人参皂甙对铝中毒大鼠学习记忆功能的保护作用及机理探讨
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摘要
目的
Alzheimer病是最常见的痴呆病因之一，发病率随年龄逐渐增高，是病因不明，渐进性进展的退行性疾病。Alzheimer病的病理特征是：老年斑，神经纤维缠结和神经元缺失。主要临床表现是：记忆力减退，认知障碍及人格改变。目前没有可以逆转或阻止Alzheimer病进展的治疗方法。因此，开发研究防治Alzheimer病的药物是非常必要的。
本实验通过Morris水迷宫测试，HE染色，免疫细胞化学染色等方法，观察大鼠行为学，海马CA1区组织病理学及免疫细胞化学的变化，研究人参皂甙对抗铝对大鼠海马神经元的损害，保护大鼠学习记忆功能，防止大鼠铝中毒后出现Alzheimer痴呆的作用，并探讨可能的作用机理。
方法
1、预防性用药 将18只Wistar大鼠(230克-250克，三月龄，雄性)随机分成三组，实验组6只；对照组6只；模型组6只。实验组大鼠每天饮水法服用人参皂甙40ml／d.kg，持续五周，模型组及对照组大鼠饮用等量的自来水。
2、侧脑室注射三氯化铝(Alcl_3)预防用药结束后，麻醉各组大鼠，参照大鼠脑立体定位仪图谱，将3％Alcl_3 10ul缓慢注入实验组及模型组大鼠左侧侧脑室，对照组大鼠相应部位注入等量的生理盐水。
3、Morris水迷宫检测 术后第四天，全部大鼠进行Morris水迷宫检测。
4、HE染色Morris水迷宫测试结束后，灌流，固定，取材(大鼠海马)，包埋，切片(片厚10um)，HE染色。
5、免疫组化染色 取材，包埋，切片，用兔抗鼠β-APP抗体和SABC试剂盒及DAB进行免疫组化染色。
6、统计学处理 用SPSS统计软件对Morris水迷宫测试数据进行分析处理。
结果
1、行为学与对照组比较，实验组大鼠的逃避潜伏期无明显延长，两者
之间无显著性差异(P>0.05)，表明实验组大鼠的学习记忆功能在侧脑室
注铝后无明显减退。模型组大鼠的逃避潜伏期与对照组比较明显延长，两
者之间有显著性差异(P<0.05)，表明模型组大鼠的学习记忆功能在侧脑
室注铝后明显减退。
2、组织病理学与对照组比较，实验组大鼠海马CA:区无明显神经元缺
失;模型组大鼠海马CA:区与对照组比较有明显的神经元缺失。
3、免疫细胞化学实验组大鼠与对照组大鼠海马CA:区p一App免疫反
应均呈弱阳性。模型组大鼠海马CA:区p一人PP反应呈强阳性。
结论
1、人参皂贰具有对抗铝对大鼠海马神经元的损害，保护大鼠的学习记
忆功能，防止大鼠铝中毒后出现Ahhe加er型痴呆的作用。
2、人参皂贰的作用机理可能是通过抑制铝中毒后大鼠脑内p一APP的
过度表达，从而抑制和清除海马p一AP的沉积来实现的。
Alzheimers disease, by far the single most frequent cause of dementia, becomes increasingly common with advancing age. A slowly progssive degenerative disorder of unknown cause, it is characterized by typical histopathoplogic features; neurofibrillary tangles, neuritic plaques, and neuronal loss. Its typical clinical foundings are impairment of memory, congective dysfunction and personality change. No currently available treatment has been known unequivocally to reverse existing deficits or arrest the disease progression. Therefore, it is very necessary to explore treatments that can prevent from developing Alzheimers disease.
In this experimental, the behavioral change, histopathologic and immuno-histochemical changes were observed by Morris water maze investigation histopathologic staining and immunohistochemical staining , in order to study the function of Ginseng Saponin of resisting the damage to hippocampus of rats following Alcl3 injection, protecting the ability of learning and memory, preventing from developing Alzheimers dementia after aluminum poisoning and explore the possible mechanism of the function.
Methods
1. Pretreatment.18 Wistar rats (230 -250g,three months,all male) were
randomly divided into 3 groups; experimental group (6) ; control group (6) ; model group (6). Experimental group rats were pretreated with Ginseng Sa-ponion (40ml/d. kg) for five weeks, whereas the model and control group rats drank the same amount of water.
2. Lateralcerebroventricular injection of Alcl3 After pretreatment, each rat was anesthetized. Then 10u13% Alcl3 was injected respectively into lateralcerebroventricular of experimental and model group rats by microliter syringes according to The Stereotaxic Atlas of The Rat Brain. , whereas the same amount of normal sadine was injected into lateralcerebroventricular of control group rats.
