不同电针对SAMP8小鼠海马神经元保护机制的实验研究

【作者】 唐银杉；

【导师】 李志刚；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 1背景阿尔茨海默氏病(Alzheimer’s Disease, AD),又称“老年性痴呆”,是一种以近事记忆力下降,空间、语言和行为障碍,或可伴有抑郁及其它人格改变的中枢神经退行性病交,属于中医“呆病”的范围。到目前为止,AD的发病机制尚不清楚,但其主要的病理特征可包括：神经纤维缠结(neuron fibrillary tangles, NFTs),由tau蛋白在细胞内异常聚集而成；老年斑(senile plaques, SP),由β淀粉样蛋白(β-amyloid protein, Aβ)在细胞外大量沉淀所形成；神经元的丢失。2目的本研究拟以国际上公认的SAMP8快速老龄化痴呆小鼠模型,从中医整体观基础理论出发,选取与AD神经元生存与凋亡密切相关的PI3K/AKT信号转导通路,探讨不同电针神经元保护的疗效差异,寻找针刺抗痴呆的新靶点,进一步揭示音乐电针治疗老年性痴呆的可能作用机制,为临床治疗AD提供一定的理论依据。3研究方法选用8月龄,雄性,SAMR1正常老化小鼠10只；SAMP8快速老龄化小鼠60只,随机分为模型组、非穴位组、音乐组、盐酸多奈哌齐组、脉冲电针组和音乐电针组(n=10)。脉冲电针组,针柄连接HANS-LH202型电针仪,选取“百会”、“印堂”,“人中”点刺放血,20min/日,共15天；非穴位组,在小鼠尾部距尾根1/3处给予同样的刺激。音乐电针组,选取与脉冲电针组小鼠相同的穴位,采用抗痴呆的音乐处方,针柄连接ZJ-12H音乐电针仪。音乐组,在音乐电针组干预时抓取一次,同时和音乐电针组聆听音乐,以保证处理条件相同。多奈哌齐组,采用灌胃给药15天。正常组和模型组均不给予治疗,只是在其他治疗组干预的同时抓取1次。15天后,采用mirrors水迷宫对各组小鼠进行行为学评价。采用尼氏染色和苏木精-伊红染色观察每组小鼠海马CA1区神经元的数目和组织形态的病理改变；采用酶联免疫吸附(ELISA)法检测每组小鼠血清中Aβ40和Aβ42蛋白水平表达情况；采用免疫组化、western bolt和Real-time PCR检测各组小鼠海马Tau、P-Tau(serl99/202)、AKT、P-AKT(ser473)、GSK-3p、P-GSK-3p(ser9)和GFAP蛋白和基因表达水平。4研究结果(1)Morris水迷宫试验结果显示：与SAMR1正常老化小鼠相比,SAMP8模型小鼠学习记忆能力明显降低,表现为逃避潜伏期延长、游泳距离增加、跨平台次数减少和目标象限停留时间缩短(P<0.01)；与SAMP8模型组小鼠相比,非穴位组学习记忆能力与之相当,无统计学差异(P>0.05)；音乐组小鼠的逃避潜伏期时间和游泳距离有下降趋势,穿越平台次数有增多趋势,目标象限停留时间有延长趋势,但无统计学差异(P>0.05)；盐酸多奈哌齐组、脉冲电针组和音乐电针组小鼠学习记忆能力显著改善,这反映在逃避潜伏期和游泳距离减少(P<0.05)、跨平台次数和目标象限停留时间频率增加(P<0.05,P<0.05,P<0.01)。(2)光镜下,各组小鼠HE染色的结果显示：SAMP8模型组中,海马CA1区的细胞排列紊乱,细胞层数显著减少,细胞核仁不清楚,可有核固缩的现象；非穴位组和音乐组小鼠海马CA1区细胞排列错乱,神经元明显丢失,细胞层数减少；盐酸多奈哌齐组、脉冲电针组和音乐电针组小鼠海马CAI区细胞排列较为规则整齐,核仁清晰呈圆形或椭圆形,细胞层数较模型组明显增加。(3)各组小鼠尼氏染色结果表明：与SAMRl正常老化小鼠对比,SAMP8模型组小鼠海马CA1区尼氏体浅染,神经元胞体形态排列不规则,尼氏体数量明显减少(P<0.01)。与SAMP8模型组相比,非穴位组和音乐组小鼠海马CA1区神经元数量和病理形态改变相似；盐酸多奈哌齐组、脉冲电针组和音乐电针组小鼠均能改善SAMP8小鼠海马CA1区神经元减少和丢失现象,起到保护神经元的作用。(4)酶联免疫吸附(ELISA)结果表明：与SAMR1正常老化小鼠相比,模型组小鼠血清A β40和A β42蛋白表达均显著增高(P<0.01)。与SAMP8模型组相比,非穴位组血清Aβ蛋白表达相似,无显著差异(P>0.05),音乐组血清Aβ蛋白表达呈减少趋势,但仍无显著差异(P>0.05)；盐酸多奈哌齐组、脉冲电针组和音乐电针组小鼠血清A β40降低,有统计学差异(P<0.05)；三组在降低A β42蛋白水平上有显著性差异(P<0.05,P<0.05,P<0.01)。与脉冲电针组相比,音乐电针组小鼠血清中A β42蛋白水平显著下降,有统计学差异(P<0.05)。(5)不同电针干预SAMP8小鼠海马P13K/AKT信号转导通路的影响：1)免疫组织化学法(HIC)：总AKT和总GSK-3p蛋白含量在各组小鼠海马内变化不大,无显著差异(P>0.05)。与正常SAMR1小鼠相比,SAMP8模型小鼠P-AKT(ser473). P-GSK-3β(ser9)蛋白表达均减少(P<0.01)；与SAMP8模型小鼠比较,非穴位组和音乐组P-AKT(ser473). P-GSK-3β(ser9)蛋白表达无显著性差异(P>0.05)；盐酸多奈哌齐组、脉冲电针组和音乐电针组小鼠海马P-AKT(ser473)、P-GSK-3β(ser9)蛋白增高(P<0.05,P<0.05,P<0.01)。2)Western bolt结果显示：与免疫组化结果一致。3)Real-time PCR结果显示：与正常组比较,模型组小鼠海马AKT mRNA和GSK-3β mRNA表达显著性降低(P<0.01)；非穴位组AKT mRNA和GSK-3β mRNA表达较SAMP8模型组无显著性差异(P>0.05)；音乐组该基因表达有上升趋势,但无统计学差异(P>0.05)；盐酸多奈哌齐组、脉冲电针组和音乐电针组均能一定程度上改善小鼠海马AKT mRNA和GSK-3β mRNA的基因表达,有统计学差异(P<0.05,P<0.05,P<0.01)。(6)对比不同电针干预SAMP8小鼠海马tau蛋白表达水平的影响：1)免疫组织化学法(HIC)：总Tau蛋白在各组小鼠海马内无统计学差异(P>0.05)。与正常组相比,模型组小鼠海马P-Tau(ser199/202)蛋白表达显著增多,有统计学差异(P<0.01)。与模型组相比,非穴位组和音乐组小鼠海马P-Tau(serl99/202)蛋白表达无显著变化(P>0.05);盐酸多奈哌齐组、脉冲电针组和音乐电针组均能降低SAMP8小鼠海马P-Tau(ser199/202)蛋白含量(P<0.05,P<0.05,P<0.01)。2)Western bolt和Real-time PCR结果显示：各指标表达趋势同免疫组化。(7)对比不同电针干预SAMP8小鼠海马星形胶质细胞GFAP的影响：1)免疫组织化学法(HIC)：与SAMR1正常小鼠比较,GFAP蛋白在SAMP8模型组小鼠海马区表达显著增加(P<0.01)。与模型组小鼠相比,非穴位组和音乐组GFAP表达无统计学差异(P>0.05)；盐酸多奈哌齐组、脉冲电针组和音乐电针组均能明显降低SAMP8小鼠海马GFAP蛋白含量,有统计学差异(P<0.05,P<0.05,P<0.01)。2)Western bolt和Real-time PCR结果显示：各指标表达趋势同免疫组化。5结论(1)SAMP8快速老化痴呆小鼠存在着明显的学习记忆认知障碍,并且也出现了tau蛋白和Ap蛋白明显增加的病理表现,同时还伴随着神经元的损伤和减少,是AD的理想模型之一。(2)电针能较好的保护小鼠海马神经元,脉冲电针和音乐电针可改善痴呆小鼠神经元的丢失；在提高海马CA1区神经元的数量以及改善SAMP8小鼠神经元损伤方面,音乐电针优于脉冲电针。(3)电针可明显改善SAMP8快速老化小鼠学习记忆认知障碍,其中在改善认知障碍方面,音乐电针组仅有优于脉冲电针组的趋势,无统计学差异;但其在学习记忆力保持方面优于脉冲电针。(4)电针抗痴呆的作用机制之一可能是通过PI3K/AKT信号转导通路的上调,增加AKT的活性,促进GSK-3p (ser9)磷酸化,从而抑制GSK-3β活性,进而降低Aβ蛋白的沉积和tau蛋白过度磷酸化,最终达到减少神经毒性,保护神经元的目的。其中,音乐电针在调节PI3K/AKT信号转导通路的关键蛋白方面优于脉冲电针。(5)在改善SAMP8小鼠海马内星形胶质细胞GFAP表达方面,音乐电针能明显减少其表达,抑制炎症因子的释放,减轻神经元的损伤,且明显优于脉冲电针组,这可能是音乐电针抗老年痴呆症的机制之一。更多还原

