人参皂苷Rg1对阿尔兹海默病大鼠认知功能障碍的作用及机制研究
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摘要
阿尔兹海默病(Alzheimers disease, AD)是一种严重的中枢神经系统退行性疾病,主要临床表现为进行性的认知功能减退,预后不良,患者一般死于肺栓塞和继发感染。阿尔兹海默病常散发,其临床发病率女性是男性的1.5～3倍。神经病理学特征主要包括β样淀粉样蛋白(β3-amyloid protein, Aβ)沉积形成的细胞外老年斑(senile plaque, SP)、tau蛋白的过度磷酸化形成的神经细胞内神经原纤维缠结以及神经元的丢失伴随着胶质细胞的增生等。其中不溶性淀粉样蛋白杂乱无序的聚集被认为是AD发生发展的关键环节。淀粉样蛋白淀粉样前体蛋白(amyloid precusor protein, APP)水解产生：正常情况下,淀粉样前体蛋白主要被α-水解酶水解生成s-APPα和C-端片段C83,这两种水解产物均可通过正常途径代谢；而在AD大脑中,淀粉样前体蛋白则主要由β-水解酶水解生成β-APP多肽和C-端片段C99,随后产物被γ-分泌酶水解生成一些长度为36-43个氨基酸的不溶性片段,即Aβ。Aβ1-42是淀粉样蛋白最常见的形式之一,也是形成不溶性淀粉样蛋白聚集的主要分型之一,与AD疾病的发生发展密切相关。人脑中α-分泌酶的主要类型是解整合素样金属蛋白酶(α-secretase a disintegrin and metallopeptidase domain10, ADAM10)和ADAM17,而分布于神经元中的主要是ADAM10;β-分泌酶的主要类型是β位淀粉样前体蛋白裂解酶-1(β-site-APP cleavage enzyme-1, BACE-1),另一种类型BACE-2在神经系统中分布较少。研究发现,AD患者大脑中α-分泌酶较正常大脑分泌减少,而β-分泌酶则分泌增多或者活性增强,这一改变影响淀粉样前体蛋白的水解过程,引起AD患者Aβ异常增多。
雌激素与女性AD密切相关。研究发现女性绝经后记忆障碍的出现较较绝经前明显增多,另外的研究也证实女性绝经后雌激素水平的降低是AD发病的高风险因素之一。研究发现,绝经后的妇女使用激素替代疗法(estrogen replacement therapy, ERT)能够明显推迟甚至阻止AD的发生。雌激素影响AD发生发展的确切机制尚不明确,但研究发现雌激素能够通过调节体外培养的神经元和胶质细胞的淀粉样前体蛋白水解过程来减少Aβ的产生,并且,雌激素已被证实在缺血休克和包括AD在内的许多神经退行性疾病中发挥着神经保护作用,提示雌激素可能通过影响淀粉样前体蛋白水解过程及其神经保护作用发挥抗AD的作用。然而,临床研究发现,雌激素替代疗法有着不可忽视的副作用。比如有研究发现使用雌激素替代疗法的女性患者患子宫内膜癌和乳腺癌的风险明显增加。因此,亟需寻找一种有效且安全的替代疗法,而人参皂苷Rg1有可能符合这一要求而替代雌激素。
人参皂苷Rg1是从中药人参中提取的中药有效单体之一,其分子式为C42H72O14,分子量为801.01,具有水溶性,且易溶于甲醇、乙醇。依据大样本的临床研究,人参皂苷Rg1是安全的植物雌激素,并不能带来当前药用雌激素所引起的生殖系统癌症高发病率的风险。有研究发现,人参皂苷Rg1与雌激素在诸如抗氧化、抑制凋亡等方面具有类似的功效,并且两者的上述作用均是是通过胰岛素样生长因子受体(insulin-like growth factor receptor, IGF-IR) I途径和雌激素受体(estrogen receptor, ER)途径实现的。此外,人参皂苷Rg1还可能影响Aβ的生成与代谢过程。由此可见,人参皂苷Rg1可能作为雌激素的替代疗法。
本研究中,我们首先探讨了利用去卵巢结合D-半乳糖腹腔注射建立大鼠AD模型的可行性,发现去卵巢结合D-半乳糖腹腔注射处理的大鼠行为学改变以及海马病理学改变均符合AD疾病情况下的变化,提示造模成功。然后,我们研究了人参皂苷Rg1对AD大鼠模型学习和记忆的影响。机制方面,主要研究了AD模型大鼠海马中ADAM10和BACE1的改变情况,以明确人参皂苷Rg1对淀粉样蛋白水解过程的影响。同时,我们还研究了活化的含半胱氨酸的天冬氨酸蛋白水解酶caspase3的变化情况,以明确人参皂苷Rg1对AD模型大鼠神经元凋亡的影响。
目的
本实验研究首先验证利用去卵巢手术联合D-半乳糖腹腔注射建立大鼠AD模型的可行性。然后,以雌激素为对照,观察人参皂苷Rg1对AD大鼠模型记忆功能障碍的改善作用,并进一步探讨其作用机制。
方法
1.研究对象及分组：将96只Wistar大鼠随机分为两组,一组72只行去卵巢手术,另一组24只行假手术,术后行抗炎处理。将假手术组大鼠随机等分为两组：一组按1ml/kg/d在大鼠腹部皮下注射生理盐水(CT),另一组按100mg/kg/d腹腔注射D-半乳糖(D)；去卵巢手术组大鼠随机等分为六组：第一组按1ml/kg/d的剂量在去卵巢手术大鼠的腹部皮下注射生理盐水(O),第二组的去卵巢大鼠同时注射D-半乳糖(O+D),第三组是腹腔注射D-半乳糖的去卵巢大鼠按100μg/kg/d的剂量在皮下注射雌激素(O+D+E),第四组是腹腔注射D-半乳糖的去卵巢大鼠腹腔注射5mg/kg/d剂量的人参皂苷Rgl,为人参皂苷低剂量处理组(O+D+L),第五组是腹腔注射D-半乳糖的去卵巢大鼠腹腔注射中剂量的,即10mg/kg/d的人参皂苷Rg1(O+D+M),第六组是腹腔注射D-半乳糖的去卵巢大鼠用高剂量即腹腔注射20mg/kg/d的人参皂苷Rgl(O+D+H)。所有液体均按照每天需要注射的剂量稀释,以使每天为大鼠注射的液体量保持在300μl左右,用药6周。
2.研究内容：(1) Morris水迷宫实验检测各组大鼠的空间学习与记忆的能力;(2)Elisa实验检测各组大鼠海马组织中Apl-42的含量；(3)免疫组织化学法检测各组大鼠海马细胞中ADAM10和BACE1勺表达情况；(4)实时定量PCR检测大鼠海马中ADAM10、BACE1、caspase3mRNA的表达情况；(5) Western blot检测大鼠海马中ADAM10、活化的caspase3蛋白的表达情况。
结果
1.去卵巢结合D-半乳糖腹腔注射能够显著损害大鼠的认知功能,引起大鼠海马内Aβ1-42的高表达。
(1)造模后各组大鼠Morris水迷宫的实验结果：通过水迷宫实验,我们发现去卵巢术、D-半乳糖的腹腔注射都能够明显延长大鼠寻找平台的潜伏期时间(P<0.05),而去卵巢术结合D-半乳糖腹腔注射组大鼠的潜伏期时间最长,较前两者的差异有显著性(P<0.05)。而且,卵巢切除术、D-半乳糖的腹腔注射都能够明显降低大鼠第七天穿越平台的次数,而卵巢切除术结合D-半乳糖腹腔注射组大鼠的第七天平均穿越平台的次数最少,其中,两者结合组的平均穿越平台数是对照组的40%,约是卵巢切除术后组、D-半乳糖腹腔注射组的70%。
(2)造模后各组大鼠海马中Aβ1-42的浓度测定：Elisa的实验结果显示去卵巢术结合D-半乳糖的腹腔注射能够显著提高大鼠海马中Aβ1-42的水平(P<0.05),较对照组提高了约110%,而卵巢切除术、D-半乳糖的腹腔注射则具有相对较弱的作用。
2.人参皂苷Rg1和雌激素能够显著改善模型大鼠的学习和记忆能力
(1)各组大鼠Morris水迷宫的实验结果：使用人参皂苷Rg1和雌激素处理的各组大鼠,都明显减少了造模所引起的潜伏期延长(P<0.05)。三种不同剂量人参皂苷的处理组中,中剂量和高剂量人参皂苷Rg1处理组的大鼠在缩短平均潜伏期方面明显优低剂量组(P<0.05)。同时,各组大鼠的游泳速度没有显著变化,证明了各种处理因素没有明显影响大鼠的运动能力。并且,使用人参皂苷Rg1和雌激素治疗的大鼠都能有效防止造模所引起的大鼠穿越平台次数的减少,治疗后的大鼠穿越平台数目明显增多(P<0.05)。而且与学习阶段所得到的结果一致,中剂量和高剂量的人参皂苷Rgl处理组的大鼠穿越平台的次数明显多于低剂量组(P<0.05)。
