补肾益智方对老年性痴呆大鼠学习记忆及胆碱能系统的影响
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摘要
研究背景
阿尔茨海默病(Alzheimer's disease，AD)是一种以损害大脑记忆和认知功能为主的中枢神经系统退行性疾病，AD主要病理改变为老年斑形成、神经纤维缠结及大量的神经元丢失。海马和新皮层胆碱乙酰转移酶(ChAT)及乙酰胆碱(Ach)显著减少引起皮层胆碱能神经元递质紊乱，被认为是记忆障碍和其他认知功能障碍的原因之一，ChAT减少与痴呆严重性、老年斑、神经纤维缠结数量增多有关。在AD的发病机制中，胆碱能损伤学说是目前较为公认的学说。
在老年人群中，AD已成为继心脏病、肿瘤和中风之后的第四位死亡原因，给个人、家庭及社会带来沉重的负担和痛苦，重视老年痴呆的防治，具有重要意义。而中医药对延缓神经系统退行性变、促进脑神经功能的恢复在临床与科研中日益得到肯定。补肾益智方是以广州中医药大学赖世隆教授为首的科研小组针对老年性痴呆的主要病理机制、总结长期临床经验并结合现代药理学研究成果设计而成，由蛇床子、枸杞子、女贞子、人参、制首乌、丹皮、冰片等中药组成。具有填精补髓，益气养血，活血通络，开窍的功效。
研究目的
本实验研究以IBO致老年性痴呆大鼠为动物模型，研究补肾益智方对老年性痴呆大鼠模型的学习记忆能力、病理形态学及乙酰胆碱脂酶的影响，探讨补肾益智方防治老年性痴呆的作用机理。
方法
把3月龄的SD大鼠260只按体重随机法分为6组：正常组、模型组、阳性药组和补肾益智方低、中、高剂量组，采用基底前脑注射IBO的方法制造老年性痴呆模型，造模后正常组和模型组给予灌胃生理盐水，阳性药给予石杉碱甲，补肾益智方低、中、高剂量给予相应的剂量灌胃28天后对上述动物进行为期6日的Morris水迷宫测试，记录其在定位航行试验和空间探索试验的数据；水迷宫测试完成后，每组随机取6只大鼠做脑组织病理切片，其余大鼠先心脏取血，分离血清，然后断头取脑，分离两侧皮层和海马为标本。取冻存的一侧皮质进行AchE的检测。定位航行及空间探索数据结果以边际均值±标准误表示，脑组织总蛋白含量、乙酰胆碱酯酶(AchE)活性活性及神经元数目以(?)±s表示，用SPSS11.5统计软件对数据进行统计分析。统计分析方法：定位航行测试的数据采用的重复测量方差分析进行统计，空间探索试验的数据采用多因素方差分析进行统计，脑组织总蛋白含量、乙酰胆碱酯酶(AchE)活性及神经元数目采用单因素方差分析。
结果
1．大鼠学习记忆能力，包括定位航行和空间探索测试两部分。
1)定位航行试验结果：
(1)五天变化趋势：随着时间的推移，逃避潜伏期逐渐缩短；其中，后四天的逃避潜伏期同第1天比较差异有统计学意义(P＜0.01)；提示寻找平台是一个学习和记忆的过程，经过前2天的训练，第3、4、5天逃避潜伏变化趋势减小。速度的变化趋势是除了第2天有所提高外，第3天开始明显下降，第4、5恢复并达到一个稳定水平。提示大鼠的体力未受明显的影响。行程和初始角度的变化趋势是：随着时间的增加，逐渐缩短。提示游泳路程的缩短和初始角度地缩小可能与大鼠学习记忆能力改善有关。
(2)组间比较：模型组与正常组相比，逃避潜伏期时间增加(P＜0.01)，游泳路程亦增加(P＜0.01)，提示IBO致老年性痴呆模型大鼠学习记忆能力下降，造模成功。与模型组相比，补肾益智方低、高剂量组大鼠的逃避潜伏期缩短(P＜0.01)；补肾益智方低、中、高剂量组游泳路程亦缩短(P＜0.01)。
2)空间探索试验结果：模型组与正常组相比，大鼠在原平台象限停留时间减少(P＜0.05)，提示IBO致老年性痴呆模型大鼠学习记忆能力下降，造模成功。与模型组相比，补肾益智方低、中、高剂量组大鼠在原平台象限停留时间增加(P＜0.05，P＜0.01)。
上述结果提示补肾益智方低、中、高剂量组均能明显改善IBO致老年性痴呆大鼠的学习记忆能力。
2．大鼠皮质匀浆液的蛋白含量。统计分析结果相关系数R为0.991，提示标准曲线各点的浓度和读数一致性好，结果准确。
3．大鼠脑皮质乙酰胆碱脂酶活性。各组大鼠脑皮质乙酰胆碱脂酶活性：正常组＞阳性药组＞高剂量组＞低剂量组＞中剂量组＞模型组。与模型组比较，补肾益智方高剂量组、阳性药组的乙酰胆碱脂酶活性升高，差异有统计学意义(P＜0.05)。提示补肾益智方高剂量组能明显改善模型大鼠胆碱能系统的功能。
4．大鼠海马CA1区神经元数量。各组大鼠海马CA1区神经元数量：正常组＞低剂量组＞阳性药组＞高剂量组＞模型组＞中剂量组。各组与模型组比较，差异均无统计学意义。提示补肾益智方对IBO致老年性痴呆模型大鼠海马神经元的数量未产生明显影响。
结论
补肾益智方低、中、高剂量组都能明显改善IBO致老年性痴呆大鼠的学习记忆能力；补肾益智方高剂量组能改善老年性痴呆大鼠胆碱能系统的功能；补肾益智方各剂量组对老年性痴呆大鼠海马CA1区神经元的数量未产生明显的影响。
This study was designed to investigate the influences of BuShenYiZhi Decoction on cognition and memory function impaired in rat model with Alzheimer's disease, further to evaluate the molecular mechanism of brain acetylcholine esterase system and histology of the neurons in hippocampus CA1 of rat model with Alzheimer's disease.