【Abstract】 1. BackgroundAlzheimer Disease (AD) is one of central nervous system degenerative diseases, which has a main performance gradual cognitive dysfunction, bad memory and learning ability, sometimes it may also be accompanied by anxiety, depression, language barriers and other many personality changes. So far, the pathogenesis of AD is not yet clear. But the main pathological characteristics can include:neurofibrillary tangles (NFTs), gathered by the abnormal tau protein in the cell; Age spots (senile plaques, SP), by beta amyloid protein (Aβ) outside the cell formed by the large amounts of precipitation and the loss of neurons.2. PurposeThe study is based on the theory of Traditional Chinese Medicine, we choose the internationally recognized SAMP8rapidly aging dementia model mice. The selection is closely related to the AD neuron loss of PI3K/AKT signal transduction pathway, exploring the different electro-acupuncture intervention difference in the protection of neurons. Seeking new targets to reveal the mechanism of action of acupuncture treatment of senile dementia may, provide a certain theoretical basis for clinical treatment of AD.3. MethodThere were70adult male mice at8month, including10SAMR1mice and60SAMP8mice. The60SAMP8mice were randomly divided into six groups:Model group, Non-acupuncture group, Music group, Donepezil Hcl group, Pulse electric acupuncture(EA) group and Music electric acupuncture group (N=10). The EA group, the needles handle connected the HANS-LH202electric acupuncture apparatus, selecting "Bai Hui","Yin Tang" and "Ren Zhong" acupoints,20min/day,15days. The non-acupuncture group was given the same stimulus in the tail root1/3of mice. The music electric acupuncture group, selecting the same acupoints and choosing dementia music prescription, the needles handle connected the ZJ-12H musical electro-acupuncture apparatus. The Music group, in order to ensure under the same conditions, mice were catched once a time while listening to music. The Donepezil HCL group, mice were used intragastric administration for15days. The normal group and model group mice only were be catched at a time as same as other groups. After15days, using mirrors water maze(MWM) to judge the behavior of groups of mice. Choosing Nissl’s staining and HE staining to observe number of neurons and pathological changes of morphology in mice’s hippocampus CA1area. Using enzyme-linked immunosorbent (ELISA) method to detect each group mice serum A beta40 and A beta42protein expression levels. Using immunohistochemistry, western bolt and Real-time PCR test groups of mice hippocampus Tau, P-Tau (ser199/202), AKT, P-AKT (ser473), GSK-3beta, P-GSK-3beta (ser9) and GFAP protein and gene expression level.4. Results(1) MWM Results:Compared with normal group, the SAMP8mice were significantly poor in the ability of learning and memory, reflecting the escape latency period extended, swimming distance increased, the reduced the number of cross-platform and target quadrant stay a shorter time (P<0.01). Compared with the SAMP8model group mice, the learning and memory ability in non-acupuncture group is similarity to the model group (P>0.05). In music group, the escape latency time and swimming distance have a downward trend, through the platform number and the target quadrant residence time has increasing trend, but no statistical difference (P>0.05). The Donepezil HCL group, the pulse electric acupuncture group and music electric acupuncture group had a significantly improve the ability of learning and memory, this is reflected in escape latent period and reduce the swimming distance (P<0.05), the cross-platform and target quadrant time frequency increases (P<0.05, P<0.05, P<0.01).