基于恐伤肾的阿尔茨海默病肾虚证大鼠模型的建立与评价

Establishment and evaluation of a rat model of kidney deficiency syndrome combined with Alzheimer’s disease based on fear of kidney injury

目的构建和评价D-半乳糖和三氯化铝结合夹尾(恐吓)建立阿尔茨海默病肾虚证病证结合大鼠模型。方法随机将48只SD大鼠分为对照组、模型组、夹尾组和肾气丸组,每组12只。对照组给予0.9%NaCl溶液灌胃,其余组均采用D-半乳糖及三氯化铝造模,持续8周;夹尾组和肾气丸组从第5周开始,在造模的同时用夹尾(恐吓)法建立肾虚证病证结合模型,肾气丸组同时给予金匮肾气丸10g/kg灌胃。每2周观察记录1次大鼠的症状、体征变化,第8周末进行水迷宫测试。测试结束后,腹主动脉采血,离心收集血清检测下丘脑-垂体系统生化指标。取大鼠大脑,用HE染色和免疫荧光组化法对大脑的海马组织进行检测。结果夹尾组表现为精神亢奋、躁动,大便色黑干硬,毛色发暗枯槁等,造模第6周和第8周症状、体征量化评分均明显高于其他组(P均<0.05);对照组、模型组症状、体征相似,实验过程中各时间点量化评分比较差异均无统计学意义(P均>0.05)。水迷宫测试显示模型组、夹尾组、肾气丸组与对照组相比潜伏期均明显延长(P均<0.05),穿越平台次数明显减少(P均<0.05),而夹尾组比模型组出现较早。海马组织HE染色和免疫荧光组化显示模型组、夹尾组比对照组出现较多神经元细胞死亡和CHAT阳性神经元减少,而夹尾组较为严重,肾气丸组则有改善。下丘脑-垂体-肾上腺轴生化指标血清皮质醇水平模型组明显高于对照组、夹尾组、肾气丸组(P均<0.05),夹尾组明显低于肾气丸组和对照组(P均<0.05),肾气丸组与对照组比较差异无统计学意义(P>0.05)。结论大鼠经D-半乳糖和三氯化铝结合夹尾(恐吓)建立阿尔茨海默病肾虚证是比较理想病证结合模型,可能皮质醇下降是阿尔茨海默病肾虚证证候客观化评价指标之一。

Objective It is to establish and evaluate the method of clamp tail (for fright) plus combination of D-galactose and aluminum trichloride for establishing rat model of Alzheimer's disease with kidney deficiency syndrome.Methods Forty-eight SD rats were randomly divided into 4 groups: control group (A),model group (B),clip tail group (C) and Shenqiwan group (D),12 rats in each group.The rats in group A was given 0.9% NaCl solution by gavage,the ones in the other groups were all modeled with D-galactose and aluminum trichloride for eight weeks.The rats in groups C and D were frighted by tail-clamping from the fifth week to establish the model of syndrome of kidney deficiency,furthermore,the ones in group D were given 10 g/kg of Jinkui Shenqi Pill by gavage.The changes of symptoms and signs of the rats were observed and recorded every two weeks,and water maze tests were performed at the end of the eighth week.After the water maze was over,blood was collected from the abdominal aorta,and serum was collected by centrifugation to detect biochemical indicators of the hypothalamic-pituitary system.The rat brain was extracted and the hippocampus tissue was examined by HE and immunofluorescence histochemistry.Results The rats in group C were excited and agitated,their stool was black and dry,and hair was dark and dry,the scores of symptoms and signs of the 6th week and the 8th week were significantly higher than those of the other groups ( P <0.05).The symptoms and signs of group A and group B were similar,there was no significant difference in the quantitative scores at each time point during the experiment ( P <0.05).The water maze test showed that the latency of group B,group C and group D was significantly longer than that of group A ( P <0.05),and the number of crossing platforms was significantly reduced ( P <0.05),and group C was earlier compared with group B.HE staining and immunofluorescence histochemistry of hippocampus showed that more neuronal cell death and CHAT-positive neurons decrease was found in group B and group C than that in group A,while group C was more serious and that in group D was improved.The serum cortisol level in the hypothalamic-pituitary-adrenal axis biochemical index was significantly higher in group B than that in the other three groups ( P <0.05).The level in group C was significantly lower than that in group A and group D ( P <0.05),there was no significant difference between group A and group D ( P >0.05).Conclusion The rat model of Alzheimer's disease with kidney deficiency syndrome which are established by the method of clamp tail (for fright) plus combination of D-galactose and aluminum trichloride are satisfied,probably cortisol decline is one of the objective evaluation indicators for the syndrome of kidney deficiency of Alzheimer's disease.