摘要
目的
从中医学对老年记忆的生理病理认识探讨老年人轻度认知障碍的发病机理为脾肾亏虚,脑失所养。遵循中医“虚则补之”“未病先防,既病防变”的治疗原则,提出固本健脑法治疗MCI。用东莨菪碱、NaNO2、40%乙醇腹腔注射制备学习记忆获得障碍、巩固障碍、再现障碍小鼠模型,观察固本健脑法对学习记忆获得障碍、巩固障碍、再现障碍模型小鼠学习记忆能力的影响,探讨固本健脑法对学习记忆获得、巩固、再现各反面障碍的治疗效果；用D-gal与NaNO2腹腔注射制备MCI大鼠模型,通过观察固本健脑法对MCI模型大鼠学习记忆能力、海马组织病理形态、Cav-1、Syn、SNAP-25表达等相关指标的影响,探讨固本健脑法对MCI模型大鼠的治疗作用机理,为中医药治疗中老年人轻度认知障碍提供理论和实验依据。
方法
1.将3月龄昆明种小鼠90只,随机分成3组,每组又分为3个亚组,即正常组(A组)30只,其中A1、A2、A3每组各10只；固本健脑液组(B组)30只,其中B1、B2、B3每组各10只；模型组(C组)30只,其中C1、C2、C3各10只。适应性喂养一周后,各组小鼠均每日1次按10ml/kg·d-1的剂量灌胃,正常组和模型组小鼠灌服0.9%的生理盐水,固本健脑液组则灌服含生药1g/m1的固本健脑液。10天后B1、C1组腹腔注射东莨菪碱制备记忆获得障碍模型,B2、C2组腹腔注射亚硝酸钠制备记忆巩固障碍模型,B3、C3组腹腔注射40%乙醇制备记忆再现障碍模型,A1、A2、A3组腹腔注射等容量生理盐水,用跳台法实验观察实验小鼠的学习记忆能力。
2.将用Morris水迷宫法筛选出记忆能力相当的84只3月龄SD大鼠随机分为6组,即正常组、模型组、脑复康治疗组(简称脑康组)、固本健脑液低剂量组(简称固低组)、固本健脑液中剂量组(简称固中组)、固本健脑液高剂量组(简称固高组),每组14只。各组大鼠适应性喂养一周后,每日上午正常组大鼠腹腔注射生理盐水,固高组、固中组、固低组、脑康组、模型组大鼠腹腔注射D-ga1和NaNO2,连续40天以复制MCI模型,从造模13天起每日下午加以正常组、模型组大鼠灌服生理盐水,固高组、固中组、固低组、脑康组大鼠分别灌服相应药物进行预防,连续28天。40天后用水迷宫行为学试验观察各组大鼠学习记忆能力,光镜、电镜下观察各组大鼠海马组织病理形态学改变,免疫组化法检测各组大鼠海马组织Cav-1的表达,实时荧光定量PCR方法检测各组大鼠海马组织Cav-1、SNAP-25的表达,免疫印迹法检测各组大鼠血清Cav-1、Syn的表达。
结果
1.与学习记忆获得障碍、巩固障碍、再现障碍模型各组小鼠相比较,正常组潜伏期明显高于各模型组(P<0.01)、5分钟内错误次数明显低于各模型组(P<0.01)；固本健脑液治疗组潜伏期高于各模型组(P<0.05)、5分钟内错误次数低于各模型组(P<0.05)。
2.与正常组相比,固高组、固中组、固低组、脑康组、模型组大鼠精神倦怠,均不同程度的表现出饮水量、进食量、活动量减少,皮毛干枯、稀疏、脱落。
3. Morris水迷宫行为学实验,定位航行实验中各组大鼠的平均潜伏期、平台所在象限百分比,空间探索实验中各组大鼠的平均潜伏期、跨越原平台位置的次数、平台所在象限百分比的结果,正常组、固高组、固中组与模型组比较均有显著性差异(P<0.01),固低组、脑康组与模型组比较均有差异(P<0.05),固高组、固中组与脑康组比较均有差异(P<0.05),固低组与脑康组比较没有统计学意义(P>0.05)。
4.光镜、电镜病理形态上观察,正常组主要表现为海马神经元细胞饱满,细胞层数及细胞数目较多,大小一致,排列紧密、整齐,胞膜完整,突触结构丰富；模型组大鼠海马神经细胞数目明显减少,细胞排列混乱,层数减少甚至单层,胞膜、核膜界限不清,胞核空泡变性,核仁变小、裂解,胞质增多,突触数目明显减少；各治疗组介于正常组和模型组之间,与模型组相比均有不同程度的改善,以固高组、固中组的改善最为显著。
5.免疫组化检测各组大鼠海马组织Cav-1蛋白的表达,结果正常组大鼠海马内Cav-1蛋白阳性免疫产物表现最多,模型组最少,各治疗组介于正常和模型组之间,与模型组相比均有不同程度的改善,以固高组、固中组的改善最为显著。
6.实时荧光定量PCR方法检测各组大鼠海马组织Cav-1、SNAP-25的表达,结果二者在各组大鼠海马内的表达具有高度的正相关性。正常组大鼠海马内Cav-1、SNAP-25的表达明显高于模型组(P<0.01)；各治疗组大鼠海马内Cav-1、SNAP-25的表达介于正常组和模型组之间,与模型组比较均有显著性差异(P<0.01)；固高组、固中组与脑康组大鼠海马内Cav-1、SNAP-25的表达比较均有差异(P<0.05),固低组与脑康组比较没有统计学意义(P>0.05)。
7.免疫印迹法检测各组大鼠血清Cav-1、Syn的表达,结果二者在各组大鼠海马内的表达显示出高度的正相关性。正常组大鼠海马内Cav-1、Syn的表达明显优于模型组(P<0.01)；各治疗组大鼠海马内Cav-1、Syn的表达介于正常组和模型组之间,与模型组比较均有明显提高(P<0.01)；固高组、固中组大鼠海马内Cav-1、Syn的表达高于脑康组(P<0.05),固低组与脑康组大鼠海马内Cav-1、Syn的表达比较没有统计学意义(P>0.05)。
结论
1.脾肾亏虚,脑失所养是轻度认知障碍的基本病机,固本健脑法是防治MCI的有效可行方法。