(2) HE Staining results:In model group, the disordered arrangement of cells, layer number of cells significantly reduced, cell nucleolus is not clear and the phenomenon of nuclear pyknosis in hippocampus CA1area. In non-acupuncture group and music group, cells arranged disorder, loss of neurons, the cell layers reduced in hippocampal CA1region. The Donepezil HCL group, the pulse electric acupuncture group and the music electric acupuncture group, arrangement of cells rules and clear nucleoli assumes the circular or elliptic, layer number of cells increased significantly.(3) Nissl’s staining results:Compared with normal group, the model group, austenite shallow dye, the neuron cell body form is irregular, number of neuron were decreased significantly in hippocampal CA1area. Compared with SAMP8model group, number of hippocampus CA1area neurons and the pathological morphological changes are similarity in the non-acupuncture and music group. In Donepezil HCL group, the pulse electric acupuncture group and music electric acupuncture group, it could improve neurons decrease and loss phenomenon to protect the neurons in hippocampus CAl area(4) ELISA Results:Compared with normal group, A beta40and42A beta protein in serum expression were significantly increased in model group (P<0.01). Compared with model group, the serum in non-acupuncture group is similar to model group, there was no significant difference (P>0.05). A beta protein expression in serum showed A trend of decrease in music group, but there was still no significant difference (P>0.05). Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the A beta40is reduced in serum, which has a statistically significant (P<0.05), and the decreasing A beta42protein level had significant difference (P<0.05, P<0.05, P<0.01). Compared with the pulse electric acupuncture group, the A beta42protein levels in serum dropped significantly in music electric acupuncture group, which has statistically significant (P<0.05).(5) Different electric acupuncture intervention SAMP8mice hippocampus’PI3K/AKT signal transduction pathway:1) The immune histochemical method (HTC):the content of total AKT and GSK-3beta protein changed little within these seven groups, there were no significant difference (P>0.05). Compared with the normal mice, the express of P-AKT (ser473), P-GSK-3beta (ser9) protein were reduced rapidly in SAMP8(P<0.01). Compared with model of SAMP8mice, the express of P-AKT (ser473), P-GSK-3beta (ser9) protein expression there was no significant difference in the non-acupuncture and music group (P>0.05). In Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the express of P-AKT (ser473), P-GSK-3beta (ser9) protein increased (P<0.05);2) The Western Bolt results were in accordance with the immunohistochemical’s;3) Real-time PCR results:Compared with normal group, the expression AKT mRNAand GSK-3beta mRNA was significantly lower in model group (P<0.01); The non-acupuncture had no significant difference with model group (P>0.05); The expression of these genes has a rising trend in music group, but also has no statistical difference (P>0.05); In Donepezil HCL, the pulse electric acupuncture and music electric acupuncture group, there had a certain degree of improvement expression in mice hippocampal’s AKT mRNA and GSK-3beta mRNA, which had a statistically significant (P<0.05, P<0.05, P<0.01).