(2)各组大鼠海马中Aβ1-42的浓度测定：人参皂苷Rg1和雌激素的处理,都能够不同程度地降低大鼠海马中Ap1-42的含量(P<0.05),其中,模型组大鼠海马中Aβ1-42的含量是正常大鼠的2.2倍,中剂量的人参皂苷Rg1降低到模型大鼠海马中Aβ1-42含量的63%,高剂量处理组则降低到约69%,而低剂量只降低到造模组含量的85%,这说明中剂量和高剂量的治疗效果明显优于低剂量组。
3.人参皂苷Rg1和雌激素促进模型大鼠学习和记忆能力的机制研究
(1)各组大鼠海马中ADAM10和BACE1的阳性细胞表达情况：免疫组化的结果显示,在对照组大鼠的海马中ADAM10的阳性细胞率为67.4%±1.78%,而造模大鼠(O+D)的海马中ADAM10阳性细胞率仅为12±1.53%(P<0.01)。用人参皂苷和雌激素处理后,低剂量人参皂苷Rg1组海马中阳性细胞率为29.9±1.52%,中剂量人参皂苷Rg1处理组的阳性细胞率为66.45%4±1.55%,高剂量人参皂苷Rg1处理组的阳性细胞率为50.3%±4.67%,雌激素处理组的阳性细胞率为50.3±4.67%。关于BACE1,造模组中BACE1阳性神经元率为62.3%±1.55%,使用人参皂苷Rg1和雌激素处理后,中剂量人参皂苷Rg1处理后BACE1阳性细胞率为19.34%±1.03%,高剂量的Rg1处理后为25.13%±1.13%,雌激素处理后阳性细胞率则降为25.89%+1.42%(P<0.05)。
(2)实时定量检测大鼠海马中ADAM10、BACE1(?)(?)cawspase3的表达情况：结果显不,ADAM10的表达在造模组中明显减少(P<0.05),人参皂苷Rg1和雌激素能够明显改变造模大鼠的这种ADAM10的减少情况(P<0.05)。并且数据显示,中剂量的人参皂苷Rg1组的ADAM10mRNA水平是造模组的1.8倍,高剂量的人参皂苷Rg1组的是造模组的1.6倍,而低剂量组ADAM10的基因水平仅为造模组的1.1倍,这说明中高剂量的人参皂苷Rg1的治疗效果明显优于低剂量的人参皂苷。而关于BACE1的表达情况,去卵巢结合D-半乳糖能够明显促进大鼠海马中BACE1的表达,人参皂苷Rg1和雌激素的处理都能够明显抑制造模引起的这种作用,且中剂量和高剂量组在降低BACE1的表达方面明显优于低剂量组。
(3) Western blot实验检测ADAM10和caspase3的表达情况：蛋白印迹的实验结果以及对其进行的光密度分析显示ADAM10在海马中的表达因造模急剧下降,且人参皂苷Rg1和雌激素的处理都能够明显升高因造模而降低的ADAM10的表达水平(P<0.05)。而关于凋亡因子的表达情况,造模组大鼠海马组织中活化的caspase3水平明显升高(P<0.05),人参皂苷Rg1和雌激素的治疗都能够明显降低活化的casapse3的水平(P<0.05)。其中,中剂量的人参皂苷Rg1处理的大鼠活化的casapse3水平是造模组的53%,高剂量处理组活化的casapse3的水平是造模组的51%,而低剂量的人参皂苷Rg1处理后活化的casapse3水平仅降为造模组的87%,说明中剂量和高剂量的人参皂苷Rg1在降低活化的caspase3的效果方面明显优于低剂量组。
结论
1.卵巢切除术结合D-半乳糖腹腔注射组大鼠在行为学改变和海马组织的病理改变均符合AD的特征,表明卵巢切除术结合D-半乳糖腹腔注射可以用于模拟AD。
2.人参皂苷Rg1能够明显地改善利用去卵巢结合D-半乳糖腹腔注射建立的AD大鼠模型的认知功能障碍。
3.人参皂苷Rg1对AD大鼠模型学习和记忆能力的改善作用可能是通过调节淀粉样前体蛋白水解过程及其神经保护作用来实现的。
Alzheimers disease (AD) is a severe neurodegenerative disorder of the central nerve system (CNS) manifested as progressive cognition and function impairments. The neuropathological hallmarks of AD include insoluble fibrillar plaques of amyloid (3-proteins (Aβ) and neurofibrillary deposits of hyperphosphorylated tau protein. The accumulation of Aβ is considered to be the primary influence driving AD pathogenesis. Aβ is produced by the proteolytic processing of amyloid precursor protein (APP). APP is first cleaved by β-secretase to generate secreted β-APP polypeptide along with a C-terminal fragment named C99, and then subsequently cleaved by y-secretase to generate Aβ. In the alternative pathway APP is cleaved by a-secretase, such as a-secretase a disintegrin and metallopeptidase domain10(ADAM10) and ADAM17, resulting in the release of a soluble form of APP (sAPPa), which is different than Aβ. A decrease in a-secretase and an increase in β-secretase activity have been seen in patients with AD, suggesting the use of secretases as targets for therapy.
Estrogen has long been linked to AD. Estrogen has been demonstrated to have neuroprotective effects in the context of the pathophysiological process such as ischemia and neurodegenerative diseases, including AD. Moreover, estrogen can reduce Aβ through regulation of proteolytic processing in cultured human neuronal and glial cells. However, ERT displays some side effects. Thus, there is an urgent need to develop effective and safe alternative therapies. Ginsenoside Rgl may become one of the alternative candidates.
Ginsenoside Rg1, a natural product extracted from Panax ginseng C.A. Meyer, has been reported to exert notable neuroprotective activities such as anti-oxidative and anti-apoptosis effects in cultured neurons, which is comparable to that presented by estrogen. Furthermore, ginsenoside Rgl may influence Aβ formation;. However, the effect of ginsenoside Rg1on cognition capacity of AD is still poorly understood, and the underlying mechanisms remain to be fully elucidated.