2.固本健脑法能明显改善学习记忆获得、巩固、再现障碍模型大鼠学习记忆能力。
3.联合使用D-gal和NaNO2腹腔注射,可以较好地复制MCI模型,重复性高。
4.固本健脑法能有效改善MCI模型大鼠学习记忆能力；减轻MCI模型大鼠病理形态学的改变；提高MCI模型大鼠海马组织内Cav-1、Syn、SNAP-25的表达。
5.固本健脑法可能是通过作用于Cav-1蛋白,提高突触前Syn、SNAP-25蛋白表达水平,调节海马神经可塑性,从而改善年龄相关性神经可塑性下降而发挥神经保护作用,提高学习记忆能力延缓衰老。
Objective
From the theory of chinese medical science, to explore the etiological factor of Mild Cognitive Impairment (MCI) is the deficiency of spleen and kidney. Follows the chinese medical principle of "treating the asthenia-syndrome by therapy of invigoration" and "protecting the healthy body, preventing the transmission of the disease", we propose the therapy of Gubenjiannao Methods is operative on treating MCI. In the experiments, To built the three mouse models of the impairment of memory acquisition, consolidated memory disorder and recovered memory disorder caused respectively by scopolamine, NaNO2 and 40% ethanol. To built MCI model of rats by the way of intra-peritoneal injection of D-galactose and NaN02. To observe the effects of Gubenjiannao Methods on learning memory, pathological, the hippocamPal levels of Caveolin-1 (Cav-1), Synaptophysin(Syn) and Synaptosomal Associated Protein of 25 kD (SNAP-25). To provide traditional Chinese medicine with experimental accordance and clinical guidance.
Methods
1. The normal 90 3-month-old Kunming rats were randomly divided into three groups:normal group (A group), Gubenjinnao liquid group (B guoup)and model group(C group). there are 15 rats in every group. and each group was divided into three sub-groups (10 rats in every sub-groups). After feeding in ordinary ways for one week, the normal group and model group were fed with 0.9% physiological saline, the Gubenjiannao group were fed with the Gubenjinnao liquid (containing crude drug 1g/ml). Then 10 days later, the B1 and C1 group were prepared by injection of scopolamine model of memory deficits, B2 and C2 preparation of intraperitoneal injection of sodium nitrite memory consolidation Disorder, B3 and C3 intraperitoneal injection of 40% ethanol prepared by the memory representation Disorder. A1, A2, A3 groups with intraperitoneal injection of physiological saline of equal dosage. Test the method of learning and memory abilities of mice with step-down method experiment.
