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“益肾调督”电针法对Aβ1-42诱导的痴呆模型大鼠海马中 Aβ相关清除酶的影响

作者:孙梦晓 来源:本站原创 点击:134次 更新:2018-12-19

汤双红,肖佳欢,王 芸,陶一鸣,杜艳军

(1 湖北中医药大学针灸骨伤学院,武汉 430061)

[摘 要]  目的:探讨“益肾调督”电针法对阿尔茨海默症Aβ相关清除酶的影响。方法:将40只wistar雄性大鼠随机分为正常组、假手术组、模型组和电针组,每组各10只,正常组常规饲养,模型组与电针组经双侧海马各注射5 uL Aβ1-42,假手术组于双侧海马注射等量生理盐水,造模后常规饲养。电针组于造模后百会及肾俞穴电针刺激,5HZ,连续波,每次15 min,每日1次,连续治疗15 d。用Morris水迷宫实验检测各组大鼠记忆和空间探索能力,Western-blot法检测各组大鼠海马组织中脂蛋白酯酶(LPL)、胰岛素降解酶(IDE)、转甲状腺素蛋白(TTR)、载脂蛋白E(ApoE)、α2巨球蛋白(α2M)及β淀粉样蛋白1-42(Aβ1-42)的表达。结果:与假手术组比较,模型组大鼠的平均逃避潜伏期明显延长(P<0.01),跨越原平台次数和在有效区停留时间明显缩短(P<0.01);与模型组比较,治疗组大鼠平均逃避潜伏期明显缩短(P<0.01),跨越原平台次数和在有效区停留时间明显延长(P<0.01)。与假手术组相比,模型组LPL、IDE、TTR、ApoE及α2M在海马组织中的表达显著减弱(P<0.05或P<0.01),Aβ1-42在海马中的表达显著增强(P<0.01);与模型组相比,电针组LPL、IDE、TTR、ApoE及α2M在海马组织中的表达显著增强(P<0.05或P<0.01),Aβ1-42在海马中的表达显著减弱(P<0.01)。结论:“益肾调督”电针法可上调Aβ1-42诱导的AD模型大鼠海马组织中LPL、IDE、TTR、ApoE及α2M的表达,促进β-淀粉样蛋白的降解,有助于改善AD病理变化,延缓AD病程的发展。

[关键词]  “益肾调督”;电针;痴呆;β-淀粉样蛋白;脂蛋白酯酶;胰岛素降解酶;转甲状腺素蛋白;载脂蛋白E;α2-巨球蛋白

Effects of “ Supplementing Kidney and Regulating Du Meridian” with Electroacupuncture on A-beta Related Enzymes in the Hippocampus of Rats Model of Dementia Induced by A-beta 1-42

TANG Shuanghong, XIAO Jiahuan, WANG Yun, TAO Yiming, DU Yanjun

Abstract: Objective:To explore the effect of electroacupuncture therapy of ”Supplementing kidney and regulating Du Meridian” on A beta related enzymes in Alzheimer’s disease. Methods: Forty male Wistar rats were randomly divided into 4 groups: a normal group, a sham-operation group, a model group and an electroacupuncture group, 10 rats in each group. The normal group was normally bred, while both the model and electroacupuncture groups werebilaterally injected in the hippocampus with 5μL of Aβ1-42 on each side. As an injection control, the sham operation group was  bilaterally injected in the hippocampus in an identical manner with 5 μL of saline and they were otherwise normally bred after modeling. Rats in the electroacupuncture group received electroacupuncture therapy of five hertz continuous wave at both the “Băihuì (百会 GV20)”and bilateral“Shènshū (肾俞 BL23)” points for a duration of 15 minutes per treatment every day continuously for 15 days. At the conclusion of the treatments, Morris water maze test was used to detect memory and spatial exploration ability of rats in each group, the hippocampal expression levels of insulin degrading enzyme (IDE), lipoprotein (LPL), transthyretin (TTR), apolipoprotein E (ApoE) , α2 macroglobulin (α2M) and Aβ1-42 were probed by Western blot. Results Compared with the control group, the average escape latency of the model group was significantly prolonged (P < 0.01), the number of crossing the original platform and the residence time in the effective zone were significantly shortened (P < 0.01), the average escape latency was significantly shortened (P < 0.01), the number of crossing the original platform and the residence time in the effective zone were significantly prolonged (P < 0.01). Compared with the sham group, the expression levels of IDE, LPL, TTR, ApoE, and α2M in the hippocampus were significantly decreased (P <0.05 or P <0.01) and the expression  of Aβ1-42 was significantly increased (P<0.01) in model group. Compared with the model group, the expression levels of IDE, LPL, TTR, ApoE,  and α2M in the hippocampus of these rats were significantly increased (P <0.05 or P <0.01) ,the expression of Aβ1-42  was significantly decreased (P <0.01) in electroacupuncture group. Conclusion Electroacupuncture therapy by Supplementing kidney and Regulating Du Meridian can enhance the expression of IDE, LPL, TTR, ApoE, and α2M in the hippocampus of AD rats injected with Aβ1-42, and may consequently promote the degradation of Aβ1-42 to help improve the pathological  manifestations of AD and therefore delay AD progression.