It has been reported that ovariectomy combined with D-galactose (D-gal) injection serves as an ideal AD rodent model capable of mimicking pathological, neurochemical and behavioral alterations in AD. Furthermore, ovarian steroid deprivation is necessary to test the effect of17β-estradiol (E2) and its alteration candidates; D-gal injection induces neuronal damage and a decline in learning and memory capacity in the treated rats or mice. Thus, in the present study, we compared ginsenoside Rg1with E2in their promoting effects on spatial learning and memory capacity using the ovariectomized (OVX) with D-gal injected rats, which exhibited cognitive impairments and hippocampal Aβ production increases. We have found that ginsenoside Rg1and E2exerted similar effects on the improvement of cognitive performance and the regulation of APP pathway in the AD rats. Our results suggest that ginsenoside Rgl can be a potential therapy for AD in clinic.
Objective:
The research observed whether the OVX and D-galactose could minic the pathological alterations of AD. Study the effect of gisenoside Rg1working on the cognitive impairments of AD rats and explore the underlying mechanisms.
Methods:
After one week acclimatization, the rats were randomly divided into two groups:the first group of24rats received sham operation, while the other group of72rats was ovariectomized. The sham operated rats were randomly and equally divided in to two groups:the sham operated and normal saline (NS,1ml/kg/d, subcutaneous injection, i.h.) injected group (CT), the sham operated and D-gal (100mg/kg/d, i.p.) injected group (D). The OVX rats were further randomly and equally divided into six groups:the OVX and NS (1ml/kg/d, i.h.) injected group (O), the OVX and D-gal injected group (O+D), the OVX, D-gal and17β-estradiol (E2,100μg/kg/d, i.h.) injection group (O+D+E), the OVX, D-gal and low ginsenoside Rgl (5mg/kg/d, i.p.) injected group (O+D+L), the OVX, D-gal and moderate ginsenoside Rg1(10mg/kg/d, i.p.) injected group (O+D+M), and the OVX, D-gal and high ginsenoside Rg1(20mg/kg/d, i.p.) injected group (O+D+H). The volume of each injection was about300μl/d, due to the weight of the animal, and was administrated for6weeks.
After the last administration, the rats were used in the following experiments.(1) Observation of the learning and memory performances by Morris Water Maze;(2) Measurment of Aβ1-42using Elisa;(3) Detection of ADAM10-positive and BACE1-positive cells by immunohistochemistry;(4) Analysis of ADAM10, BACE1and caspase3mRNA expression using Realtime-PCR;(5) Analysis of ADAM10and caspase3protein expression by Westernblot.
Results:
1. OVX and D-galactose could minic the pathological alterations of AD.
(1) Morris Water Maze:The spatial learning and memory capacity of rats was significantly impaired in the O, D and O+D group compared with the CT group and most notably in the O+D group, as shown by prolonged escape latency and decreased platform crossing numbers (P＜0.05). In the spatial retention trial, crossing number in the O+D group was about40%of that in the CT group (P＜0.01) and about70%of that in the O and D groups (P<0.05).
(2) Analysis of Aβ1-42by Elisa assay:The ovariectomy plus D-gal increased Aβ1-42production by about110%(P＜0.01) while ovariectomy or D-gal alone displayed a much milder effect.
2. The effect of gisenoside Rgl working on the cognitive impairments of AD rats.
(1) Morris Water Maze:Ginsenoside Rg1and E2treatment decreased the OVX+D-gal-induced increase of escape latency. Among the three ginsenoside Rgl injected groups, moderate and high ginsenoside Rgl treatment exhibited more improvement compared with low one treatment (P＜0.05). The swim speeds, which are obtained by path lengths divided by swim times, is not significantly different among groups. Ginsenoside Rgl and E2prevented the spatial retention decline induced by OVX+D-gal as demonstrated by an increase in platform crossing (P＜0.05). In accordance with that in the spatial acquisition trial, the moderate and high ginsenoside Rgl treated rats exhibited the better performance (P<0.05).
(2) Analysis of Aβ1-42by Elisa assay:Ginsenoside Rgl and E2treatment reduced the OVX+D-gal-induced increase of Aβ1-42(P＜0.05), with different extents. Among the ginsenoside Rgl treatments, the moderate (63%of O+D) and high ginsenoside Rgl (69%of O+D) were more effective than the low one (85%of O+D) on reducing the OVX+D-gal-induced increase of Aβ1-42(2.2-fold over CT)(P<0.05).
3. The mechanism of gisenoside Rgl working on the cognitive impairments of AD rats.
(1) Immunohistochemistry analysis:The frequency of ADAM10-positive hippocampal neurons of OVX+D-gal AD rats (12%±1.53%) was significantly decreased in comparison to control ones (67.4%±1.78%)(P＜0.01). Ginsenoside Rgl and E2treatment significantly increased proportion of ADAM10-positive neurons in hippocampus of AD rats (P＜0.05). OVX+D-gal increased frequency (62.3%±1.55%, P＜0.05) of BACE1-positive neurons in hippocampus. Ginsenoside Rg1and E2treatment significantly decreased proportion of BACE1-positive neurons in hippocampus of AD rats (P＜0.05).
(2) Realtime-PCR analysis:ADAM10mRNA expression was inhibited by OVX+D-gal, while ginsenoside Rg1and E2treatment increased its expression in OVX+D-gal rats. Additionally, moderate and high ginsenoside Rg1exhibited more effective influences on ADAM10expression than low ginsenoside Rg1(P＜0.05). OVX+D-gal increased BACE1expression in mRNA level, while Ginsenoside Rg1and E2decreased OVX+D-gal-induced BACE1expression (P＜0.05). The moderate and the high ginsenoside Rgl were more effective on increasing the OVX+D-gal-reduced BACE1expression than the low one (P＜0.05).
(3) Western blot assay:ADAM10expression in hippocampus was obviously decreased by OVX+D-gal, while ginsenoside Rg1and E2treatment inversed OVX+D-gal reduction of ADAM10expression. Cleaved caspase3level was increased in OVX+D-gal AD rats compared with sham ones (P＜0.05). Ginsenoside Rgl and E2treated OVX+D-gal AD rats exhibited lower caspase3activity than OVX+D-gal AD rats (P＜0.05). Cleaved caspase3levels in the moderate ginsenoside Rgl treated rats and high ginsenoside Rgl treated rats were less than that in the low ones (P＜0.05).
Conclusion:
1. Ovariectomy plus D-gal induces cognitive performance impairments and Aβ production which are characteristics of AD, suggesting that the OVX combined with D-gal injected rats can serve as an AD model.
2. Ginsenoside Rg1and E2are effective for treatment of learning and memory decline in the AD rats.
3. The effect of Ginsenoside Rg1and E2might be asoociated with suppressing generation of Aβ,increasing expression of ADAM10and inhibition of BACE1expression and cleaved casapase3.
