原,Ⅳ型胶原是血管基底膜的一个核心结构成分,其降解会导致血脑屏障的破坏[19-22]。在EAE小鼠出现临床症状时,MMP-2和MMP-9的表达达到高峰,在MS患者的脑组织中也存在相同现象[23-25]。在本研究中,我们发现血脑屏障渗透性的升高与MMP-2和MMP-9的上调同时存在,即多奈哌齐能降低MMP的表达和血脑屏障的渗透性[26]

目前,多奈哌齐已被用于治疗EAE小鼠及MS患者的认知功能障碍[8-10]。我们的研究进一步表明多奈哌齐可以减轻EAE小鼠疾病的严重程度、降低EAE小鼠中血脑屏障的渗透性及MMP-2/9的表达,提示在疾病临床期予以多奈哌齐可以减轻EAE小鼠炎症及炎症介导的一系列病理生理反应,而通过抑制MMP-2/9的表达改善血脑屏障渗透性的效应可能是多奈哌齐治疗EAE的一个机制,提示多奈哌齐在MS的治疗上具有应用前景。

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