3. Morris water maze investigation Moriss. water maze was used to investigate learning and memory abilities of three groups rats, four days after operation.
4. HE staining.Piped and Fixed, then Drawed hippocampus of three groups rats respectively after Morris water maze investigation. Embed and Sliced (the thickness of tissue slides was lOura).. HE stained .
5. Immunohistochemistry staining. Immunohistochemistry staining was conducted by using B- APP rabbit anti - mouse antibody and SABC kid and DAB.
6. Statistics process .The data gained from Morris water maze investigation was processed by SPSS Microsoft.
Results
1. Behavior.The escape latency of experimental group rats did not prolong compared with that of contral group rats . There was no significant difference between the escape latency of experimental and contral group rats ( p > 0. 05), which showed the ability of learning and memory of experimental group rats did not decreased following Alcl3 injection, howver, the escape latency of model group rats prolonged compaired with that of control group rats. There was significant diference between the escape latency of model and control group rats ( p < 0. OS ) , which showed the ability of learning and memory of model group rats decreased significantly following Alcl3 injection.
2. Histopathology .There was not significant neuronol loss in hippocampal
CA, of experimental group rats compared with control group rats . Whereas , there was significant neuronal loss in hippocampal CA, of model group rats compared with contral group rats.
3. Immunocytochemistry.Immunoreaction in hippocampal CA, of experimental and control group rats was softly positive. However, immunoreaction in hippocampal CA, of model group rats was strongly positive.
Conclusions
1. Ginseng Saponin has the function of resisting damage to hippocampus of rat caused by aluminum and protecting the ability of learning and memory of rat , and preventing from developing Alzheimer s dementia after aluminum poisoning
2. The mechanism of Ginseng Saponin may be related to inhibit over expression of β- APP and deposition of β- AP.
Alzheimer病是最常见的痴呆病因之一，发病率随年龄逐渐增高，是病因不明，渐进性进展的退行性疾病。Alzheimer病的病理特征是：老年斑，神经纤维缠结和神经元缺失。主要临床表现是：记忆力减退，认知障碍及人格改变。目前没有可以逆转或阻止Alzheimer病进展的治疗方法。因此，开发研究防治Alzheimer病的药物是非常必要的。
本实验通过Morris水迷宫测试，HE染色，免疫细胞化学染色等方法，观察大鼠行为学，海马CA1区组织病理学及免疫细胞化学的变化，研究人参皂甙对抗铝对大鼠海马神经元的损害，保护大鼠学习记忆功能，防止大鼠铝中毒后出现Alzheimer痴呆的作用，并探讨可能的作用机理。
方法
1、预防性用药 将18只Wistar大鼠(230克-250克，三月龄，雄性)随机分成三组，实验组6只；对照组6只；模型组6只。实验组大鼠每天饮水法服用人参皂甙40ml／d.kg，持续五周，模型组及对照组大鼠饮用等量的自来水。
2、侧脑室注射三氯化铝(Alcl_3)预防用药结束后，麻醉各组大鼠，参照大鼠脑立体定位仪图谱，将3％Alcl_3 10ul缓慢注入实验组及模型组大鼠左侧侧脑室，对照组大鼠相应部位注入等量的生理盐水。
3、Morris水迷宫检测 术后第四天，全部大鼠进行Morris水迷宫检测。
4、HE染色Morris水迷宫测试结束后，灌流，固定，取材(大鼠海马)，包埋，切片(片厚10um)，HE染色。
5、免疫组化染色 取材，包埋，切片，用兔抗鼠β-APP抗体和SABC试剂盒及DAB进行免疫组化染色。
6、统计学处理 用SPSS统计软件对Morris水迷宫测试数据进行分析处理。
结果
1、行为学与对照组比较，实验组大鼠的逃避潜伏期无明显延长，两者
之间无显著性差异(P>0.05)，表明实验组大鼠的学习记忆功能在侧脑室
注铝后无明显减退。模型组大鼠的逃避潜伏期与对照组比较明显延长，两
者之间有显著性差异(P<0.05)，表明模型组大鼠的学习记忆功能在侧脑
室注铝后明显减退。
2、组织病理学与对照组比较，实验组大鼠海马CA:区无明显神经元缺
失;模型组大鼠海马CA:区与对照组比较有明显的神经元缺失。
3、免疫细胞化学实验组大鼠与对照组大鼠海马CA:区p一App免疫反
应均呈弱阳性。模型组大鼠海马CA:区p一人PP反应呈强阳性。
结论
1、人参皂贰具有对抗铝对大鼠海马神经元的损害，保护大鼠的学习记
忆功能，防止大鼠铝中毒后出现Ahhe加er型痴呆的作用。
2、人参皂贰的作用机理可能是通过抑制铝中毒后大鼠脑内p一APP的
过度表达，从而抑制和清除海马p一AP的沉积来实现的。
Alzheimers disease, by far the single most frequent cause of dementia, becomes increasingly common with advancing age. A slowly progssive degenerative disorder of unknown cause, it is characterized by typical histopathoplogic features; neurofibrillary tangles, neuritic plaques, and neuronal loss. Its typical clinical foundings are impairment of memory, congective dysfunction and personality change. No currently available treatment has been known unequivocally to reverse existing deficits or arrest the disease progression. Therefore, it is very necessary to explore treatments that can prevent from developing Alzheimers disease.