雌激素与女性AD密切相关。研究发现女性绝经后记忆障碍的出现较较绝经前明显增多,另外的研究也证实女性绝经后雌激素水平的降低是AD发病的高风险因素之一。研究发现,绝经后的妇女使用激素替代疗法(estrogen replacement therapy, ERT)能够明显推迟甚至阻止AD的发生。雌激素影响AD发生发展的确切机制尚不明确,但研究发现雌激素能够通过调节体外培养的神经元和胶质细胞的淀粉样前体蛋白水解过程来减少Aβ的产生,并且,雌激素已被证实在缺血休克和包括AD在内的许多神经退行性疾病中发挥着神经保护作用,提示雌激素可能通过影响淀粉样前体蛋白水解过程及其神经保护作用发挥抗AD的作用。然而,临床研究发现,雌激素替代疗法有着不可忽视的副作用。比如有研究发现使用雌激素替代疗法的女性患者患子宫内膜癌和乳腺癌的风险明显增加。因此,亟需寻找一种有效且安全的替代疗法,而人参皂苷Rg1有可能符合这一要求而替代雌激素。
人参皂苷Rg1是从中药人参中提取的中药有效单体之一,其分子式为C42H72O14,分子量为801.01,具有水溶性,且易溶于甲醇、乙醇。依据大样本的临床研究,人参皂苷Rg1是安全的植物雌激素,并不能带来当前药用雌激素所引起的生殖系统癌症高发病率的风险。有研究发现,人参皂苷Rg1与雌激素在诸如抗氧化、抑制凋亡等方面具有类似的功效,并且两者的上述作用均是是通过胰岛素样生长因子受体(insulin-like growth factor receptor, IGF-IR) I途径和雌激素受体(estrogen receptor, ER)途径实现的。此外,人参皂苷Rg1还可能影响Aβ的生成与代谢过程。由此可见,人参皂苷Rg1可能作为雌激素的替代疗法。
本研究中,我们首先探讨了利用去卵巢结合D-半乳糖腹腔注射建立大鼠AD模型的可行性,发现去卵巢结合D-半乳糖腹腔注射处理的大鼠行为学改变以及海马病理学改变均符合AD疾病情况下的变化,提示造模成功。然后,我们研究了人参皂苷Rg1对AD大鼠模型学习和记忆的影响。机制方面,主要研究了AD模型大鼠海马中ADAM10和BACE1的改变情况,以明确人参皂苷Rg1对淀粉样蛋白水解过程的影响。同时,我们还研究了活化的含半胱氨酸的天冬氨酸蛋白水解酶caspase3的变化情况,以明确人参皂苷Rg1对AD模型大鼠神经元凋亡的影响。
目的
本实验研究首先验证利用去卵巢手术联合D-半乳糖腹腔注射建立大鼠AD模型的可行性。然后,以雌激素为对照,观察人参皂苷Rg1对AD大鼠模型记忆功能障碍的改善作用,并进一步探讨其作用机制。
方法
1.研究对象及分组：将96只Wistar大鼠随机分为两组,一组72只行去卵巢手术,另一组24只行假手术,术后行抗炎处理。将假手术组大鼠随机等分为两组：一组按1ml/kg/d在大鼠腹部皮下注射生理盐水(CT),另一组按100mg/kg/d腹腔注射D-半乳糖(D)；去卵巢手术组大鼠随机等分为六组：第一组按1ml/kg/d的剂量在去卵巢手术大鼠的腹部皮下注射生理盐水(O),第二组的去卵巢大鼠同时注射D-半乳糖(O+D),第三组是腹腔注射D-半乳糖的去卵巢大鼠按100μg/kg/d的剂量在皮下注射雌激素(O+D+E),第四组是腹腔注射D-半乳糖的去卵巢大鼠腹腔注射5mg/kg/d剂量的人参皂苷Rgl,为人参皂苷低剂量处理组(O+D+L),第五组是腹腔注射D-半乳糖的去卵巢大鼠腹腔注射中剂量的,即10mg/kg/d的人参皂苷Rg1(O+D+M),第六组是腹腔注射D-半乳糖的去卵巢大鼠用高剂量即腹腔注射20mg/kg/d的人参皂苷Rgl(O+D+H)。所有液体均按照每天需要注射的剂量稀释,以使每天为大鼠注射的液体量保持在300μl左右,用药6周。
2.研究内容：(1) Morris水迷宫实验检测各组大鼠的空间学习与记忆的能力;(2)Elisa实验检测各组大鼠海马组织中Apl-42的含量；(3)免疫组织化学法检测各组大鼠海马细胞中ADAM10和BACE1勺表达情况；(4)实时定量PCR检测大鼠海马中ADAM10、BACE1、caspase3mRNA的表达情况；(5) Western blot检测大鼠海马中ADAM10、活化的caspase3蛋白的表达情况。
结果
1.去卵巢结合D-半乳糖腹腔注射能够显著损害大鼠的认知功能,引起大鼠海马内Aβ1-42的高表达。
(1)造模后各组大鼠Morris水迷宫的实验结果：通过水迷宫实验,我们发现去卵巢术、D-半乳糖的腹腔注射都能够明显延长大鼠寻找平台的潜伏期时间(P<0.05),而去卵巢术结合D-半乳糖腹腔注射组大鼠的潜伏期时间最长,较前两者的差异有显著性(P<0.05)。而且,卵巢切除术、D-半乳糖的腹腔注射都能够明显降低大鼠第七天穿越平台的次数,而卵巢切除术结合D-半乳糖腹腔注射组大鼠的第七天平均穿越平台的次数最少,其中,两者结合组的平均穿越平台数是对照组的40%,约是卵巢切除术后组、D-半乳糖腹腔注射组的70%。
(2)造模后各组大鼠海马中Aβ1-42的浓度测定：Elisa的实验结果显示去卵巢术结合D-半乳糖的腹腔注射能够显著提高大鼠海马中Aβ1-42的水平(P<0.05),较对照组提高了约110%,而卵巢切除术、D-半乳糖的腹腔注射则具有相对较弱的作用。
2.人参皂苷Rg1和雌激素能够显著改善模型大鼠的学习和记忆能力
(1)各组大鼠Morris水迷宫的实验结果：使用人参皂苷Rg1和雌激素处理的各组大鼠,都明显减少了造模所引起的潜伏期延长(P<0.05)。三种不同剂量人参皂苷的处理组中,中剂量和高剂量人参皂苷Rg1处理组的大鼠在缩短平均潜伏期方面明显优低剂量组(P<0.05)。同时,各组大鼠的游泳速度没有显著变化,证明了各种处理因素没有明显影响大鼠的运动能力。并且,使用人参皂苷Rg1和雌激素治疗的大鼠都能有效防止造模所引起的大鼠穿越平台次数的减少,治疗后的大鼠穿越平台数目明显增多(P<0.05)。而且与学习阶段所得到的结果一致,中剂量和高剂量的人参皂苷Rgl处理组的大鼠穿越平台的次数明显多于低剂量组(P<0.05)。
(2)各组大鼠海马中Aβ1-42的浓度测定：人参皂苷Rg1和雌激素的处理,都能够不同程度地降低大鼠海马中Ap1-42的含量(P<0.05),其中,模型组大鼠海马中Aβ1-42的含量是正常大鼠的2.2倍,中剂量的人参皂苷Rg1降低到模型大鼠海马中Aβ1-42含量的63%,高剂量处理组则降低到约69%,而低剂量只降低到造模组含量的85%,这说明中剂量和高剂量的治疗效果明显优于低剂量组。
3.人参皂苷Rg1和雌激素促进模型大鼠学习和记忆能力的机制研究
(1)各组大鼠海马中ADAM10和BACE1的阳性细胞表达情况：免疫组化的结果显示,在对照组大鼠的海马中ADAM10的阳性细胞率为67.4%±1.78%,而造模大鼠(O+D)的海马中ADAM10阳性细胞率仅为12±1.53%(P<0.01)。用人参皂苷和雌激素处理后,低剂量人参皂苷Rg1组海马中阳性细胞率为29.9±1.52%,中剂量人参皂苷Rg1处理组的阳性细胞率为66.45%4±1.55%,高剂量人参皂苷Rg1处理组的阳性细胞率为50.3%±4.67%,雌激素处理组的阳性细胞率为50.3±4.67%。关于BACE1,造模组中BACE1阳性神经元率为62.3%±1.55%,使用人参皂苷Rg1和雌激素处理后,中剂量人参皂苷Rg1处理后BACE1阳性细胞率为19.34%±1.03%,高剂量的Rg1处理后为25.13%±1.13%,雌激素处理后阳性细胞率则降为25.89%+1.42%(P<0.05)。
(2)实时定量检测大鼠海马中ADAM10、BACE1(?)(?)cawspase3的表达情况：结果显不,ADAM10的表达在造模组中明显减少(P<0.05),人参皂苷Rg1和雌激素能够明显改变造模大鼠的这种ADAM10的减少情况(P<0.05)。并且数据显示,中剂量的人参皂苷Rg1组的ADAM10mRNA水平是造模组的1.8倍,高剂量的人参皂苷Rg1组的是造模组的1.6倍,而低剂量组ADAM10的基因水平仅为造模组的1.1倍,这说明中高剂量的人参皂苷Rg1的治疗效果明显优于低剂量的人参皂苷。而关于BACE1的表达情况,去卵巢结合D-半乳糖能够明显促进大鼠海马中BACE1的表达,人参皂苷Rg1和雌激素的处理都能够明显抑制造模引起的这种作用,且中剂量和高剂量组在降低BACE1的表达方面明显优于低剂量组。
(3) Western blot实验检测ADAM10和caspase3的表达情况：蛋白印迹的实验结果以及对其进行的光密度分析显示ADAM10在海马中的表达因造模急剧下降,且人参皂苷Rg1和雌激素的处理都能够明显升高因造模而降低的ADAM10的表达水平(P<0.05)。而关于凋亡因子的表达情况,造模组大鼠海马组织中活化的caspase3水平明显升高(P<0.05),人参皂苷Rg1和雌激素的治疗都能够明显降低活化的casapse3的水平(P<0.05)。其中,中剂量的人参皂苷Rg1处理的大鼠活化的casapse3水平是造模组的53%,高剂量处理组活化的casapse3的水平是造模组的51%,而低剂量的人参皂苷Rg1处理后活化的casapse3水平仅降为造模组的87%,说明中剂量和高剂量的人参皂苷Rg1在降低活化的caspase3的效果方面明显优于低剂量组。
结论
1.卵巢切除术结合D-半乳糖腹腔注射组大鼠在行为学改变和海马组织的病理改变均符合AD的特征,表明卵巢切除术结合D-半乳糖腹腔注射可以用于模拟AD。
2.人参皂苷Rg1能够明显地改善利用去卵巢结合D-半乳糖腹腔注射建立的AD大鼠模型的认知功能障碍。
3.人参皂苷Rg1对AD大鼠模型学习和记忆能力的改善作用可能是通过调节淀粉样前体蛋白水解过程及其神经保护作用来实现的。