Sixty AD rats aged 3-month were randomly divided into six groups including normal group, AD model group, Hup-A group and BSYZD(low dose, middle dose, high dose) group. The rats in normal group were injected physiologic saline into basal nucleus of Meynert, but the rats in other groups were injured by 10μg ibotenic acid(IBO) injection in basal forebrain. After being treated for four weeks, we assayed the ability of cognition and memory abilities of all the rats by the test of Morris water maze test, including Place Navigation Training and the Spatial Probe Test. After finishing 6-day's Morris water maze test, we randomly selected 6 rats in each group and then took out their whole brain to detect the number and morphology of neurons by H E staining. Other rats were killed and collected their blood by sucking the heart and separating the hippocampus cortex from the brain tissue. The blood samples were centrifuged and the serum samples were pipetted into other EP tubes. All these samples were frozen in -70℃till analysis. Acetylcholine erase activity of cortex tissue was measured by TECAN Enzyme machine. The protein levels of the cortex samples were determined in a 96 well plate by a micro-method way. Place Navigation Training and Spatial Probe Test experiments data were expressed as estimated marginal means±Std. Error, other data were expressed as x±s. Statistical method: data as the escape latency, distance, average speed and primary angle were analyzed by Repeated Measures module in SPSS11. 5. Data of Spatial Probe Test were analyzed by Multivariate module in SPSS11. 5.0ther data were analyzed by one-way ANOVA in SPSS11. 5.
1. The Morris water maze test includes two parts: Place Navigation Training and the Spatial Probe Test. The results were expressed as follows:
1) Place Navigation Training
(1) Trend of the five-day changes: As time went on, the escape latencies were shorter gradually. And the later four days of the escape latency were significantly shorter than that of the first day(P＜0.01). All above indicated that the position of the platform is a process of learning and memorizing which can be trained through a repeated way. Trend of the changes in speed showed the increasement on the second day and decreasement since the third day, rebound a little and became stable on the forth and the fifth day, which implied the physical vigor had not been influenced. Trend of the changes in distance and primary angel showed that the distance and primary angel became shorter and smaller as time went on, which perhaps connect with improvement of rats' learning and memory ability.