In this experimental, the behavioral change, histopathologic and immuno-histochemical changes were observed by Morris water maze investigation histopathologic staining and immunohistochemical staining , in order to study the function of Ginseng Saponin of resisting the damage to hippocampus of rats following Alcl3 injection, protecting the ability of learning and memory, preventing from developing Alzheimers dementia after aluminum poisoning and explore the possible mechanism of the function.
Methods
1. Pretreatment.18 Wistar rats (230 -250g,three months,all male) were
randomly divided into 3 groups; experimental group (6) ; control group (6) ; model group (6). Experimental group rats were pretreated with Ginseng Sa-ponion (40ml/d. kg) for five weeks, whereas the model and control group rats drank the same amount of water.
2. Lateralcerebroventricular injection of Alcl3 After pretreatment, each rat was anesthetized. Then 10u13% Alcl3 was injected respectively into lateralcerebroventricular of experimental and model group rats by microliter syringes according to The Stereotaxic Atlas of The Rat Brain. , whereas the same amount of normal sadine was injected into lateralcerebroventricular of control group rats.
3. Morris water maze investigation Moriss. water maze was used to investigate learning and memory abilities of three groups rats, four days after operation.
4. HE staining.Piped and Fixed, then Drawed hippocampus of three groups rats respectively after Morris water maze investigation. Embed and Sliced (the thickness of tissue slides was lOura).. HE stained .
5. Immunohistochemistry staining. Immunohistochemistry staining was conducted by using B- APP rabbit anti - mouse antibody and SABC kid and DAB.
6. Statistics process .The data gained from Morris water maze investigation was processed by SPSS Microsoft.
Results
1. Behavior.The escape latency of experimental group rats did not prolong compared with that of contral group rats . There was no significant difference between the escape latency of experimental and contral group rats ( p > 0. 05), which showed the ability of learning and memory of experimental group rats did not decreased following Alcl3 injection, howver, the escape latency of model group rats prolonged compaired with that of control group rats. There was significant diference between the escape latency of model and control group rats ( p < 0. OS ) , which showed the ability of learning and memory of model group rats decreased significantly following Alcl3 injection.
2. Histopathology .There was not significant neuronol loss in hippocampal
CA, of experimental group rats compared with control group rats . Whereas , there was significant neuronal loss in hippocampal CA, of model group rats compared with contral group rats.
3. Immunocytochemistry.Immunoreaction in hippocampal CA, of experimental and control group rats was softly positive. However, immunoreaction in hippocampal CA, of model group rats was strongly positive.
Conclusions
1. Ginseng Saponin has the function of resisting damage to hippocampus of rat caused by aluminum and protecting the ability of learning and memory of rat , and preventing from developing Alzheimer s dementia after aluminum poisoning
2. The mechanism of Ginseng Saponin may be related to inhibit over expression of β- APP and deposition of β- AP.
引文
1．1．徐淑去，卞如濂等主编．药理实验方法学．第三版．人民卫生出版社 2002，849，828．
2. G. Paxinos C. Watson The Rat Brain in Stereotaxic Coordinates 1986
3．贾建平 2003神经病学新进展 人民卫出版社 2003，249-25
4．赵宪林，方秀斌，李东培．大鼠血管性痴呆模型制作中国医科大学学报 2002；31(3)：166-167．
5. Katzmon R, Alzheimer disease N Engl JMed. 1986; 314: 9647
6．郑观成．脑老化与老年痴呆，第一卷，第一版，上海，上海科学技术出版社，1994，185．
7．秦俊法．微量元素与脑功能．第一版，北京．原子能出版社．1994，52．
8. Klatzo I, Wishie wisk H, Streicher E. Experimental production of neurofibrillary degeneration. J Nneuropath Exp Neural, 1965, 24: 187.
9. Crapper DG. Krishnan SS, Dakton AJ. Brain alcuminum distribution in Alzheimer's disease and ecperimental neurofibrillary degeneratin Science 1973, 180(4085): 511.
