电针调控SAMP8小鼠血脑屏障增强盐酸多奈哌齐对β-淀粉样蛋白的清除

Effect of electroacupuncture combined with Donepezil on learning-memory ability and expression of hippocampal β-amyloid clearance-related genes in SAMP8 mice

目的:观察电针对快速老化(SAMP8)小鼠血脑屏障中β-淀粉样蛋白(Aβ)代谢途径相关因子基质金属蛋白酶-9(MMP-9)、低密度脂蛋白受体相关蛋白-1(LRP-1)、P糖蛋白(Pgp)、紧密连接蛋白Claudin-5的调控,探讨电针加强盐酸多奈哌齐清除Aβ的机制。方法:30周龄雄性SAMP8小鼠随机分为模型组、药物组、针药组,每组6只,以6只同周龄抗快速老化SAMR1小鼠作为对照组。药物组采用盐酸多奈哌齐灌胃,1.3 mg·kg^-1·d^-1,连续4周;针药组在药物组的基础上予"印堂""百会"穴电针治疗,每次15 min,频率2 Hz,电流1 mA,每日1次,每周6次,连续4周。运用Morris水迷宫实验观察小鼠学习记忆能力及空间探索能力的变化;HE染色观察各组小鼠海马区神经元形态;实时荧光定量PCR法检测小鼠海马区MMP9、LRP-1、Pgp、Claudin-5、AβmRNA相对表达量。结果:与对照组相比,模型组小鼠的逃避潜伏期明显延长(P<0.01),穿越原平台次数明显减少(P<0.01),海马神经元结构形态发生明显改变,MMP-9、AβmRNA表达水平明显增加(P<0.01),LRP-1、Pgp、Claudin-5 mRNA表达水平明显下降(P<0.01)。与模型组相比,药物组、针药组逃避潜伏期均明显缩短(P<0.01,P<0.05),穿越原平台次数明显增加(P<0.01),海马神经元细胞排列规则整齐、细胞层数明显增加,AβmRNA表达水平明显下降(P<0.01),LRP-1、Pgp mRNA表达水平明显上调(P<0.05,P<0.01);针药组MMP-9 mRNA表达水平明显下降(P<0.01),Claudin-5 mRNA表达水平明显上调(P<0.01)。与药物组相比,针药组逃避潜伏期从第4天开始明显缩短(P<0.05,P<0.01),穿越原平台次数明显增高(P<0.01),海马神经元损伤减轻,MMP-9、AβmRNA表达水平明显下降(P<0.01),LRP-1、Pgp、Claudin-5 mRNA表达水平明显上调(P<0.01)。结论:电针可能通过下调MMP-9,上调LRP-1、Pgp、Claudin-5增强盐酸多奈哌齐对Aβ在血脑屏障中的转运,从而增强盐酸多奈哌齐治疗阿尔茨海默病的效果。

Objective To investigate the effect of electroacupuncture(EA) combined with Donepezil on learning-memory ability and gene expression of β-amyloid(Aβ) clearance-related factors in the hippocampus in senescence-accelerated mouse prone 8(SAMP8) mice, so as to explore their synthetic effect in improving dementia of Alzheimer’s disease(AD).Methods Male SAMP8 mice(30-week-old) were randomly divided into model, medication and EA+medication groups(n=6 mice in each group), and other 6 senescence-resistant 1(SAMR1) mice were used as the control group. Mice of the medication and EA+medication group received gavage of Donepezil(1.3 mg·kg^-1·d^-1) once daily for 4 weeks. EA(2 Hz, 1 mA) was applied to "Baihui"(GV20) and "Yintang"(EX-HN3) for 15 min, once daily, 6 days a week for 4 weeks for rats in the EA+medication group. The Morris water maze(MWM) task(including place navigation tests and space exploration trials) was used to assess the mouse’s learning-memory ability. Histopathological changes of hippocampus tissue were observed by H.E. staining. The expression levels of matrix metalloprotein 9(MMP-9), low density lipoprotein receptor-related protein-1(LRP-1), P-glycoprotein(Pgp, an important drug transporter responsible for multidrug resistance), Claudin-5(a component of tight junction strands that serves as a physical barrier to prevent solutes and water from passing freely through the paracellular space between epithelial or endothelial cell sheets of blood-brain barrier, BBB) and Aβ mRNAs of the hippocampus tissue were detected by quantitative real-time PCR.Results Compared with the control group, the average escape latency of place navigation tests, and the expression levels of MMP-9 and Aβ mRNAs were significantly increased(P<0.01), and the number of platform quadrant-crossing times of space exploration trials, and the expression levels of LRP-1, Pgp and Claudin-5 mRNAs considerably decreased in the model group(P<0.01). After the intervention, the learning-memory ability was significantly improved in the medication and EA+medication groups(P<0.01,P<0.05), the expression levels of Aβ mRNAs in the medication and EA+medication groups and MMP-9 mRNA in the EA+medication group were obviously down-regulated(P<0.01), and those of LRP-1 and Pgp mRNAs in the medication and EA+medication groups and Claudin-5 mRNA in the EA+medication group were remarkably up-regulated(P<0.05, P<0.01). The therapeutic effect of EA+medication was apparently superior to that of simple medication in shortening the escape latency(P<0.05,P<0.01) and in down-regulating the expression of MMP-9 and Aβ mRNAs(P<0.01), and in increasing the number of platform quadrant-crossing times(P<0.01), and expression levels of LRP-1, Pgp and Claudin-5 mRNAs(P<0.01). H.E. staining showed scatted and loose arrangement of neurons in the hippocampus, with reduction of number of cell layers and unclear nucleoli, which was relatively milder in the medication and EA+medication groups. Conclusion EA can enhance the effect of Donepezil in improving learning-memory ability in AD mice possibly by regulating expression of MMP-9, LRP-1, Pgp and Claudin-5 mRNAs and strengthening the effect of Donepezil in transporting Aβ via BBB.