艾灸“得气”对阿尔茨海默病模型大鼠脑海马Aβ受体介导转运和酶降解相关蛋白的影响

Effect of moxibustion with deqi on Aβ-receptor mediated transport and enzymatic degradation in hippocampus in rats with Alzheimer’s disease

目的:观察艾灸“得气”对阿尔茨海默病(Alzhemier’s disease, AD)模型大鼠的疗效及脑内β淀粉样蛋白(Aβ)转运和降解相关酶蛋白表达的影响,探讨其改善认知功能的分子机制。方法:将60只SPF级SD雄性大鼠随机分为空白组(8只)、假手术组(8只)和造模组(44只),造模组采用双侧脑室注射Aβ1-42建立AD模型。38只造模成功的大鼠随机取8只作为模型组,剩余大鼠于“大椎”穴行温和灸,每天1次,每次40 min,共28 d。根据“得气”出现与否及出现早晚分为艾灸得气组(12只)、艾灸迟发得气组(10只)和艾灸非得气组(8只)。干预后各组大鼠行Morris水迷宫试验检测认知功能;HE染色观察大鼠脑组织形态;Western blot法检测大鼠海马Aβ及其受体介导转运相关蛋白[低密度脂蛋白受体相关蛋白(LRP)1、高级糖化终产物受体(RAGE)、载脂蛋白E(Apo E)]和降解相关酶[中性内肽酶(NEP)、胰岛素降解酶(IDE)、内皮素转换酶(ECE)-1和血管紧张素转换酶(ACE)2]的蛋白表达。结果:与假手术组比较,模型组逃避潜伏期延长(P<0.01),平台穿越次数、平台象限与总时间之比减少(P<0.01);脑组织损伤较重;海马Aβ、RAGE蛋白表达升高(P<0.01),海马LRP1、Apo E、NEP、IDE、ECE-1、ACE2蛋白表达降低(P<0.01)。与模型组比较,艾灸得气组逃避潜伏期缩短(P<0.05,P<0.01),艾灸迟发得气组和艾灸非得气组第2~5天逃避潜伏期缩短(P<0.05,P<0.01),艾灸得气组和艾灸迟发得气组平台穿越次数、平台象限与总时间之比增加(P<0.01,P<0.05);各艾灸组脑损伤均减轻,其中艾灸得气组损伤程度最轻,艾灸迟发得气组次之,艾灸非得气组稍有改善;各艾灸组Aβ、RAGE蛋白表达均降低(P<0.01,P<0.05),LRP1、IDE蛋白表达升高(P<0.01,P<0.05),艾灸得气组和艾灸迟发得气组Apo E蛋白表达升高(P<0.01,P<0.05),艾灸得气组NEP表达增加(P<0.05),艾灸得气组、艾灸迟发得气组ECE-1和ACE2蛋白表达增加(P<0.05)。与艾灸迟发得气组、艾灸非得气组比较,艾灸得气组第3~5天逃避潜伏期缩短(P<0.05),平台穿越次数、平台象限与总时间之比增加(P<0.05,P<0.01);与艾灸非得气组比较,艾灸得气组Aβ降低(P<0.05),LRP1和Apo E蛋白表达升高(P<0.05);艾灸得气组NEP表达高于艾灸迟发得气组、艾灸非得气组(P<0.05)。结论:艾灸“得气”较非“得气”能更好地促进Aβ转运和降解,从而降低脑内Aβ水平,改善AD模型大鼠认知功能。

Objective To observe the clinical effect of moxibustion with deqi on Alzheimer’s disease(AD) rats, and evaluate its effect on β-amyloid(Aβ) transport and enzymatic degradation proteins, to explore its molecular mechanism for improving cognitive function. Methods Sixty SPF-grade male SD rats were randomly divided into a blank group(8 rats), a sham-operation group(8 rats) and a model establishment group(44 rats). The rats in the model establishment group were injected with Aβ1-42 at bilateral ventricles to establish AD model. Among the 38 rats with successful model establishment, 8 rats were randomly selected as the model group, and the remaining rats were treated with mild moxibustion at "Dazhui"(GV 14), once a day, 40 min each time, for 28 days. According to whether deqi appeared and the occurrence time of deqi, the rats were divided into a deqi group(12 rats), a delayed deqi group(10 rats) and a non-deqi group(8 rats). After the intervention, the Morris water maze test was applied to evaluate the cognitive function;the HE staining was applied to observe the brain morphology;the Western blot method was applied to measure the protein expression of Aβ and its receptor mediated transport [low-density lipoprotein receptor-related protein(LRP) 1, receptor for advanced glycation end products(RAGE), apolipoprotein E(Apo E)] and enzymatic degradation [neprilysin(NEP), insulin degrading enzyme(IDE), endothelin converting enzyme(ECE)-1 and angiotensin converting enzyme(ACE) 2]. Results Compared with the sham-operation group, in the model group, the escape latency was prolonged(P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were reduced(P<0.01);the brain tissue was seriously damaged;the expression of hippocampal Aβ and RAGE was increased(P<0.01), and the expression of hippocampal LRP1, Apo E, NEP, IDE, ECE-1 and ACE2 was decreased(P<0.01). Compared with the model group, the escape latency was shortened in the deqi group(P<0.05, P<0.01), and the escape latency in the delayed deqi group and the non-deqi group was shortened from Day 2 to Day 5(P<0.05, P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were increased in the deqi group and the delayed deqi group(P<0.01, P<0.05);the brain damage in each moxibustion group was reduced, which was smallest in the deqi group, followed by the delayed deqi group and the non-deqi group;the expression of Aβ and RAGE was decreased(P<0.01, P<0.05) and the expression of LRP1 and IDE was increased in each moxibustion group(P<0.01, P<0.05);the expression of Apo E was increased in the deqi group and the delayed deqi group(P<0.01, P<0.05);the expression of NEP was increased in deqi group(P<0.05), and the expression of ECE-1 and ACE2 was increased in the deqi group and the delayed deqi group(P<0.05). Compared with the delayed deqi group and the non-deqi group, the escape latency in the deqi group was shortened from Day 3 to Day 5(P<0.05), and the times of platform crossing and the ratio of platform quadrant to total time were increased(P<0.05, P<0.01). Compared with the non-deqi group, the expression of Aβ was reduced(P<0.05), the expression of LRP1 and Apo E was increased in the deqi group(P<0.05). The expression of NEP in the deqi group was higher than that in the delayed deqi group and the non-deqi group(P<0.05). Conclusion Compared with non-deqi, moxibustion with deqi could promote Aβ transport and degradation, thereby reducing Aβ level in the brain and improving cognitive function for AD rats.