2. Filtering 84 3-month-old SD rats of memory with Morris water maze(MWM), randomly dividing into six groups:normal group, MCI model group, piracetam treated group, high-dose of Gubenjiannao Liquid group (HGG for short), medium-dose of Gubenjiannao Liquid group (MGG for short), low-dose of Gubenjiannao Liquid group (LGG for short), there are 14 rats in every group. After adaptively feeding all rats for one week, injecting physiological saline into abdomen cavity of mormal guoup every morning, injecting D-galactose and NaNO2 into abdomen cavity of model group, piracetam treated group, HGG, MGG and LGG every morning,40 days continuous to copy the MCI model. From the 13th model-copying days on, Filling physiological saline with normal group and model group every afternoon, piracetam treated group, HGG, MGG and LGG of rats fed with the appropriate drugs prevention every afternoon, for consecutively 28 days. Furthermore Morris water maze(MWM) was used to observing the ability of space learning memory of each group. Use the light microscope and electron microscope to observe the changes of morphology and structure in the hippocampus. Test Cav-1 expression by immunohistochemical staining, real-time PCR method was used to detect the hippocampus Cav-1, SNAP-25 expression, hippocampus Cav-1, Syn expression was detected by Western blot.
Results
1. Compared with rat in the model groups of learning and memory disorder acquisition, disorder strengthening and disorder representation, the level of incubation period of the rats in the normal group are higher than that of the rest groups (P<0.01), while their level of making mistakes within 5 minutes are lower than that of the model groups (P<0.01); incubation period level of the Gubenjiannao Liquor group is higher than the other moder groups (P<0.05) and their mistake-making rate within 5 minutes is lower the rest groups (P<0.05).
2. Compared with normal group, the test 5 group all show different degrees characterized by decline in water intake, food intake and activition, and fur dry shriveling, thinning and falling.
3. In the Morris water maze behavior experiment, the average incubation period, percentage of quadrant in the learning ability gain, the average incubation period, percentage of quadrant, the frequency in span of platform localization in space research. The results show that has significant differences between the model group and normal group, HGG and MGG(p<0.01), compared with model guoup, LGG and piracctam treated group (p<0.05), there aren't differences between the LGG and piracctam treated group(p>0.05); there are significant differences between the HGG and piracctam treated group (p<0.01).
4. By the light microscopy and electrical microscopy observation, the neuron of model group was less than that of normal group, death cell could be seen clearly, the neuron decreased and deformed in model group rats, pellet vacuole denaturation, cell nucleus lessen, the mitochondria swelling and vacuole, the endoplasmic reticulum dilatation, amount of synapse decrease, the HGG can ameliorate this pathological changes obviously, there are significant differences between the HGG and piracctam treated group.
5. The results of immunohistochemical staining, the hippocampus of rats treated Cav-1 expression between the normal group and model group, which shows different signs of improvements, the HGG and the MGG being the most remarkable.
6. In testing the hippocampal Cav-1, SNAP-25 expression by the Real-time quantitative PCR method, the results is that in each group they show high positive revelance. The results of the normal group is significantly higher that that of the model group (P<0.01); the hippocampus of rats treated Cav-1, SNAP-25 expression between the normal group and model group, with a notable difference over the model group (P<0.01); there is a difference between the piracctam treated group and the HGG as welll as the MGG(P<0.05); while the comparison between the LGG and the piracctam treated group has no statistical meaning (P>0.05).
7. In testing the blood serum Cav-1, Syn by the Western blotting, the results is that in each group they show high positive revelance. The result of the normal group is better than that of the model group(P<0.01); the hippocampus of rats treated Cav-1, Syn expression between the normal and model groups, with a notable improvement over the model group(P<0.01); there is a difference between the piracctam treated group and the HGG as welll as the MGG(P<0.05); while the comparison between the LGG and the piracctam treated group has no statistical meaning (P>0.05).
Conclusion
1. Spleen and kidney deficiency is the basic etiological factor for MCI, the therapy of Gubenjiannao Method is a effective and feasible cure.
2. The Gubenjiannao Method can notably improve the modeling rats' learning and memorizing ability in the learning and memory acquisition, strengthening and representation disorder.
3. By combining the D-gal intraperitoneal injection and NaNO2 intraperitoneal injection, the MCI model is comparatively easier, and better to make, with a high repetition..
4. The Gubenjiannao Method can effectively improve the learning and memorizing ability of the MCI rats, lighten the pathomorphological changes of the MCI rats and better the Cav-1, Syn, SNAP-25 expression in the hippocampal sections of the MCI rats.
5. The Gubenjiannao Method may increased levels of presynaptic proteins such as Syn and SNAP-25 expression, regulating neuronal plasticity by working on the Cav-1 protein, thus to lower the decline of the age-related nerve formation, to protect the nerves, better the learning and memorizing ability and to delay senility.
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