基于“肾脑相济”理论探讨艾灸对阿尔茨海默病大鼠海马AMPK/mTOR信号通路的影响

目的 观察艾灸对阿尔茨海默病（Alzheimer’s disease, AD）大鼠腺苷酸活化蛋白激酶(adenosine 5-monophosphate activated protein kinase, AMPK)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路相关递质表达的影响，探讨艾灸治疗AD的作用机制。方法 将SD大鼠按照随机数字表法分为正常组8只、模型组32只，采取侧脑室注射β淀粉样蛋白(amyloid β-protein，Aβ)25-35的方法建立大鼠AD模型。将模型复制成功的大鼠随机分为模型组、药物组、艾灸组，每组8只。艾灸组大鼠用艾条灸“百会”“肾俞”“三阴交”，每次15 min，同时按3 mg/kg灌胃蒸馏水；药物组大鼠按3 mg/kg灌胃盐酸多奈哌齐；对照组及模型组大鼠按3 mg/kg灌胃蒸馏水。采用Morris水迷宫法检测大鼠行为学表现，苏木精—伊红染色法观察大鼠海马病理组织改变，Western blot法检测大鼠海马磷酸化雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin, p-mTOR)、核糖体蛋白S6 激酶(ribosomal protein S6 kinase p70,P70S6K)、 自噬相关基因5(autophagy-related gene 5,ATG5)、磷酸化腺苷酸活化蛋白激酶(phosphorylated adenosine 5-monophosphate activated protein kinase,p-AMPK)、微管相关蛋白1轻链3B（microtubule associated protein light chain 3B,LC3B）-Ⅱ／LC3B-Ⅰ的表达水平。结果 苏木精—伊红染色结果表明，模型组海马神经元萎缩明显，与模型组比较，药物组和艾灸组海马神经元形态及分化程度均有明显改善。与正常组比较，模型组大鼠的逃避潜伏期显著延长（P＜0.05），p-mTOR及P70S6K 表达水平均显著升高（P＜0.05），ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著降低（P＜0.05）。与模型组比较，药物组和艾灸组大鼠的逃避潜伏期均显著缩短（P＜0.05）， p-mTOR及P70S6K 表达水平均显著下降（P＜0.05），ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著上升（P＜0.05）。与药物组比较，艾灸组大鼠逃避潜伏期显著缩短（P＜0.05）；p-mTOR及P70S6K表达水平显著降低（P＜0.05），ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著上升（P＜0.05）。结论 艾灸能够调控AMPK/mTOR信号通路，诱导细胞自噬，阻断脑内Ａβ表达，从而改善认知功能。

Effect of Moxibustion on the Adenosine 5-Monophosphate Activated Protein Kinase/Mammalian Target of Rapamycin Signaling Pathway in the Hippocampus of Rats with Alzheimers Disease:A Study Based on the Theory of Mutual Help Between the Kidney and the Brain

Objective To investigate the effect of moxibustion on the expression of mediators associated with the adenosine 5-monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in rats with Alzheimers disease (AD),as well as the mechanism of action of moxibustion in the treatment of AD.Methods Sprague-Dawley rats were divided into normal group with 8 rats and model group with 32 rats using a random number table.Intracerebroventricular injection of β-amyloid 25-35 (Aβ25-35) was performed to establish a rat model of AD.After modeling,the rats were randomly divided into moxibustion group,model group,and medication group,with 8 rats in each group.The rats in the moxibustion group were given moxa stick moxibustion at Baihui,Shenshu,and Sanyinjiao for 15 minutes each time and 3 mg/kg distilled water by gavage,those in the medication group were given donepezil hydrochloride at a dose of 3 mg/kg by gavage,and those in the control group and the model group were given 3 mg/kg distilled water by gavage.The Morris water maze test was used to observe the behavioristics of rats;HE staining was used to observe the histopathological changes of the hippocampus;Western blot was used to measure the expression levels of phosphorylated mTOR (p-mTOR),70-kDa ribosomal protein S6 kinase (P70S6K),autophagy-related gene 5(ATG5),phosphorylated AMPK (p-AMPK),and microtubule-associated protein light chain 3B-Ⅱ (LC3B-Ⅱ )/microtubule-associated protein light chain 3B-Ⅰ(LC3B-Ⅰ) in the hippocampus of rats.Results HE staining showed that the model group had marked atrophy of hippocampal neurons,and the medication group and the moxibustion group had significant improvements in the morphology and differentiation of hippocampal neurons compared with the model group.Compared with the normal group,the model group had a significantly longer escape latency (P<0.05),significant increases in the expression levels of p-mTOR and P70S6K (P<0.05),and significant reductions in the expression levels of ATG5,LC3B-Ⅱ/LC3B-Ⅰ,and p-AMPK (P<0.05).Compared with the model group,the medication group and the moxibustion group had a significant reduction in escape latency (P<0.05),significant reductions in the expression levels of p-mTOR and P70S6K (P<0.05),and significant increases in the expression levels of ATG5,LC3B-Ⅱ/LC3B-Ⅰ,and p-AMPK (P<0.05).Compared with the medication group,the moxibustion group had a significant reduction in escape latency (P<0.05),significant reductions in the expression levels of p-mTOR and P70S6K (P<0.05),and significant increases in the expression levels of ATG5,LC3B-Ⅱ/LC3B-Ⅰ,and p-AMPK (P<0.05).Conclusion Moxibustion can improve cognitive function by regulating the AMPK/mTOR signaling pathway,inducing cell autophagy,and blocking the expression of Aβ in brain.