Keywords:“Supplementing kidney and Regulating Du Meridian”, Electroacupuncture, dementia, beta amyloid protein, lipoprotein, insulin degrading enzyme, transthyretin, apolipoprotein E, alpha-2 macroglobulin

       阿尔茨海默病(Alzhemier’s disease, AD)是一种常见的神经退行性疾病,主要表现为进行性的认知功能障碍及人格的改变。随着社会人口老龄化,AD患病人群逐渐增加,严重影响着老年人的生存质量,成为公共健康面对的最大挑战之一[1]。β淀粉样蛋白(β-amyloid,Aβ)沉积是引起AD病理损伤的中心环节及关键因素[2、3],除了抑制Aβ的产生,促进Aβ的清除也是减轻AD病理损伤的有效途径之一。Aβ在脑内的清除主要有三大途径:经血脑屏障(BBB)转运、细胞内吞作用和经Aβ酶降解系统降解。脑啡肽酶(NEP)和胰岛素降解酶(Insulin-Degrading,IDE)是Aβ降解酶系统中最主要的酶[4、5],α2巨球蛋白(α2-macroglobulin,α2M)、转甲状腺素蛋白(Transthyretin,TTR)、脂蛋白酯酶(lipoprotein ,LPL)及载脂蛋白E(apolipoprotein E,ApoE)则是与Aβ内吞途径密切相关的酶。课题组前期初步证实了针灸能够促进AD大鼠海马脑啡肽酶和前序列蛋白酶的表达[6],参与了Aβ的降解。但其对Aβ其他降解酶和内化酶的影响尚不清楚,故本研究拟通过观察大鼠海马中LPL、IDE、TTR、ApoE、α2M及Aβ1-42的变化,来进一步探讨针灸治疗AD的更多机制。

1.材料与方法

1.1 主要试剂与仪器

       Aβ1-42(美国Sigma公司);多聚甲醛、水合氯醛(固体)、青霉素粉剂及氯化钠固体、牙托粉(国药集团化学试剂有限公司);BCA蛋白质定量试剂盒(碧云天);兔抗大鼠Aβ1-42单克隆抗体、兔抗大鼠IDE单克隆抗体、兔抗大鼠α2M单克隆抗体、兔抗大鼠TTR单克隆抗体、兔抗大鼠LPL单克隆抗体及兔抗大鼠ApoE单克隆抗体(Abcam公司);PBS缓冲液(武汉谷歌生物公司);RIPA裂解液、Bradford蛋白浓度测定试剂盒(碧云天);PVDF膜(Millipore);β-actin抗体(塞维尔生物);牙科钻(成都康发医疗器械有限公司);大鼠脑立体定位仪(成都泰盟科技有限公司); Morris水迷宫(成都泰盟);微量注射器(宁波市镇海玻璃仪器厂);针灸针(0.30×25 mm,苏州环球针灸医疗器械有限公司)。

