乌鸡白凤丸有效成份与雌激素对脑内多巴胺能神经元的保护作用及其机制探讨
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摘要
大量研究业已揭示，性腺类固醇激素尤其是雌激素在神经系统退行变性性疾病如帕金森病(PD)中具有神经保护作用。雌激素可以抵抗神经毒性药物如1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)的作用，而雄激素无此作用。另有研究报道，植物性雌激素、中药以及针灸对中枢神经系统均具有神经保护作用。乌鸡白凤丸(BFP)是一种传统的中药复方，广泛应用于妇产科疾病的预防与治疗，被认为具有雌激素样作用，并对中枢神经系统有保护作用。为探讨雌激素及BFP有效成份对中枢神经系统尤其是多巴胺(DA)能神经元功能的影响，本研究应用聚合酶链免疫技术(RT-PCR)检测内源性雌激素对MPTP诱发的PD模型小鼠的酪氨酸羟化酶(TH)表达的影响；观察BFP有效成份与17β雌二醇预处理的去卵巢PD模型小鼠多个脑区如纹状体、中脑、皮层及小脑的TH和多巴胺转运蛋白(DAT)表达情况；比较BFP有效成份对雌性与雄性PD模型小鼠的TH和DAT表达的作用；实验还应用点阵列技术观察BFP有效成份以及雌激素预处理的PD模型小鼠中脑与下丘脑的1176种基因的表达特性。此外，本实验观察了MPTP对体外培养的PC12细胞的作用，并应用RT—PCR技术，以促凋亡基因Bax为实验指标，观察BFP有效成份与MPTP对Bax mRNA水平的影响；并应用流式细胞技术观察细胞经MPTP与BFP有效成份处理后的细胞凋亡状况。另外，实验应用快速周期伏安法(FCV)在体观察了BFP有效成份对电刺激诱发雌、雄鼠中央杏仁核DA释放的影响。实验结果表明：
中文.摘要
.雌性PD模型鼠的中脑TH基因表达明显高于去卵巢雌鼠，而且经BFP有效成份
(3叭g，Zw，po.)预处理的PD模型雌鼠的中脑TH表达水平也明显高于去卵巢
PD模型雌鼠。
2.MPTP明显减少去卵巢PD模型小鼠中脑、纹状体和小脑的TH与DAT基因表达
(P 体TH与DAT基因表达的影响勿<0.01)。17p一雌二醇(sopg/kg，Zw，i.p.)可拮抗
MPTP对中脑与纹状体DAT的基因表达的损伤，而对TH无影响。
3.MPTP明显减少雌性与雄性C57BL/6小鼠的中脑与纹状体的TH和DAI，基因表
达水平。与对照组相比，BFP有效成份对于雌、雄PD模型小鼠的TH和DAT
基因表达均有保护作用。
4.MPTP(l 000 pM，24h)增加体外培养PclZ细胞的促凋亡基因Bax的表达，而
BFP有效成份预处理(l 00op留nil，6h)可拮抗MPTP毒性对Bax表达的影响。
5.应用流式细胞仪经PI染色结果显示:与对照组(0.82%)相比，MPTP明显增加
体外培养PC12细胞的凋亡细胞数目(1 1 .8%);BFP有效成份预处理可明显减弱
MPTP对细胞活性的影响，凋亡细胞比率为5 .3%。
6.点阵列结果表明，在1176种cDNA中，BFP有效成份处理组的中脑与下丘脑组
织的40种基因发生明显变化，17p一雌二醇处理组的93种基因发生显著改变，
两组结果表明，35种基因发生类似改变。
7.快速周期伏安法检测显示:侧脑室注射BFP有效成份(0 .75林g，1 .5林g)可增加雌
性与去卵巢雌性大鼠的杏仁核DA释放量。注射5分钟后，DA的释放开始增加，
并持续40分钟，而同法注射BFP有效成份对雄性大鼠杏仁核的DA释放无影响。
中文摘要
上述结果表明，BFP有效成份作为一种具有雌激素样作用的传统中药，有类似
于雌激素的神经保护作用，表现在保护DA能神经元并拮抗神经毒性药物的毒性作
用。然而BFP有效成份的作用与雌激素有所不同，例如BFP有效成份可有效预防
MPTP对TH表达的影响，而雌激素对TH表达的作用不明显。点阵列结果显示，
BFP有效成份对多数基因的作用强度弱于雌激素，两者对既定基因的调控作用不完
全一致。对BFP有效成份作用的机制研究显示，BFP的作用至少与其抗凋亡机制相
关。此外，在体FCV结果显示BFP有效成份可以调节DA能神经元的代谢，侧脑
室注射BFP可以增强中脑边缘结构杏仁核的DA释放，且作用具有性别差异。总之，
BFP有效成份对中枢DA能神经系统的作用不完全与雌激素相同。本实验中研究的
BFP有效成份对DA能神经元的调节和保护作用以及对其可能机制的初步探讨，为
帕金森病的预防与治疗提供新的可能方案，为传统中药的新用途和进一步开发为可
用于临床防治PD的新药提供实验依据。
It has been well-known that gonadal steroid hormones, estrogen especially, plays an important role in protecting the brain from neurodegenerative processes, including Parkinson's disease (PD). Estrogen, but not androgen, has been reported to prevent l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine(MPTP)-induced dopamine (DA) depletion. Phytoestrogen and Chinese medicine, as well as acupuncturing without the use of any drug have also been demonstrated to have neuroprotective effects in PD. As a well-known gynaecological tonic in China, Bak Foong Pill (BFP, also known as Bai Feng Wan) has long been recognized to have estrogen-like activities, has also been reported to exert beneficial effects on various functional systems including central nervous system. For exploring possible neuroprotective effect of BFP extracts and estrogen, and related mechanism(s) underlying the actions, we observed the actions of BFP extracts on tyrosine hydroxylase (TH) gene expression in the midbrain of ovariectomized (OVX) and normal fe
