探讨电针对阿尔茨海默病小鼠模型学习记忆能力的影响及作用机制

Exploring the impact and mechanisms of electroacupuncture on the ability of learning and memory in a mouse model of Alzheimer’s disease

目的探讨电针对β-淀粉样蛋白25-35(Aβ_(25-35))诱导的阿尔茨海默病(AD)小鼠模型学习记忆能力和核苷酸寡聚化结构域样受体蛋白3(NLRP3)/半胱氨酸蛋白酶-1(caspase-1)通路的影响。方法将30只2月龄雄性C57BL/6小鼠按体质量随机分为对照组、模型组和电针组,每组10只。双侧侧脑室内立体定位注射Aβ_(25-35)诱导建立AD小鼠模型。分别对对照组(侧脑室内注射无菌生理盐水)、模型组(侧脑室内注射Aβ_(25-35))和电针组(侧脑室内注射Aβ_(25-35),电针“百会”和“大椎”)进行干预。每侧侧脑室注射的Aβ_(25-35)和生理盐水均为3μL。注射1次,7 d后进行电针治疗,2个疗程,每疗程6 d(每天1次,每次15 min),2个疗程中间休息1 d,共计治疗13 d。采用Morris水迷宫、Y迷宫和旷场实验检测各组小鼠学习记忆能力。酶联免疫吸附测定(ELISA)法检测各组小鼠海马组织白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)表达水平。实时荧光PCR检测小鼠海马组织NLRP3、caspase-1和IL-1β的mRNA水平。蛋白质印迹法检测NLRP3、caspase-1和IL-1β蛋白表达水平。结果与对照组相比,模型组小鼠在Morris水迷宫的第4、5天逃避潜伏期延长(P<0.05),穿越目标象限平台次数和停留时间百分比降低(P<0.05);Y迷宫自发交替准确率下降(P<0.05);旷场中理毛次数减少(P<0.05),中心区域停留时间延长(P<0.05);海马组织内IL-1β和TNF-α蛋白表达上调(P<0.05);NLRP3、caspase-1和IL-1β的mRNA及蛋白表达水平均上调(P<0.05)。与模型组相比,电针组第5天逃避潜伏期缩短(P<0.05),穿越目标象限平台次数和停留时间百分比增加(P<0.05);Y迷宫自发交替准确率上升(P<0.05);旷场中理毛次数增多(P<0.05),中心区域停留时间缩短(P<0.05);海马组织内IL-1β和TNF-α蛋白表达下调(P<0.05)。NLRP3、caspase-1和IL-1β的mRNA及蛋白表达水平均下调(P<0.05)。结论电针“百会”和“大椎”可改善Aβ_(25-35)诱导建立的AD小鼠模型学习记忆能力,这可能与电针通过NLRP3/caspase-1通路减轻海马炎症反应相关。

Objective We aimed to investigate the effects of electroacupuncture on learning and memory and on the NOD-like receptor protein 3(NLRP3)/cysteine-aspartic proteases-1(caspase-1)pathway in a mouse model of Alzheimer’s disease(AD)induced by Aβ_(25-35).Methods Thirty 2-month-old male C57BL/6 mice were divided into control,model,and electroacupuncture groups according to the principle of random assignment,with ten mice per group.The AD mouse model was induced by stereotaxic injection of Aβ_(25-35)into the bilateral lateral ventricles.The interventions were conducted in the control group(intralateral ventricular injection of sterile saline),model group(intralateral ventricular injection of Aβ_(25-35)),and electroacupuncture group(intralateral ventricular injection of Aβ_(25-35)and electroacupuncture of GV20 and GV14)respectively.Aβ_(25-35)and saline were injected into each lateral ventricle at 3μL.One dose of injection was given and seven days later electroacupuncture was administered.Treatment was carried out for 2 sessions and 6 days per session(1 session of 15 min once per day)with 1 day of rest in between,for a total of 13 days.The Morris water maze,Y-maze,and open field experiments were performed to evaluate the learning and memory abilities of the mice in each group.The expression levels of the inflammatory factors interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the hippocampus of each group were measured by enzyme linked immunosorbent assay(ELISA).The mRNA levels of NLRP3,caspase-1,and IL-1βin the hippocampus of mice were analyzed by real-time PCR.The protein expression of NLRP3,caspase-1,and IL-1βwas investigated with Western blotting.ResultsCompared to the control group,the mice in the model group had a prolonged escape latency on Day 4 and 5 of the Morris water maze(P<0.05)and a reduced number of platform crossings and percentage of time spent in the target quadrant(P<0.05).The spontaneous alternation accuracy of the mice in the Y-maze was significantly reduced(P<0.05).Mice in the open field test had a remarkably lower number of grooming incidents(P<0.05)and spent more time in the central area(P<0.05).The expression of inflammatory factors IL-1βand TNF-αwas elevated in the hippocampal tissue of the model group by ELISA(P<0.05).The mRNA and protein expression of NLRP3,Caspase-1 and IL-1βwere upregulated in the model group(P<0.05).Compared to the model group,the mice in the electroacupuncture group had a shorter escape latency on Day 5 of the Morris water maze(P<0.05),an increased number of platform crossings and percentage of time spent in the target quadrant(P<0.05),improved accuracy of spontaneous alternation in the Y-maze(P<0.05),a higher number of grooming incidents in the open field(P<0.05),and a shorter time spent in the central area(P<0.05).The expression of IL-1βand TNF-αwas reduced in the hippocampal tissue of the electroacupuncture group(P<0.05).The mRNA and protein expression of NLRP3,Caspase-1,and IL-1βwere all downregulated in the electroacupuncture group(P<0.05).Conclusion Electroacupuncture at the GV20 and GV14 acupuncture points enhanced learning and memory in the Aβ_(25-35)-induced AD mouse model,which may be related to the alleviation of hippocampal inflammatory responses by electroacupuncture via the NLRP3/caspase-1 pathway.