(2) Comparisons between the six groups. Compare with normal group, the escape latency and swimming distance of rats in AD model group were all longer significantly(P＜0.01), which meant the AD rat model was successful. Compare with BSYZD(low dose, high dose) group, the escape latency of rats in AD model group were significantly shorter(P＜0.01). Compare with BSYZD(low dose, middle dose, high dose) group, the swimming distance of rats in AD model group were significantly shorter(P＜0.01).
2) Spatial Probe Test
Compare with normal group, the rats of AD model group in the fourth quadrant(the position of the platform) were significantly shorter(P＜0.05). In addition, compared with BSYZD(low dose, middle dose, high dose) group, the time of rats in AD model group in the forth quadrant were significantly longer(P＜0.05, P＜0.01), which eluciated that memory for the position of the platform had been strengthened.
These results concluded that BSYZD(low dose, middle dose, high dose) could improve the cognition and memory ability of AD rat model to some extent.
2. Measuement of protein in the cortex. The correlation coefficient R result was 0.991, which meant a good consistency among the points of the standard curve and good accuracy of the results.
3. Acetylcholine erases activity of cortex tissue. The result was: Normal group＞Hup-A group＞high dose BSYZD group＞low dose BSYZD group＞middle dose BSYZD group＞AD model group. Compare with those rat in AD model group, the rats in Hup-A group and high dose BSYZD group's acetyleholinest erase activity were significantly enhanced(P＜0.05), which meant the AchE activity had been recuperated.
4. The number and morphology of neurons by H E staining showed that no significant change observed among the groups.
This study concluded that BSYZD(low dose, middle dose, high dose) could improve the cognition and memory ability of AD rat model to some extent. We also found that the acetylcholine erase activity of rats in High dose BSYZD group were significantly increased(P＜0.05) compared to those in AD model group, which showed that the Ache activity were recuperated. The number and morphology of neurons by H E staining showed that no Significant difference among the groups.