male mice. TH and dopamine transporter (DAT) mRNA levels in the striatum, midbrain, cortex and cerebellum have been detected in BFP extracts- and estrogen-pretreated PD model mice. Effects of BFP extracts on DAT and TH gene expressions have also been measured in both female and male PD mice. In addition, we compared the effects of BFP extracts and estrogen by observing the 1176 mouse cDNAs using microarray technique. The mechanisms underlying the effects of BFP extracts have been detected by measuring the pro-apoptotic gene-Bax expression by employing RT-PCR and observing the apoptotic cells by using flow cytometry. In vivo DA release from the central amygdaloid nucleus have also been measured with fast cyclic voltammetry (FCV). Results were as follows:
1. TH mRNA levels are significantly higher in the MPTP-induced female PD model mice with/without BFP extracts pretreatment (3g/kg, 2 weeks), compared with OVX female mice.
2. In the OVX C57BL/6 mice, MPTP significantly decreased DAT and TH mRNA levels in the striatum, midbrain and cerebellum, but not the cortex of the mice (p<0.001). However, MPTP-challenge with BFP extracts pretreatment demdnstrated reduced neurotoxicity, with DAT and TH mRNA levels either not affected by MPTP or affected to a significantly lesser extent in the midbrain and striatum as compared to the MPTP treated controls. 17B-estradiol treatment prevented MPTP-induced reduction of DAT expression in striatum and midbrain, but failed to alter TH expression.
3. MPTP significantly decreased TH and DAT mRNA levels in the striatum and midbrain of both female and male C57BL/6 mice. While, MPTP challenge with BFP extracts pretreatment showed reduced neurotoxicity, with TH and DAT mRNA levels in the midbrain and striatum higher than that of the vehicle treated animals.
4. To explore anti-apoptotic action of BFP extracts, we measured the expression level of Bax, a pro-apoptotic gene, in a dopamine-secreting neuroendocrine cell line, PC 12, by semi-quantitative RT-PCR. The results showed that Bax mRNA level was significantly increased by MPTP administration (1000 for 24 h). BFP extracts (1000 ug/ml) pretreatment for 6 h attenuated MPTP-elevated expression level of Bax.
5. Furthermore, we measure apoptotic cell population in PC 12 cells by propidium iodide (PI) staining with flow cytometry. The number of apoptotic cells was greatly
enhanced by MPTP, an averaged 11.8% of apoptotic cells as compared to 0.8 % in the control. However, PC 12 cells grew in the medium containing BFP significantly reduced the MPTP-induced apoptotic cell population, with only an averaged 5.3 % of apoptotic cells observed.
6. Among the 1176 mouse cDNAs, 40 genes expression changed in the midbrain and hypothalamus of PD model mice fed with BFP extracts, and 93 individual genes were regulated significantly in the mice injected with 17B -estradiol, by employing the microarray technique. Noteworthy, 35 genes were the same as the BFP-treated group.