Alzheimers disease (AD) is a severe neurodegenerative disorder of the central nerve system (CNS) manifested as progressive cognition and function impairments. The neuropathological hallmarks of AD include insoluble fibrillar plaques of amyloid (3-proteins (Aβ) and neurofibrillary deposits of hyperphosphorylated tau protein. The accumulation of Aβ is considered to be the primary influence driving AD pathogenesis. Aβ is produced by the proteolytic processing of amyloid precursor protein (APP). APP is first cleaved by β-secretase to generate secreted β-APP polypeptide along with a C-terminal fragment named C99, and then subsequently cleaved by y-secretase to generate Aβ. In the alternative pathway APP is cleaved by a-secretase, such as a-secretase a disintegrin and metallopeptidase domain10(ADAM10) and ADAM17, resulting in the release of a soluble form of APP (sAPPa), which is different than Aβ. A decrease in a-secretase and an increase in β-secretase activity have been seen in patients with AD, suggesting the use of secretases as targets for therapy.
Estrogen has long been linked to AD. Estrogen has been demonstrated to have neuroprotective effects in the context of the pathophysiological process such as ischemia and neurodegenerative diseases, including AD. Moreover, estrogen can reduce Aβ through regulation of proteolytic processing in cultured human neuronal and glial cells. However, ERT displays some side effects. Thus, there is an urgent need to develop effective and safe alternative therapies. Ginsenoside Rgl may become one of the alternative candidates.
Ginsenoside Rg1, a natural product extracted from Panax ginseng C.A. Meyer, has been reported to exert notable neuroprotective activities such as anti-oxidative and anti-apoptosis effects in cultured neurons, which is comparable to that presented by estrogen. Furthermore, ginsenoside Rgl may influence Aβ formation;. However, the effect of ginsenoside Rg1on cognition capacity of AD is still poorly understood, and the underlying mechanisms remain to be fully elucidated.
It has been reported that ovariectomy combined with D-galactose (D-gal) injection serves as an ideal AD rodent model capable of mimicking pathological, neurochemical and behavioral alterations in AD. Furthermore, ovarian steroid deprivation is necessary to test the effect of17β-estradiol (E2) and its alteration candidates; D-gal injection induces neuronal damage and a decline in learning and memory capacity in the treated rats or mice. Thus, in the present study, we compared ginsenoside Rg1with E2in their promoting effects on spatial learning and memory capacity using the ovariectomized (OVX) with D-gal injected rats, which exhibited cognitive impairments and hippocampal Aβ production increases. We have found that ginsenoside Rg1and E2exerted similar effects on the improvement of cognitive performance and the regulation of APP pathway in the AD rats. Our results suggest that ginsenoside Rgl can be a potential therapy for AD in clinic.
Objective:
The research observed whether the OVX and D-galactose could minic the pathological alterations of AD. Study the effect of gisenoside Rg1working on the cognitive impairments of AD rats and explore the underlying mechanisms.
Methods:
After one week acclimatization, the rats were randomly divided into two groups:the first group of24rats received sham operation, while the other group of72rats was ovariectomized. The sham operated rats were randomly and equally divided in to two groups:the sham operated and normal saline (NS,1ml/kg/d, subcutaneous injection, i.h.) injected group (CT), the sham operated and D-gal (100mg/kg/d, i.p.) injected group (D). The OVX rats were further randomly and equally divided into six groups:the OVX and NS (1ml/kg/d, i.h.) injected group (O), the OVX and D-gal injected group (O+D), the OVX, D-gal and17β-estradiol (E2,100μg/kg/d, i.h.) injection group (O+D+E), the OVX, D-gal and low ginsenoside Rgl (5mg/kg/d, i.p.) injected group (O+D+L), the OVX, D-gal and moderate ginsenoside Rg1(10mg/kg/d, i.p.) injected group (O+D+M), and the OVX, D-gal and high ginsenoside Rg1(20mg/kg/d, i.p.) injected group (O+D+H). The volume of each injection was about300μl/d, due to the weight of the animal, and was administrated for6weeks.
After the last administration, the rats were used in the following experiments.(1) Observation of the learning and memory performances by Morris Water Maze;(2) Measurment of Aβ1-42using Elisa;(3) Detection of ADAM10-positive and BACE1-positive cells by immunohistochemistry;(4) Analysis of ADAM10, BACE1and caspase3mRNA expression using Realtime-PCR;(5) Analysis of ADAM10and caspase3protein expression by Westernblot.
