目的 探讨不同频率电针治疗阿尔茨海默病(AD)大鼠神经性病变的机制,为临床上采用电针治疗AD提供理论依据.方法 2016年3—6月,选择健康的SPF级Wistar雄性大鼠90只,适应性饲养1周后进行Morris水迷宫实验,筛选合格后按照随机数字表法分为正常组、假手术组、模型组、2 Hz电针治疗组、30 Hz电针治疗组和50 Hz电针治疗组(每组15只).正常组不做任何处理.模型组、2 Hz电针治疗组、30 Hz电针治疗组和50 Hz电针治疗组注射寡聚体Aβ1-42溶液建造AD模型.假手术组采用0.9%氯化钠溶液建造假手术模型,并再次进行Morris水迷宫实验确认造模成功.造模完成后,2 Hz电针治疗组采用G6805-Ⅱ型电针治疗仪针刺百会穴、肾俞穴进行治疗,30 Hz电针治疗组和50 Hz电针治疗组除电针频率分别选择30 Hz和50 Hz外,其他所有参数和操作同2 Hz电针治疗组.正常组、模型组、假手术组与2 Hz、30 Hz、50 Hz电针治疗组同样进行抓取,但不做任何处理后放回.治疗结束后处死各组大鼠,取大脑海马区、皮质区组织待测.采用Western blotting法检测各组大脑海马区、皮质区Tau蛋白及其磷酸化〔Tau(pS262)、Tau(pS396)、Tau(pS404)〕、14-3-3ζ、α氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)表达水平.结果 模型组大脑海马区Tau表达水平低于正常组,Tau(pS262)、Tau(pS396)表达水平高于正常组(P<0.05);50 Hz电针治疗组Tau表达水平高于模型组、2 Hz电针治疗组、30 Hz电针治疗组,Tau(pS262)、Tau(pS396)表达水平低于模型组、2 Hz电针治疗组、30 Hz电针治疗组(P<0.05).模型组大脑皮质区Tau表达水平低于正常组,Tau(pS262)、Tau(pS404)表达水平高于正常组(P<0.05);50 Hz电针治疗组Tau表达水平高于模型组、2 Hz电针治疗组、30 Hz电针治疗组,Tau(pS404)表达水平低于模型组、2 Hz电针治疗组、30 Hz电针治疗组(P<0.05).模型组大脑海马区、皮质区14-3-3ζ表达水平高于正常组,海马区AMPA表达水平低于正常组(P<0.05);50 Hz电针治疗组大脑海马区、皮质区14-3-3ζ表达水平低于模型组、2 Hz电针治疗组、30 Hz电针治疗组,海马区AMPA表达水平高于模型组、2 Hz电针治疗组、30 Hz电针治疗组(P<0.05).结论 不同电针治疗AD大鼠可能是通过调节Tau蛋白及其磷酸化水平以及相关蛋白(14-3-3ζ、AMPA受体蛋白)的表达水平来改善AD大鼠的神经性病变,进而达到治疗的效果.%Objective To explore the mechanism of different frequencies of eletroacupuncture in the treatment of neuropathy in Alzheimer′ s disease (AD ) rats,so as to provide a theoretical basis for clinical treatment of AD with electroacupuncture. Methods This study was conducted between March and June 2016. Ninety healthy male Wistar rats (SPF) were selected and qualified rats were screened by Morris water maze after 1 week of adaptive feeding. Then the qualified rats were divided into normal group,sham operation group,model group,2 Hz,30 Hz and 50 Hz electroacupuncture treatment groups according to the random number table method with 15 in each. The normal group was not given any treatment. The AD rats were established by injecting Aβ1 - 42 oligomer into the lateral cerebral ventricle in model group,2 Hz,30 Hz and 50 Hz electroacupuncture treatment groups,whereas the rats in the sham operation group were constructed with 0. 9% sodium chloride solution. And the success of modeling was confirmed by Morris water maze test again. After that, the rats in 2 Hz electroacupuncture treatment group were treated by G6805 - Ⅱ type electroacupuncture instrument stimulation at Baihui and Shenshu acupoints with the frequency of 2 Hz. The rats in the 30 Hz and 50 Hz electroacupuncture treatment groups received the same treatment with the same parameters and operations as the 2 Hz electroacupuncture treatment group received except for electroacupuncture frequencies,30 Hz was selected for the 30 Hz electroacupuncture treatment group,and 50 Hz for the 50 Hz electroacupuncture treatment group. The rats in normal group,model group and sham operation group were grabbed in the same way as those rats in the electroacupuncture treatment groups but were put back without any treatment. The rats were sacrificed after intervention and samples of brain hippocampus,cortical tissues were collected. The expression levels of Tau protein,levels of phosphorylated Tau protein at serine 262,369,404 sites 〔Tau (pS262),Tau (pS396),Tau (pS404)〕,14 - 3 - 3ζ and AMPA receptor protein in hippocampus and cortical tissue of rats were detected by Western blotting. Results Compared with the normal group,the expression levels of Tau protein in hippocampus tissue of AD rats in model group significantly decreased (P <0. 05),whereas the levels of Tau (pS262)and Tau (pS396)increased obviously (P < 0. 05). The 50 Hz electroacupuncture treatment group had higher expression level of Tau protein but lower expression levels of Tau (pS262)and Tau (pS396)in the hippocampus tissue than model group,2 Hz and 30 Hz electroacupuncture treatment groups (P < 0. 05). Compared with the normal group,the model group had lower expression level of Tau protein and higher expression levels of Tau (pS262)and Tau (pS404)in the cerebral cortex (P < 0. 05). The 50 Hz electroacupuncture treatment group had higher expression level of Tau protein whereas lower expression level of Tau (pS404 ) in the cerebral cortex than the model group,2 Hz and 30 Hz electroacupuncture treatment groups (P < 0. 05). The expression levels of 14 - 3 - 3ζ in the hippocampus and cortex of the model group were higher than those in the normal group,whereas the expression level of AMPA receptor protein in hippocampus was lower than that in the normal group (P < 0. 05). The 50 Hz electroacupuncture treatment group had lower expression levels of 14 - 3 - 3ζ in hippocampus and cortex but higher expression level of AMPA receptor protein in hippocampus than the model group,2 Hz and 30 Hz electroacupuncture treatment groups (P < 0. 05 ) . Conclusion Different frequencies of electroacupuncture treatment probably improved the neuropathy of AD rats by regulating the expression levels of Tau protein and phosphorylated Tau protein and related proteins (14 - 3 - 3ζ and AMPA),and thus achieved the effect of the treatment of AD.
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