7. Intracerebroventricular
中文.摘要
.雌性PD模型鼠的中脑TH基因表达明显高于去卵巢雌鼠，而且经BFP有效成份
(3叭g，Zw，po.)预处理的PD模型雌鼠的中脑TH表达水平也明显高于去卵巢
PD模型雌鼠。
2.MPTP明显减少去卵巢PD模型小鼠中脑、纹状体和小脑的TH与DAT基因表达
(P
MPTP对中脑与纹状体DAT的基因表达的损伤，而对TH无影响。
3.MPTP明显减少雌性与雄性C57BL/6小鼠的中脑与纹状体的TH和DAI，基因表
达水平。与对照组相比，BFP有效成份对于雌、雄PD模型小鼠的TH和DAT
基因表达均有保护作用。
4.MPTP(l 000 pM，24h)增加体外培养PclZ细胞的促凋亡基因Bax的表达，而
BFP有效成份预处理(l 00op留nil，6h)可拮抗MPTP毒性对Bax表达的影响。
5.应用流式细胞仪经PI染色结果显示:与对照组(0.82%)相比，MPTP明显增加
体外培养PC12细胞的凋亡细胞数目(1 1 .8%);BFP有效成份预处理可明显减弱
MPTP对细胞活性的影响，凋亡细胞比率为5 .3%。
6.点阵列结果表明，在1176种cDNA中，BFP有效成份处理组的中脑与下丘脑组
织的40种基因发生明显变化，17p一雌二醇处理组的93种基因发生显著改变，
两组结果表明，35种基因发生类似改变。
7.快速周期伏安法检测显示:侧脑室注射BFP有效成份(0 .75林g，1 .5林g)可增加雌
性与去卵巢雌性大鼠的杏仁核DA释放量。注射5分钟后，DA的释放开始增加，
并持续40分钟，而同法注射BFP有效成份对雄性大鼠杏仁核的DA释放无影响。
中文摘要
上述结果表明，BFP有效成份作为一种具有雌激素样作用的传统中药，有类似
于雌激素的神经保护作用，表现在保护DA能神经元并拮抗神经毒性药物的毒性作
用。然而BFP有效成份的作用与雌激素有所不同，例如BFP有效成份可有效预防
MPTP对TH表达的影响，而雌激素对TH表达的作用不明显。点阵列结果显示，
BFP有效成份对多数基因的作用强度弱于雌激素，两者对既定基因的调控作用不完
全一致。对BFP有效成份作用的机制研究显示，BFP的作用至少与其抗凋亡机制相
关。此外，在体FCV结果显示BFP有效成份可以调节DA能神经元的代谢，侧脑
室注射BFP可以增强中脑边缘结构杏仁核的DA释放，且作用具有性别差异。总之，
BFP有效成份对中枢DA能神经系统的作用不完全与雌激素相同。本实验中研究的
BFP有效成份对DA能神经元的调节和保护作用以及对其可能机制的初步探讨，为
帕金森病的预防与治疗提供新的可能方案，为传统中药的新用途和进一步开发为可
用于临床防治PD的新药提供实验依据。
It has been well-known that gonadal steroid hormones, estrogen especially, plays an important role in protecting the brain from neurodegenerative processes, including Parkinson's disease (PD). Estrogen, but not androgen, has been reported to prevent l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine(MPTP)-induced dopamine (DA) depletion. Phytoestrogen and Chinese medicine, as well as acupuncturing without the use of any drug have also been demonstrated to have neuroprotective effects in PD. As a well-known gynaecological tonic in China, Bak Foong Pill (BFP, also known as Bai Feng Wan) has long been recognized to have estrogen-like activities, has also been reported to exert beneficial effects on various functional systems including central nervous system. For exploring possible neuroprotective effect of BFP extracts and estrogen, and related mechanism(s) underlying the actions, we observed the actions of BFP extracts on tyrosine hydroxylase (TH) gene expression in the midbrain of ovariectomized (OVX) and normal fe
male mice. TH and dopamine transporter (DAT) mRNA levels in the striatum, midbrain, cortex and cerebellum have been detected in BFP extracts- and estrogen-pretreated PD model mice. Effects of BFP extracts on DAT and TH gene expressions have also been measured in both female and male PD mice. In addition, we compared the effects of BFP extracts and estrogen by observing the 1176 mouse cDNAs using microarray technique. The mechanisms underlying the effects of BFP extracts have been detected by measuring the pro-apoptotic gene-Bax expression by employing RT-PCR and observing the apoptotic cells by using flow cytometry. In vivo DA release from the central amygdaloid nucleus have also been measured with fast cyclic voltammetry (FCV). Results were as follows:
1. TH mRNA levels are significantly higher in the MPTP-induced female PD model mice with/without BFP extracts pretreatment (3g/kg, 2 weeks), compared with OVX female mice.