Results:
1. OVX and D-galactose could minic the pathological alterations of AD.
(1) Morris Water Maze:The spatial learning and memory capacity of rats was significantly impaired in the O, D and O+D group compared with the CT group and most notably in the O+D group, as shown by prolonged escape latency and decreased platform crossing numbers (P＜0.05). In the spatial retention trial, crossing number in the O+D group was about40%of that in the CT group (P＜0.01) and about70%of that in the O and D groups (P<0.05).
(2) Analysis of Aβ1-42by Elisa assay:The ovariectomy plus D-gal increased Aβ1-42production by about110%(P＜0.01) while ovariectomy or D-gal alone displayed a much milder effect.
2. The effect of gisenoside Rgl working on the cognitive impairments of AD rats.
(1) Morris Water Maze:Ginsenoside Rg1and E2treatment decreased the OVX+D-gal-induced increase of escape latency. Among the three ginsenoside Rgl injected groups, moderate and high ginsenoside Rgl treatment exhibited more improvement compared with low one treatment (P＜0.05). The swim speeds, which are obtained by path lengths divided by swim times, is not significantly different among groups. Ginsenoside Rgl and E2prevented the spatial retention decline induced by OVX+D-gal as demonstrated by an increase in platform crossing (P＜0.05). In accordance with that in the spatial acquisition trial, the moderate and high ginsenoside Rgl treated rats exhibited the better performance (P<0.05).
(2) Analysis of Aβ1-42by Elisa assay:Ginsenoside Rgl and E2treatment reduced the OVX+D-gal-induced increase of Aβ1-42(P＜0.05), with different extents. Among the ginsenoside Rgl treatments, the moderate (63%of O+D) and high ginsenoside Rgl (69%of O+D) were more effective than the low one (85%of O+D) on reducing the OVX+D-gal-induced increase of Aβ1-42(2.2-fold over CT)(P<0.05).
3. The mechanism of gisenoside Rgl working on the cognitive impairments of AD rats.
(1) Immunohistochemistry analysis:The frequency of ADAM10-positive hippocampal neurons of OVX+D-gal AD rats (12%±1.53%) was significantly decreased in comparison to control ones (67.4%±1.78%)(P＜0.01). Ginsenoside Rgl and E2treatment significantly increased proportion of ADAM10-positive neurons in hippocampus of AD rats (P＜0.05). OVX+D-gal increased frequency (62.3%±1.55%, P＜0.05) of BACE1-positive neurons in hippocampus. Ginsenoside Rg1and E2treatment significantly decreased proportion of BACE1-positive neurons in hippocampus of AD rats (P＜0.05).
(2) Realtime-PCR analysis:ADAM10mRNA expression was inhibited by OVX+D-gal, while ginsenoside Rg1and E2treatment increased its expression in OVX+D-gal rats. Additionally, moderate and high ginsenoside Rg1exhibited more effective influences on ADAM10expression than low ginsenoside Rg1(P＜0.05). OVX+D-gal increased BACE1expression in mRNA level, while Ginsenoside Rg1and E2decreased OVX+D-gal-induced BACE1expression (P＜0.05). The moderate and the high ginsenoside Rgl were more effective on increasing the OVX+D-gal-reduced BACE1expression than the low one (P＜0.05).
(3) Western blot assay:ADAM10expression in hippocampus was obviously decreased by OVX+D-gal, while ginsenoside Rg1and E2treatment inversed OVX+D-gal reduction of ADAM10expression. Cleaved caspase3level was increased in OVX+D-gal AD rats compared with sham ones (P＜0.05). Ginsenoside Rgl and E2treated OVX+D-gal AD rats exhibited lower caspase3activity than OVX+D-gal AD rats (P＜0.05). Cleaved caspase3levels in the moderate ginsenoside Rgl treated rats and high ginsenoside Rgl treated rats were less than that in the low ones (P＜0.05).
Conclusion:
1. Ovariectomy plus D-gal induces cognitive performance impairments and Aβ production which are characteristics of AD, suggesting that the OVX combined with D-gal injected rats can serve as an AD model.
2. Ginsenoside Rg1and E2are effective for treatment of learning and memory decline in the AD rats.
3. The effect of Ginsenoside Rg1and E2might be asoociated with suppressing generation of Aβ,increasing expression of ADAM10and inhibition of BACE1expression and cleaved casapase3.
引文
1. Escribano L, Simon AM, Gimeno E, Cuadrado-Tejedor M, Lopez de Maturana R, Garcia-Osta A, Ricobaraza A, Perez-Mediavilla A, Del Rio J, Frechilla D Rosiglitazone rescues memory impairment in Alzheimers transgenic mice: mechanisms involving a reduced amyloid and tau pathology. Neuropsychopharmacology 35:1593-1604.2010.
2. Hardy J, Selkoe DJ The amyloid hypothesis of Alzheimers disease:progress and problems on the road to therapeutics. Science 297:353-356.2002.
3. Tanzi RE, Bertram L.Twenty years of the Alzheimers disease amyloid hypothesis: a genetic perspective. Cell 120:545-555.2005.
4. Hua X, Lei M, Zhang Y, Ding J, Han Q, Hu G, Xiao M.Long-term D-galactose injection combined with ovariectomy serves as a new rodent model for Alzheimers disease. Life Sci 80:1897-1905.2007.
5. Cui X, Zuo P, Zhang Q, Li X, Hu Y, Long J, Packer L, Liu J.Chronic systemic D-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice:protective effects of R-alpha-lipoic acid. J Neurosci Res 84:647-654.2006a.
6.王得新.哈里森临床神经病学.人民卫生处版社,2010
7. Liu M, Zhang JT.Effect of ginsenoside Rgl on c-fos gene expression and cAMP levels in rat hippocampus. Acta Pharm Sinica 1996; 17(2):171-174.
8. Wang XY, Chen J, Zhang JT.[Effect of ginsenoside Rgl on learning and memory impairment induced by beta-amyloid peptide(25-35) and its mechanism of action]. Yao Xue Xue Bao 36:1-4.2001.
9. Jiang LF, Liao HL, Huang HM, Zhou LX, Li L, Cheng SX, Du CZ.Potential Prevention and Treatment of Maifanite for Alzheimers Disease Based on Behavior Test, Oxidative Stress Assay, and Trace Element Analysis in Hippocampus of Aβ(25-35)-Induced AD Rats. Biological Trace Element Research Epub 2013.
10. Li N, Wang LM.[Effect of ginsenoside Rgl on Alzheimer s disease of model rats]. BMU Journal 30:325-329.2007.
11. Wang M, Tian D, Tan W, Wu J, Li C,Rang WQ.[Effect of ginsenoside Rgl on learning and memory of mice exposure to plumbum]. J Toxicol 26:28-30.2012.
12. Li WZ, Li WP, Zhang W, Yin YY, Sun XX, Zhou SS, Xu XQ, Tao CR.Protective effect of extract of Astragalus on learning and memory impairments and neurons apoptosis induced by glucocorticoids in 12-month-old male mice. Anat Rec (Hoboken) 294:1003-1014.2011.
13. Wang Q, Sun LH, Jia W, Liu XM, Dang HX, Mai WL, Wang N, Steinmetz A, Wang YQ, Xu CJ.Comparison of ginsenosides Rgl and Rbl for their effects on improving scopolamine-induced learning and memory impairment in mice. Phytother Res 24:1748-1754.2010.
14. Qi D, Zhu Y, Wen L, Liu Q, Qiao H.Ginsenoside Rgl restores the impairment of learning induced by chronic morphine administration in rats. J Psychopharmacol 23:74-83.2009.