1.2 实验动物分组

      Wistar大鼠40只,清洁级,(10±2)月龄,雄性,[许可证号:SCXK(鄂)2015-0018],体重(350±30)g,由湖北省实验动物研究中心提供。所有大鼠置于安静、清洁的环境中分笼饲养,室内温度控制在(24±2)℃,定时给予清洁饮水和专用饲料。适应性喂养一周后,无不良反应,饮食、饮水正常。随机分为正常组、假手术组、模型组和电针组,每组10只。整个实验过程遵守国家科技部2006年发布的《关于善待实验动物的指导性意见》中的相关规定。

1.3 模型制备

       大鼠称重,腹腔注射10%的水合氯醛(350mg/kg)麻醉后,俯卧固定于手术台上,使用大鼠脑立体定位仪固定头部。麻醉成功后无菌环境下颅顶区备皮,消毒,沿大鼠颅顶正中线剪开一长约2 cm的切口,钝性分离皮下组织、骨膜,充分暴露前囟。参照《大鼠脑立体定位图谱》[7]定位:前囟后3.3 mm,中线左右各旁开1.5 mm,深度约为3.0 mm,使用微型牙科钻于两侧冠状缝后钻出小孔,取出碎骨屑,保持硬脑膜完整。通过微量注射器缓慢均匀地于每侧海马区域注射寡聚态Aβ1-425 uL,进针深度约为3.0 mm,按1 uL/min的速度5 min内注射完,注射后留针3 min,按1.0 mm/min的速度缓慢出针,以防拔针时药物溢出。术后适量牙托粉封闭颅骨创口,无菌缝合头皮,连续3日肌注10万单位青霉素以防感染。

1.4 处理方法

(1)正常组:常规饲养,不做任何处理。

(2)假手术组:处理方法同模型组,仅注射等量生理盐水。

(3)模型组:模型复制成功后不予任何处理,常规饲养。

(4)电针组:模型复制成功后,参照《实验针灸学》取 “百会”(顶骨正中)和“肾俞”(第2腰椎下两旁)穴。先用0.30×25 mm不锈钢毫针针刺百会穴(向前平刺3~5 mm)及肾俞穴(稍向内斜刺3~5 mm)后,接G6805-II型电针治疗仪,肾俞穴左右交替使用,选择连续波,频率为5 Hz,刺激强度以大鼠腿部轻微抽动为度,留针15 min,每日1次,连续治疗15 d。

1.5 Morris 水迷宫(Morris water maze,MWM)实验

      治疗结束后,所有大鼠进行定位航行实验和空间探索实验,测试在实验中逃避潜伏期的长短和跨越原平台位置的次数,了解大鼠的学习和记忆能力。定位航行实验:实验前将大鼠放入水池自由游泳 2min,以适应水迷宫环境。正式水迷宫定位航行实验连续 5 天。实验第一天,将平台置于一个固定象限,每个时间段将大鼠分别从池壁四个起始点面向池壁放入水池,记录每次在 2min 内找到平台的时间(即逃避潜伏期,以秒为单位计时)。若大鼠在 120 s 内找不到平台,将其引上平台,其潜伏期记为120s,每次间隔 5min后再行下次测试。空间探索实验:第6天撤除平台进行空间探索实验,将大鼠从任选的非平台象限入水点面向池壁放入水中,记录 120 s内跨越原平台位置的次数及有效区的停留时间。

1.6 组织取材与处理

      各组大鼠腹腔注射10%的水合氯醛(350mg/kg)进行麻醉,麻醉成功后在干冰上将大鼠快速断头处死,剥离海马,分别置于冻存管中,储存于-80℃冰箱中保存。

1.7 指标检测

      将每组小鼠的海马按照每 1 mg 组织加入 10 μL 裂解液的比例加入裂解液在冰上进行彻底匀浆,然后于4℃12000 r / min离心 3 min,取上清液,测定其蛋白质的浓度,然后用细胞裂解液统一各组蛋白浓度,确定 Western-blot 上样量。制备 SDS-PAGE 胶、上样电泳、转膜/染色、封闭/脱色、一抗孵育(1:1000,兔抗体,Abcam公司)、洗膜、二抗孵育(1:3000,兔抗体,Abcam公司)、洗膜、DAB显色、曝光。所有印迹实验至少重复3次。用 Image J 分析软件分析各组 Western-blot 条带整合灰度比例( inte-grated density,ID) 与内参 ( β-actin) 比较,得到相对百分数 ( ID/内参 ID × 100% ) 。