(6) The different electric acupuncture influence of tau protein in hippocampus region:1) The immune histochemical method (HTC):the content of total tau protein changed little within these seven groups, there were no significant difference (P>0.05). Compared with the normal mice, the express of P-tau (ser199/202) protein were increased rapidly in SAMP8(P<0.01). Compared with model group, the express of P-tau (serl99/202) protein expression there was no significant difference in the non-acupuncture and music group (P>0.05). In Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the express of P-tau (ser199/202) protein decreased (P<0.05);2) The Western Bolt and Real-time PCR results were in accordance with the immuno histochemical’s.(7) The different electric acupuncture influence of GFAP protein in hippocampus region:1) Compared with the normal mice, the express of GFAP protein were increased rapidly in SAMP8(P<0.01). Compared with model group, the express of GFAP protein expression there was no significant difference in the non-acupuncture and music group (P>0.05). In Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the express of GFAP protein decreased rapidly (P<0.05, P<0.05, P<0.01).2) The Western Bolt and Real-time PCR results were in accordance with the immunohistochemical’s.5. Conclusion(1) The SAMP8mice had a obvious cognitive impairment of learning and memory, the pathological form of tau protein and A beta protein increased significantly, accompanied by neurons damaged and reduced, which is one of the ideal model of AD.(2) Electric acupuncture Have a good adjustment function for neuron protection, including pulse electric acupuncture and music electric acupuncture can increase the number of neurons in hippocampal CA1area, improving SAMP8mice on neuronal damage, and the music electric acupuncture is superior to the pulse.(3) Electric acupuncture could obviously improve cognitive impairment of learning and memory in the SAMP8rapid aging mice. In terms of improving cognitive impairment, the music electric acupuncture group is superior to the pulse; But the music electric acupuncture on learning memory keep better than the pulse.(4) One of the curative mechanism of action of dementia may be through the PI3K/AKT signal transduction pathway, increasing the activity of AKT, promoting the phosphorylation of GSK-3beta (ser9), thus to restrain the activity of GSK-3beta.-and thus A beta protein in deposition and abnormal tau protein phosphorylation, neurotoxicity, reduced finally protect the neurons. The music electric acupuncture in regulating the PI3K/AKT signal transduction pathways is superior to the pulse.(5) Improving the aspect of hippocampal astrocytes GFAP in SAMP8mice, the music electric acupuncture can significantly reduce its expression, inhibiting the release of inflammatory cytokines, relieving neuron damage, which is obviously better than plus. This may be one of the mechanisms of music electric acupuncture treating Alzheimer’s disease.更多还原