15. Liu C, Min S, Wei K, Liu D, Dong J, Luo J, Li P, Liu XB.[2,6-Diisopropylphenol, ginsenoside Rg-1 and lithium protects against the impairment of learning-memory induced by electroconvulsive shock in depressed rats]. Chinese Pharmacological Bulletin 28:1125-1130.2012.
16. Shen L, Zhang J.Ginsenoside Rgl increases ischemia-induced cell proliferation and survival in the dentate gyrus of adult gerbils. Neurosci Lett 344:1-4.2003.
17. Hu SW, Hu WP, Zhou H. [Effect of ginsenoside Rgl on the spatial learning and memory induced by chronic stress in rats]. Anat Res 26:29-31.2004.
18. Shi YQ, Huang TW, Chen LM, Pan XD, Zhang J, Zhu YG, Chen XC.Ginsenoside Rgl attenuates amyloid-beta content, regulates PKA/CREB activity, and improves cognitive performance in SAMP8 mice. J Alzheimers Dis 19:977-989.2010.
19. Fang F, Chen X, Huang T, Luddy JS, Yan SS. Multi-faced neuroprotective effects of Ginsenoside Rgl in an Alzheimer mouse model. Biochim Biophys Acta.2011.
20. Li H, Li SL, Wu ZH, Gong L, Wang JL, Li YZ.Effect of traditional Chinese herbal Bu-Wang-San on synaptic plasticity in ovariectomised rats. J Pharm Pharmacol 61:95-101.2009.
1. Escribano, L., et al., Rosiglitazone rescues memory impairment in Alzheimers transgenic mice:mechanisms involving a reduced amyloid and tau pathology. Neuropsychopharmacology,2010.35(7):p.1593-604.
2. Hardy, J. and D.J. Selkoe, The amyloid hypothesis of Alzheimers disease: progress and problems on the road to therapeutics. Science,2002.297(5580): p.353-6.
3. Tanzi, R.E. and L. Bertram, Twenty years of the Alzheimers disease amyloid hypothesis:a genetic perspective. Cell,2005.120(4):p.545-55.
4. Selkoe, D.J., The origins of Alzheimer disease-A is for amyloid. Jama-Journal of the American Medical Association,2000.283(12):p.1615-1617.
5. Bi, B.T., et al., Promotion of beta-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimers disease. Neurochem Int,2012.60(3):p.257-66.
6. Hartmann, T., et al., Distinct sites of intracellular production for Alzheimers disease A beta40/42 amyloid peptides. Nat Med,1997.3(9):p.1016-20.
7. Gao, S., et al., The relationships between age, sex, and the incidence of dementia and Alzheimer disease:a meta-analysis. Arch Gen Psychiatry,1998. 55(9):p.809-15.
8. Ryan, J., et al., Hormonal treatment, mild cognitive impairment and Alzheimers disease. Int Psychogeriatr,2008.20(1):p.47-56.
9. Nord, L.C., et al., Analysis of oestrogen regulation of alpha-, beta-and gamma-secretase gene and protein expression in cultured human neuronal and glial cells. Neurodegener Dis,2010.7(6):p.349-64.
10. Habauzit, D., et al., Effects of estrogens and endocrine-disrupting chemicals on cell differentiation-survival-proliferation in brain:contributions of neuronal cell lines. J Toxicol Environ Health B Crit Rev,2011.14(5-7):p. 300-27.
11. Hammond, C.B., Womens concerns with hormone replacement therapy--compliance issues. Fertil Steril,1994.62(6 Suppl 2):p.157S-160S.
12. Grady, D., et al., Hormone replacement therapy and endometrial cancer risk: a meta-analysis. Obstet Gynecol,1995.85(2):p.304-13.
13. Chen, F., E.A. Eckman, and C.B. Eckman, Reductions in levels of the Alzheimers amyloid beta peptide after oral administration of ginsenosides. FASEB J,2006.20(8):p.1269-71.
14. Henderson, V.W., Cognitive changes after menopause:influence of estrogen. Clin Obstet Gynecol,2008.51(3):p.618-26.
15. Liu, Q., J.P. Kou, and B.Y. Yu, Ginsenoside Rgl protects against hydrogen peroxide-induced cell death in PCl2 cells via inhibiting NF-kappaB activation. Neurochem Int,2011.58(1):p.119-25.
16. Gao, Q.G., et al., Ginsenoside Rgl protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways. J Neurochem,2009.109(5):p.1338-47.
17. Shi, Y.Q., et al., Ginsenoside Rgl Attenuates Amyloid-beta ontent, Regulates PKA/CREB Activity, and Improves Cognitive Performance in SAMP8 Mice. J Alzheimers Dis,2009.
18. Wang, Y.H. and G.H. Du, Ginsenoside Rgl inhibits beta-secretase activity in vitro and protects against Abeta-induced cytotoxicity in PCl2 cells. J Asian Nat Prod Res,2009.11(7):p.604-12.
19. 王永炎,李明富,戴锡盟.中医内科学.上海科学技术出版社,2000.
20. 王卫霞,王巍,陈可冀.人参皂苷对动物脑神经保护作用及其机理研究进展.中国中西医结合杂志,2005.25(1)：p.89-93.
21. 纪荣芳,牛建昭,许树强,等.从数据挖掘角度看中医药治疗健忘与痴呆.中日友好医院学报.2006,20(6)：p.337-340.
22. 杨迎,张均田,田成璋,等.人参皂什Rbl和RGl促智作用机制的探讨-对小鼠脑神经发育的影响.药学学报,1994.29(4)：p.241-245.
23. 刘态,张均田.人参皂苷Rg1对老年大鼠行为活动的改善作用.中国药学杂志,1996.31(8)：p.464-467.
24. 王晓英,陈霁,张均田.人参皂苷Rg1对p-淀粉样肽(25-35)侧脑室注射所致小鼠学习记忆障碍的改善作用及其机制.药学学报,2001.36(1)：p.1-4.
25. Wei-Zu Li,Wang-Yang Wu, Da-Ke Huang, Yan-Yan Yin,Hong-Wei Kan, XinWang,Yu-You Yao, Wei-Ping Li. Protective effects of astragalosides on dexamethasone and Aβ25-35 induced learning and memory impairments due to decrease amyloid precursor protein expression in 12-month male rats. Food and Chemical Toxicology,2012.50(6):p.1883-90.
26. Lawrence C H Wang,Lee T. Effects of ginseng saponins in energy metaliolism,memory and antineurotoxicity. In The Korean Society of Ginseng Advances in ginseng research.2002:27-46.
27. 申丽红,张均田.人参皂苷Rg1对脑缺血沙土鼠神经干细胞存活率和学习记忆能力的影响.中南药学.2004.2(1)：p.6-9.
28. 刘超,闵苏,魏珂,刘东,董军,罗洁,黎平,刘小滨.2,6-二异丙基苯酚、人参皂苷Rgl和氯化锂逆转电休克后嗅球切除抑郁大鼠学习记忆障碍.中国药理学通报,2012.28(8)：p.1125-30.
29. 胡圣望,胡旺平,周红.人参皂苷Rgl对慢性应激大鼠空间学习记忆能力的影响.解剖学研究,2004.26(1)：p.29-31.
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31. Gao, Q.G., et al., Ginsenoside Rgl protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways. J Neurochem,2009.109(5):p.1338-47.
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2. Hardy J, Selkoe DJ The amyloid hypothesis of Alzheimers disease:progress and problems on the road to therapeutics. Science 297:353-356.2002.
3. Tanzi RE, Bertram L.Twenty years of the Alzheimers disease amyloid hypothesis: a genetic perspective. Cell 120:545-555.2005.