阿尔茨海默病(Alzheimer's disease，AD)是一种以损害大脑记忆和认知功能为主的中枢神经系统退行性疾病，AD主要病理改变为老年斑形成、神经纤维缠结及大量的神经元丢失。海马和新皮层胆碱乙酰转移酶(ChAT)及乙酰胆碱(Ach)显著减少引起皮层胆碱能神经元递质紊乱，被认为是记忆障碍和其他认知功能障碍的原因之一，ChAT减少与痴呆严重性、老年斑、神经纤维缠结数量增多有关。在AD的发病机制中，胆碱能损伤学说是目前较为公认的学说。
在老年人群中，AD已成为继心脏病、肿瘤和中风之后的第四位死亡原因，给个人、家庭及社会带来沉重的负担和痛苦，重视老年痴呆的防治，具有重要意义。而中医药对延缓神经系统退行性变、促进脑神经功能的恢复在临床与科研中日益得到肯定。补肾益智方是以广州中医药大学赖世隆教授为首的科研小组针对老年性痴呆的主要病理机制、总结长期临床经验并结合现代药理学研究成果设计而成，由蛇床子、枸杞子、女贞子、人参、制首乌、丹皮、冰片等中药组成。具有填精补髓，益气养血，活血通络，开窍的功效。
研究目的
本实验研究以IBO致老年性痴呆大鼠为动物模型，研究补肾益智方对老年性痴呆大鼠模型的学习记忆能力、病理形态学及乙酰胆碱脂酶的影响，探讨补肾益智方防治老年性痴呆的作用机理。
方法
把3月龄的SD大鼠260只按体重随机法分为6组：正常组、模型组、阳性药组和补肾益智方低、中、高剂量组，采用基底前脑注射IBO的方法制造老年性痴呆模型，造模后正常组和模型组给予灌胃生理盐水，阳性药给予石杉碱甲，补肾益智方低、中、高剂量给予相应的剂量灌胃28天后对上述动物进行为期6日的Morris水迷宫测试，记录其在定位航行试验和空间探索试验的数据；水迷宫测试完成后，每组随机取6只大鼠做脑组织病理切片，其余大鼠先心脏取血，分离血清，然后断头取脑，分离两侧皮层和海马为标本。取冻存的一侧皮质进行AchE的检测。定位航行及空间探索数据结果以边际均值±标准误表示，脑组织总蛋白含量、乙酰胆碱酯酶(AchE)活性活性及神经元数目以(?)±s表示，用SPSS11.5统计软件对数据进行统计分析。统计分析方法：定位航行测试的数据采用的重复测量方差分析进行统计，空间探索试验的数据采用多因素方差分析进行统计，脑组织总蛋白含量、乙酰胆碱酯酶(AchE)活性及神经元数目采用单因素方差分析。
结果
1．大鼠学习记忆能力，包括定位航行和空间探索测试两部分。
1)定位航行试验结果：
(1)五天变化趋势：随着时间的推移，逃避潜伏期逐渐缩短；其中，后四天的逃避潜伏期同第1天比较差异有统计学意义(P＜0.01)；提示寻找平台是一个学习和记忆的过程，经过前2天的训练，第3、4、5天逃避潜伏变化趋势减小。速度的变化趋势是除了第2天有所提高外，第3天开始明显下降，第4、5恢复并达到一个稳定水平。提示大鼠的体力未受明显的影响。行程和初始角度的变化趋势是：随着时间的增加，逐渐缩短。提示游泳路程的缩短和初始角度地缩小可能与大鼠学习记忆能力改善有关。
(2)组间比较：模型组与正常组相比，逃避潜伏期时间增加(P＜0.01)，游泳路程亦增加(P＜0.01)，提示IBO致老年性痴呆模型大鼠学习记忆能力下降，造模成功。与模型组相比，补肾益智方低、高剂量组大鼠的逃避潜伏期缩短(P＜0.01)；补肾益智方低、中、高剂量组游泳路程亦缩短(P＜0.01)。
2)空间探索试验结果：模型组与正常组相比，大鼠在原平台象限停留时间减少(P＜0.05)，提示IBO致老年性痴呆模型大鼠学习记忆能力下降，造模成功。与模型组相比，补肾益智方低、中、高剂量组大鼠在原平台象限停留时间增加(P＜0.05，P＜0.01)。
上述结果提示补肾益智方低、中、高剂量组均能明显改善IBO致老年性痴呆大鼠的学习记忆能力。
2．大鼠皮质匀浆液的蛋白含量。统计分析结果相关系数R为0.991，提示标准曲线各点的浓度和读数一致性好，结果准确。
3．大鼠脑皮质乙酰胆碱脂酶活性。各组大鼠脑皮质乙酰胆碱脂酶活性：正常组＞阳性药组＞高剂量组＞低剂量组＞中剂量组＞模型组。与模型组比较，补肾益智方高剂量组、阳性药组的乙酰胆碱脂酶活性升高，差异有统计学意义(P＜0.05)。提示补肾益智方高剂量组能明显改善模型大鼠胆碱能系统的功能。
4．大鼠海马CA1区神经元数量。各组大鼠海马CA1区神经元数量：正常组＞低剂量组＞阳性药组＞高剂量组＞模型组＞中剂量组。各组与模型组比较，差异均无统计学意义。提示补肾益智方对IBO致老年性痴呆模型大鼠海马神经元的数量未产生明显影响。
结论
补肾益智方低、中、高剂量组都能明显改善IBO致老年性痴呆大鼠的学习记忆能力；补肾益智方高剂量组能改善老年性痴呆大鼠胆碱能系统的功能；补肾益智方各剂量组对老年性痴呆大鼠海马CA1区神经元的数量未产生明显的影响。
This study was designed to investigate the influences of BuShenYiZhi Decoction on cognition and memory function impaired in rat model with Alzheimer's disease, further to evaluate the molecular mechanism of brain acetylcholine esterase system and histology of the neurons in hippocampus CA1 of rat model with Alzheimer's disease.