1.8 统计分析 

       数据用均数±标准差(X±S)表示;数据进行正态分布及方差齐性检验,组间数据比较用单因素方差分析,用SPSS17.0统计软件进行分析,两两比较用LSD检验。以P < 0.05 表示有显著性差异,P < 0.01 表示有非常显著性差异。

2 结果

2.1 各组大鼠的Morris 水迷宫行为学比较

      如图1所示,定位航行试验中各组大鼠都随着训练次数的增加逃避潜伏期呈现出逐渐下降的趋势,说明在历次学习训练中各组大鼠可逐渐学会寻找平台,但学习记忆能力差异不同。图2、图3所示,在空间探索试验中,各组大鼠跨越原平台次数和在有效区停留时间均不同,跨越平台次数和有效区停留时间长,反映大鼠对原平台位置的记忆越好。实验结果显示:模型组与对照组比较,差异具有统计学意义 (P<0.01),证明该模型制作是成功的,治疗组与模型组比较,差异具有统计学意义 (P<0.01),证明针灸治疗可提高大鼠的学习记忆能力。

1.png

注:与对照组比较,## P <O.01;与模型组比较,▲▲P <0.01。

QQ截图20181219152444.png

QQ截图20181219152451.png

2.2  各组大鼠Aβ相关清除酶蛋白表达的比较

如表1、图1、图4、图5、图6所示:与假手术组相比,模型组海马组织中LPL、IDE、TTR、ApoE、α2M的表达明显减弱(P<0.05或P<0.01),Aβ1-42的表达显著增强(P<0.01);与模型组相比,电针组海马组织中LPL、IDE、TTR、ApoE及α2M的表达显著增强(P<0.05或P<0.01),Aβ1-42在海马中的表达显著减弱(P<0.01)。

QQ截图20181219152613.png



QQ截图20181219152652.png

QQ截图20181219152701.png

QQ截图20181219152739.png

3 讨论

       阿尔茨海默病俗称痴呆,属中医“癫证”、“健忘”、“呆病”等疾病范畴。中医认为该病的病位在脑,根本病机是肾精亏虚、髓海不足。《素问》:“诸髓者,皆属于脑”,脑为髓海,又为元神之府。肾主藏精、生髓,肾精不足则髓海渐空,导致元神失养,发为痴呆。《医学心悟》:“肾主智,肾虚则智不足,故善忘其前言”,指出肾虚为痴呆发生的关键。王清任在《医林改错》里指出“小儿无记性者,脑髓未满,高龄无记性者,脑髓渐空”,认为痴呆的发生与髓海不足密切相关。现代文献研究也表明众多学者支持本病的根本病机以肾虚为主[8、9]。“毒损脑络”是近年来提出的老年性痴呆新病机[10],认为具有神经元毒性的病理产物属于“内生毒邪”,如Aβ1-42、Aβ1-40、Aβ25-35等,如何清毒则需从痰、从瘀、从虚而治。本实验采用“益肾调督”电针法,穴取百会、肾俞。针刺百会可益气调神,补髓益智。另外,百会为督脉之要穴,位于巅顶,也体现了“经脉所通,主治所及”的治疗原则。另一方面,肾俞为足少阴肾经之背俞穴,是肾中经气输注和汇聚之所,针刺肾俞可补肾益精、培元固本。二穴相配共奏补肾益髓,通督调神之效。

       众所周知,Aβ沉积是引起AD病理损伤的中心环节及关键因素。正常情况下,Aβ的产生和清除处于动态平衡中,当Aβ产生过多或清除过慢时,则会大量聚集形成老年斑,产生神经毒性,引起神经元变性、坏死[11];且沉积的Aβ可通过激活星形胶质细胞和小胶质细胞诱导神经炎性因子的产生,引发脑内炎性级联反应[12、13];另外,Aβ在聚集过程中产生大量的活性氧,可导致线粒体功能障碍,突触损伤,诱导神经元凋亡,通过各种途径直接或间接地促进AD的发展[14、15]。因此抑制Aβ的产生或促进其清除是治疗AD的重要靶点。

