化瘀通络灸对血管性痴呆大鼠延迟回忆及海马神经新生的影响

Effect of Moxibustion on Delayed Memory and Expression of Hippocampal Nestin and Doublecortin Proteins in Dementia Rats

目的:观察化瘀通络灸对血管性痴呆(VD)大鼠神经干细胞(NSCs)、内皮祖细胞(EPCs)移植后延迟回忆及海马巢蛋白(Nestin)、双皮质素(DCX)表达的影响,并探索其与脑内神经新生的关系。方法:Wistar大鼠采用Morris水迷宫筛选后随机分为假手术组、VD模型组、共植体艾灸组、共植体空白组。采用改良的缺血再灌注后结扎剪断法复制VD模型,NSCs、EPCs细胞培养后进行共植体构建,共植体艾灸组及共植体空白组大鼠于模型鉴定后3d进行共植体右侧侧脑室移植。共植体艾灸组于移植后3d进行化瘀通络灸治疗,取"百会""大椎""神庭"悬灸20min,每天1次,7次为1个疗程,共3个疗程,每疗程结束后休息1d。运用Morris水迷宫测试各组大鼠延迟回忆成绩,免疫荧光双标激光共聚焦检测大鼠右侧海马Nestin、DCX的表达。结果:Morris水迷宫结果显示,治疗前VD模型组、共植体空白组、共植体艾灸组逃避潜伏期均较假手术组延长,120s内穿越原平台次数减少(P<0.008)。治疗后除VD模型组大鼠逃避潜伏期较治疗前无明显变化外(P>0.05),假手术组、共植体艾灸组、共植体空白组大鼠逃避潜伏期均较治疗前缩短(P<0.05);且共植体艾灸组大鼠120s内穿越原平台次数较治疗前增多(P<0.05)。与假手术组相比,造模后大鼠逃避潜伏期延长,穿越原平台次数减少(P<0.008);共植体艾灸组大鼠逃避潜伏期较VD模型组缩短,穿越原平台次数较VD模型组增多(P<0.008);与共植体空白组相比,共植体艾灸组逃避潜伏期缩短(P<0.008)。免疫荧光结果显示,共植体艾灸组治疗1、3个疗程后海马区Nestin的阳性表达多于共植体空白组(P<0.05);Nestin、DCX分别在治疗1、2个疗程后表达最多(P<0.05)。结论:化瘀通络灸可提高VD大鼠NSCs、EPCs移植后的延迟回忆,并通过上调海马区Nestin、DCX的表达,促进脑内神经新生。

Objective To observe the effect of ＂Huayu Tongluo＂（Blood-stasis Dispersing and Meridian-collateral Dred- ging） moxibustion on the delayed memory and expression of Nestin and Doubiecortin （DCX） proteins in the hippocampus in vascu- lar dementia （VD） rats in the view of neurogenesis produced by intracerebral transplantation of neural stem cells （NSCs） and endothelial progenitor cells （EPCs）. Methods Healthy male Wistar rats were randomized into control group, VD model group, NSCs-I-EPCs group and NSCs＋ EPCs moxibustion group. The VD model was established by using a modified 2-vessels occlusion method, and neurogenesis was produced by transplantation of NSCs-I-EPCs （2 x 108ce11/10 I^L） into the lateral ventricle for rats of the NSCs＋ EPCs groups 3 days after successful VD-modeling. Moxibustion was applied to ＂Dazhui＂ （GV 14）, ＂Baihui＂ （GV 20） and ＂Shenting＂ （GV 24） once daily for 21 days with an interval of one day between every two 7 days. The Morris Water Maze was used to test the rat＇s delayed memory ability before and 24 h after the treatment. The expression of Nestin and DCX proteins in the hippocampus tissues was detected using double-labeled immunofluorescence technique. Results Following modeling,Morris Water Maze tests showed that the average escape latency of location navigation task was significantly prolonged in VD rats （P〈0. 008）and the times of target platform crossing （spatial probing task） within 120 s were remarkably reduced in VD rats （P〈0. 008）. Compared with pre-treatment in the same one group, the escape latency of NSCs＋ EPCs and NSCs-I-EPCs moxi- bustion groups were considerably reduced （P〈0.05）, and the average times of target platform crossing of the NSCs-I-EPCs moxibustion group were markedly increased（P〈0.05）. The effect of NSCs＋ EPCs moxibustion was evidently superior to that of simple NSCs＋ EPCs in shortening the escape latency （P〈0. 008）. The expression levels of Nestin protein were significantly higher in the NSOs-I-EPCs moxibustion group after 1 and 3 period treatment than those in the NSCs＋ EPCs group （P^0.05）. Cenclusion Moxibustion intervention is able to improve the delayed memory in VD rats, which may be related to its effect in up-regulating the expression of hippocampal Nestin and DCX proteins within 15 days via accelerating neurogenesis.