4. Hua X, Lei M, Zhang Y, Ding J, Han Q, Hu G, Xiao M.Long-term D-galactose injection combined with ovariectomy serves as a new rodent model for Alzheimers disease. Life Sci 80:1897-1905.2007.
5. Cui X, Zuo P, Zhang Q, Li X, Hu Y, Long J, Packer L, Liu J.Chronic systemic D-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice:protective effects of R-alpha-lipoic acid. J Neurosci Res 84:647-654.2006a.
6.王得新.哈里森临床神经病学.人民卫生处版社,2010
7. Liu M, Zhang JT.Effect of ginsenoside Rgl on c-fos gene expression and cAMP levels in rat hippocampus. Acta Pharm Sinica 1996; 17(2):171-174.
8. Wang XY, Chen J, Zhang JT.[Effect of ginsenoside Rgl on learning and memory impairment induced by beta-amyloid peptide(25-35) and its mechanism of action]. Yao Xue Xue Bao 36:1-4.2001.
9. Jiang LF, Liao HL, Huang HM, Zhou LX, Li L, Cheng SX, Du CZ.Potential Prevention and Treatment of Maifanite for Alzheimers Disease Based on Behavior Test, Oxidative Stress Assay, and Trace Element Analysis in Hippocampus of Aβ(25-35)-Induced AD Rats. Biological Trace Element Research Epub 2013.
10. Li N, Wang LM.[Effect of ginsenoside Rgl on Alzheimer s disease of model rats]. BMU Journal 30:325-329.2007.
11. Wang M, Tian D, Tan W, Wu J, Li C,Rang WQ.[Effect of ginsenoside Rgl on learning and memory of mice exposure to plumbum]. J Toxicol 26:28-30.2012.
12. Li WZ, Li WP, Zhang W, Yin YY, Sun XX, Zhou SS, Xu XQ, Tao CR.Protective effect of extract of Astragalus on learning and memory impairments and neurons apoptosis induced by glucocorticoids in 12-month-old male mice. Anat Rec (Hoboken) 294:1003-1014.2011.
13. Wang Q, Sun LH, Jia W, Liu XM, Dang HX, Mai WL, Wang N, Steinmetz A, Wang YQ, Xu CJ.Comparison of ginsenosides Rgl and Rbl for their effects on improving scopolamine-induced learning and memory impairment in mice. Phytother Res 24:1748-1754.2010.
14. Qi D, Zhu Y, Wen L, Liu Q, Qiao H.Ginsenoside Rgl restores the impairment of learning induced by chronic morphine administration in rats. J Psychopharmacol 23:74-83.2009.
15. Liu C, Min S, Wei K, Liu D, Dong J, Luo J, Li P, Liu XB.[2,6-Diisopropylphenol, ginsenoside Rg-1 and lithium protects against the impairment of learning-memory induced by electroconvulsive shock in depressed rats]. Chinese Pharmacological Bulletin 28:1125-1130.2012.
16. Shen L, Zhang J.Ginsenoside Rgl increases ischemia-induced cell proliferation and survival in the dentate gyrus of adult gerbils. Neurosci Lett 344:1-4.2003.
17. Hu SW, Hu WP, Zhou H. [Effect of ginsenoside Rgl on the spatial learning and memory induced by chronic stress in rats]. Anat Res 26:29-31.2004.
18. Shi YQ, Huang TW, Chen LM, Pan XD, Zhang J, Zhu YG, Chen XC.Ginsenoside Rgl attenuates amyloid-beta content, regulates PKA/CREB activity, and improves cognitive performance in SAMP8 mice. J Alzheimers Dis 19:977-989.2010.
19. Fang F, Chen X, Huang T, Luddy JS, Yan SS. Multi-faced neuroprotective effects of Ginsenoside Rgl in an Alzheimer mouse model. Biochim Biophys Acta.2011.
20. Li H, Li SL, Wu ZH, Gong L, Wang JL, Li YZ.Effect of traditional Chinese herbal Bu-Wang-San on synaptic plasticity in ovariectomised rats. J Pharm Pharmacol 61:95-101.2009.
1. Escribano, L., et al., Rosiglitazone rescues memory impairment in Alzheimers transgenic mice:mechanisms involving a reduced amyloid and tau pathology. Neuropsychopharmacology,2010.35(7):p.1593-604.
2. Hardy, J. and D.J. Selkoe, The amyloid hypothesis of Alzheimers disease: progress and problems on the road to therapeutics. Science,2002.297(5580): p.353-6.
3. Tanzi, R.E. and L. Bertram, Twenty years of the Alzheimers disease amyloid hypothesis:a genetic perspective. Cell,2005.120(4):p.545-55.
4. Selkoe, D.J., The origins of Alzheimer disease-A is for amyloid. Jama-Journal of the American Medical Association,2000.283(12):p.1615-1617.
5. Bi, B.T., et al., Promotion of beta-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimers disease. Neurochem Int,2012.60(3):p.257-66.
6. Hartmann, T., et al., Distinct sites of intracellular production for Alzheimers disease A beta40/42 amyloid peptides. Nat Med,1997.3(9):p.1016-20.
7. Gao, S., et al., The relationships between age, sex, and the incidence of dementia and Alzheimer disease:a meta-analysis. Arch Gen Psychiatry,1998. 55(9):p.809-15.
8. Ryan, J., et al., Hormonal treatment, mild cognitive impairment and Alzheimers disease. Int Psychogeriatr,2008.20(1):p.47-56.
9. Nord, L.C., et al., Analysis of oestrogen regulation of alpha-, beta-and gamma-secretase gene and protein expression in cultured human neuronal and glial cells. Neurodegener Dis,2010.7(6):p.349-64.
10. Habauzit, D., et al., Effects of estrogens and endocrine-disrupting chemicals on cell differentiation-survival-proliferation in brain:contributions of neuronal cell lines. J Toxicol Environ Health B Crit Rev,2011.14(5-7):p. 300-27.
11. Hammond, C.B., Womens concerns with hormone replacement therapy--compliance issues. Fertil Steril,1994.62(6 Suppl 2):p.157S-160S.
12. Grady, D., et al., Hormone replacement therapy and endometrial cancer risk: a meta-analysis. Obstet Gynecol,1995.85(2):p.304-13.
13. Chen, F., E.A. Eckman, and C.B. Eckman, Reductions in levels of the Alzheimers amyloid beta peptide after oral administration of ginsenosides. FASEB J,2006.20(8):p.1269-71.
14. Henderson, V.W., Cognitive changes after menopause:influence of estrogen. Clin Obstet Gynecol,2008.51(3):p.618-26.
15. Liu, Q., J.P. Kou, and B.Y. Yu, Ginsenoside Rgl protects against hydrogen peroxide-induced cell death in PCl2 cells via inhibiting NF-kappaB activation. Neurochem Int,2011.58(1):p.119-25.
16. Gao, Q.G., et al., Ginsenoside Rgl protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways. J Neurochem,2009.109(5):p.1338-47.
17. Shi, Y.Q., et al., Ginsenoside Rgl Attenuates Amyloid-beta ontent, Regulates PKA/CREB Activity, and Improves Cognitive Performance in SAMP8 Mice. J Alzheimers Dis,2009.
18. Wang, Y.H. and G.H. Du, Ginsenoside Rgl inhibits beta-secretase activity in vitro and protects against Abeta-induced cytotoxicity in PCl2 cells. J Asian Nat Prod Res,2009.11(7):p.604-12.
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