       IDE是一种富含巯基的多功能的锌金属蛋白酶,分子量约为110 kD,是降解Aβ的关键酶[16]。IDE不仅能够与Aβ特异性结合形成稳定的复合物[17],影响Aβ的聚集,减少AD患者脑内老年炎性斑的形成,减轻Aβ对神经元的毒性作用,还能清除Aβ前体蛋白APP的胞质产物[18]。目前,动物实验已经证实,在具有AD病理特征的小鼠的大脑中,IDE水平的与Aβ40及Aβ42的水平呈负相关,而IDE基因敲除小鼠的原代培养的神经元降解Aβ的能力明显下降[19]。Tanzi[20]等在研究中发现IDE基因敲除的小鼠脑内Aβ水平显著升高。基因治疗实验也表明,Aβ在脑内的水平随IDE表达的增多而减少[21]。

      α2M是一种高度保守的分泌蛋白,是血浆分子量最大的蛋白质,分子量约为720 KD,是一种广谱的非特异性的蛋白酶抑制剂。研究表明,α2M可通过各种途径发挥其对Aβ的清除作用。一方面,激活状态的α2M能够识别Aβ,并通过其C末端的1314-1365氨基酸序列特异性地与Aβ结合[22],抑制细胞外可溶性Aβ的沉积,直接减轻其神经毒性;另一方面,活化的α2M的受体结合区暴露,通过激活α2M-R/LRP受体介导的细胞内吞作用,参与Aβ在溶酶体内的内化和清除等过程[23]。此外,α2M能够结合并清除一些炎症因子和细胞因子如IL-1β、IL-6、TNF-α、INF-γ[24],从而减轻AD神经炎性病变,在一定程度上延缓AD进程。LPL是与Aβ内化密切相关的酶,在AD脑内,LPL与老年斑共定位,并且它作为一种桥连分子与表面硫酸粘多糖分子相互作用而促进Aβ的内化[28]。Nishitsuji发现,在AD小鼠脑内,Aβ能与星型胶质细胞的LPL直接结合,促进Aβ在溶酶体的内吞[25]。ApoE 是中枢神经系统内主要的载脂蛋白,在脑内主要由星型胶质细胞产生,具有介导脂质转运、参与神经修复、对抗神经炎症和调节免疫应答等作用[26]。ApoE 已被证实可加速Aβ的清除,并能逆转因Aβ聚集引起的记忆缺陷[27]。ApoE还可以通过调节胆固醇的表达水平来影响Aβ在溶酶体中的内化和降解,实验表明,经ApoE处理后,AC内胆固醇的表达水平降低,加快了Aβ转运到溶酶体的速度,促进了Aβ的清除[28]。TTR是一种高度保守的分泌蛋白,由4个相同的亚基组成,每个亚基包括127个氨基酸,其相对分子质量为55×103。目前,转基因实验已经证实了TTR基因能够抑制AD相关基因行为及神经病理学改变[29]。Gloeckner[30]在研究中发现,AD患者CFS中TTR减少,且AD患者的TTR与Aβ水平及老年斑数量呈负相关。

      课题组前期研究表明,“益肾调督”针灸法能够改善AD大鼠学习记忆功能[31]、减少或抑制AD大鼠血清Aβ水平[32、33]、促进AD大鼠海马脑啡肽和前序列蛋白酶的表达[6]、改善海马神经元线粒体能量代谢[34]及减少轴突损伤,促进轴突再生[35],能在一定程度上延缓AD进程。本次实验显示,经Aβ1-42诱导的AD模型大鼠海马组织中LPL、IDE、TTR、ApoE及α2M的表达明显减弱(P<0.05或P<0.01),Aβ1-42的表达显著增强(P<0.01),经“益肾调督”电针法治疗后,大鼠海马组织中LPL、IDE、TTR、ApoE及α2M的表达明显增强(P<0.05或P<0.01),Aβ1-42的表达显著减弱(P<0.01)。提示“益肾调督”电针法可明显上调Aβ1-42诱导的AD模型大鼠海马组织中LPL、IDE、TTR、ApoE及α2M蛋白水平的表达,从而促进Aβ的清除,有助于改善AD病理变化,延缓其病理进程,其具体作用机制有待进一步研究。

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