10. Perl DP. Brady AR. Alzheimer's disease: X-ray spectrometric evidence ok alcuminum accumulation in meurofibrillary tangle bearing neurons. Scieme,1980, 208(18): 297.
11. Morris R G M. Garrud P, Raulins J NP, et al. Place natigation impaired in rats with hippocampal lesions [J] Nature, 1982, 297: 681.
12. Squire L R. Zola-Morgan S. Memory brain system and behavior[J] Trends in Neuro Science, 1988, 11: 170.
13．姚志彬．海马—研究神经科学的理想模型．广东解剖学通报 1989，11：17-21．
14．马天才，于庆海．人参对学习记忆的药理学影响．中草药，1994；21(7)：38-40．
15. Petkov VD, Gao Y, Todorov, Lazarova M, et al. Behavioral effects al stemleaves extract from panax ginseng CA Meyer, Acta physiol pharmacol Bulg, 1992;18(2): 41-8.
16．胡海涛，钱亦华等．淀粉样前体蛋白在痴呆大鼠背海马结构内的表达．解剖学杂志 1997，20(5)444-445．
17. Simmons LK, May PC, Tomaselli KJ, et al. Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. Molecular Pharmacologycal, 1993; 45: 373-379
18．陈俊抛 林昱 痴呆治疗学 人民卫生出版社，2002，269
19. Glenner GG, Wong CW. Alzheimer s disease and Down s sydrome: Sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Cmmun, 1983; 122: 1131-1135
20．吴正治等 清肝泻火对老年痴呆鼠模型学习记忆及中枢单胺神经递质的影响．中国中医基础医学杂志，1998．12．4(12)，136-137
21．贾怡昌等 铝中毒痴呆鼠海马脂质过氧化和谷氨酰胺合成酶活性变化．中华老年医学杂志，2002，21(2)，136-137
2. G. Paxinos C. Watson The Rat Brain in Stereotaxic Coordinates 1986
3．贾建平 2003神经病学新进展 人民卫出版社 2003，249-25
4．赵宪林，方秀斌，李东培．大鼠血管性痴呆模型制作中国医科大学学报 2002；31(3)：166-167．
5. Katzmon R, Alzheimer disease N Engl JMed. 1986; 314: 9647
6．郑观成．脑老化与老年痴呆，第一卷，第一版，上海，上海科学技术出版社，1994，185．
7．秦俊法．微量元素与脑功能．第一版，北京．原子能出版社．1994，52．
8. Klatzo I, Wishie wisk H, Streicher E. Experimental production of neurofibrillary degeneration. J Nneuropath Exp Neural, 1965, 24: 187.
9. Crapper DG. Krishnan SS, Dakton AJ. Brain alcuminum distribution in Alzheimer's disease and ecperimental neurofibrillary degeneratin Science 1973, 180(4085): 511.
10. Perl DP. Brady AR. Alzheimer's disease: X-ray spectrometric evidence ok alcuminum accumulation in meurofibrillary tangle bearing neurons. Scieme,1980, 208(18): 297.
11. Morris R G M. Garrud P, Raulins J NP, et al. Place natigation impaired in rats with hippocampal lesions [J] Nature, 1982, 297: 681.
12. Squire L R. Zola-Morgan S. Memory brain system and behavior[J] Trends in Neuro Science, 1988, 11: 170.
13．姚志彬．海马—研究神经科学的理想模型．广东解剖学通报 1989，11：17-21．
14．马天才，于庆海．人参对学习记忆的药理学影响．中草药，1994；21(7)：38-40．
15. Petkov VD, Gao Y, Todorov, Lazarova M, et al. Behavioral effects al stemleaves extract from panax ginseng CA Meyer, Acta physiol pharmacol Bulg, 1992;18(2): 41-8.
16．胡海涛，钱亦华等．淀粉样前体蛋白在痴呆大鼠背海马结构内的表达．解剖学杂志 1997，20(5)444-445．
17. Simmons LK, May PC, Tomaselli KJ, et al. Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. Molecular Pharmacologycal, 1993; 45: 373-379
18．陈俊抛 林昱 痴呆治疗学 人民卫生出版社，2002，269
19. Glenner GG, Wong CW. Alzheimer s disease and Down s sydrome: Sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Cmmun, 1983; 122: 1131-1135
20．吴正治等 清肝泻火对老年痴呆鼠模型学习记忆及中枢单胺神经递质的影响．中国中医基础医学杂志，1998．12．4(12)，136-137
21．贾怡昌等 铝中毒痴呆鼠海马脂质过氧化和谷氨酰胺合成酶活性变化．中华老年医学杂志，2002，21(2)，136-137