Sixty AD rats aged 3-month were randomly divided into six groups including normal group, AD model group, Hup-A group and BSYZD(low dose, middle dose, high dose) group. The rats in normal group were injected physiologic saline into basal nucleus of Meynert, but the rats in other groups were injured by 10μg ibotenic acid(IBO) injection in basal forebrain. After being treated for four weeks, we assayed the ability of cognition and memory abilities of all the rats by the test of Morris water maze test, including Place Navigation Training and the Spatial Probe Test. After finishing 6-day's Morris water maze test, we randomly selected 6 rats in each group and then took out their whole brain to detect the number and morphology of neurons by H E staining. Other rats were killed and collected their blood by sucking the heart and separating the hippocampus cortex from the brain tissue. The blood samples were centrifuged and the serum samples were pipetted into other EP tubes. All these samples were frozen in -70℃till analysis. Acetylcholine erase activity of cortex tissue was measured by TECAN Enzyme machine. The protein levels of the cortex samples were determined in a 96 well plate by a micro-method way. Place Navigation Training and Spatial Probe Test experiments data were expressed as estimated marginal means±Std. Error, other data were expressed as x±s. Statistical method: data as the escape latency, distance, average speed and primary angle were analyzed by Repeated Measures module in SPSS11. 5. Data of Spatial Probe Test were analyzed by Multivariate module in SPSS11. 5.0ther data were analyzed by one-way ANOVA in SPSS11. 5.
1. The Morris water maze test includes two parts: Place Navigation Training and the Spatial Probe Test. The results were expressed as follows:
1) Place Navigation Training
(1) Trend of the five-day changes: As time went on, the escape latencies were shorter gradually. And the later four days of the escape latency were significantly shorter than that of the first day(P＜0.01). All above indicated that the position of the platform is a process of learning and memorizing which can be trained through a repeated way. Trend of the changes in speed showed the increasement on the second day and decreasement since the third day, rebound a little and became stable on the forth and the fifth day, which implied the physical vigor had not been influenced. Trend of the changes in distance and primary angel showed that the distance and primary angel became shorter and smaller as time went on, which perhaps connect with improvement of rats' learning and memory ability.
(2) Comparisons between the six groups. Compare with normal group, the escape latency and swimming distance of rats in AD model group were all longer significantly(P＜0.01), which meant the AD rat model was successful. Compare with BSYZD(low dose, high dose) group, the escape latency of rats in AD model group were significantly shorter(P＜0.01). Compare with BSYZD(low dose, middle dose, high dose) group, the swimming distance of rats in AD model group were significantly shorter(P＜0.01).
2) Spatial Probe Test
Compare with normal group, the rats of AD model group in the fourth quadrant(the position of the platform) were significantly shorter(P＜0.05). In addition, compared with BSYZD(low dose, middle dose, high dose) group, the time of rats in AD model group in the forth quadrant were significantly longer(P＜0.05, P＜0.01), which eluciated that memory for the position of the platform had been strengthened.
These results concluded that BSYZD(low dose, middle dose, high dose) could improve the cognition and memory ability of AD rat model to some extent.
2. Measuement of protein in the cortex. The correlation coefficient R result was 0.991, which meant a good consistency among the points of the standard curve and good accuracy of the results.
3. Acetylcholine erases activity of cortex tissue. The result was: Normal group＞Hup-A group＞high dose BSYZD group＞low dose BSYZD group＞middle dose BSYZD group＞AD model group. Compare with those rat in AD model group, the rats in Hup-A group and high dose BSYZD group's acetyleholinest erase activity were significantly enhanced(P＜0.05), which meant the AchE activity had been recuperated.
4. The number and morphology of neurons by H E staining showed that no significant change observed among the groups.
This study concluded that BSYZD(low dose, middle dose, high dose) could improve the cognition and memory ability of AD rat model to some extent. We also found that the acetylcholine erase activity of rats in High dose BSYZD group were significantly increased(P＜0.05) compared to those in AD model group, which showed that the Ache activity were recuperated. The number and morphology of neurons by H E staining showed that no Significant difference among the groups.
引文
[1] 王维治．神经病学．人民卫生出版社，2002．249-50．
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