2. In the OVX C57BL/6 mice, MPTP significantly decreased DAT and TH mRNA levels in the striatum, midbrain and cerebellum, but not the cortex of the mice (p<0.001). However, MPTP-challenge with BFP extracts pretreatment demdnstrated reduced neurotoxicity, with DAT and TH mRNA levels either not affected by MPTP or affected to a significantly lesser extent in the midbrain and striatum as compared to the MPTP treated controls. 17B-estradiol treatment prevented MPTP-induced reduction of DAT expression in striatum and midbrain, but failed to alter TH expression.
3. MPTP significantly decreased TH and DAT mRNA levels in the striatum and midbrain of both female and male C57BL/6 mice. While, MPTP challenge with BFP extracts pretreatment showed reduced neurotoxicity, with TH and DAT mRNA levels in the midbrain and striatum higher than that of the vehicle treated animals.
4. To explore anti-apoptotic action of BFP extracts, we measured the expression level of Bax, a pro-apoptotic gene, in a dopamine-secreting neuroendocrine cell line, PC 12, by semi-quantitative RT-PCR. The results showed that Bax mRNA level was significantly increased by MPTP administration (1000 for 24 h). BFP extracts (1000 ug/ml) pretreatment for 6 h attenuated MPTP-elevated expression level of Bax.
5. Furthermore, we measure apoptotic cell population in PC 12 cells by propidium iodide (PI) staining with flow cytometry. The number of apoptotic cells was greatly
enhanced by MPTP, an averaged 11.8% of apoptotic cells as compared to 0.8 % in the control. However, PC 12 cells grew in the medium containing BFP significantly reduced the MPTP-induced apoptotic cell population, with only an averaged 5.3 % of apoptotic cells observed.
6. Among the 1176 mouse cDNAs, 40 genes expression changed in the midbrain and hypothalamus of PD model mice fed with BFP extracts, and 93 individual genes were regulated significantly in the mice injected with 17B -estradiol, by employing the microarray technique. Noteworthy, 35 genes were the same as the BFP-treated group.
7. Intracerebroventricular
引文
1. Miller DB, Ali SF, O'Callaghan JP, Laws SC. The impact of gender and estrogen on striatal dopamine neurotoxicity. Ann NY Acad Sci 1998,844,153-156.
2. Dluzen DE, Disshon KA, Mcdermott JL. Estrogen as a modulator of striatal dopaminergic neurotoxicity. In Recent Advances in Neurodegenerative Disorders, Marwah J and Tietelbaum H., Eds. 1998, pp. 149-192, Prominent Press, Scottsdale, AZ.
3. Baldereschi M, Di Carlo A, Rocca WA, Vanni P, Maggi S, Perissinotto E, Grigoletto F, Amaducci L, Inzitari D. For the ILSA Working Group: Parkinson's disease and parkinsonism in a longitudinal study two-fold higher incidence in men. Neurology 2000, 55: 1358-1363.
4. Bower JH, Maraganore DM, McDonnell SK, Rocca WA. Influence of strict, intermediate, and broad diagnostic criteria on the age-and sex-specific incidence of Parkinson's disease. Mov Disord 2000,15,819-825.
5. Saunders-Pullman R, Gordon-Elliott J, Parides M, Fahn S, Saunders HR, Bressman S. The effect of estrogen replacement on early Parkinson's disease. Neurology. 1999 Apr 22;52(7):1417-21.
6．李志龙，乌鸡白凤丸新用，陕西中医，1987，8(6)：264。
7．李祥舒，乌鸡白凤丸治疗中风后痴呆例近期观察，北京中医，1993，12(6)：49。
8．余萌，陈世健等，乌鸡白凤丸的临床应用与药理分析，时珍国药研究，2001(8)：736—7。
9．朱令元，万阜昌，复方乌鸡白凤丸口服液治疗月经不调疗效观察，现代诊断和治疗，1994，5(3)：140—2。
10．沈鸿等，乌鸡白凤丸口服液与丸剂对动物性激素样作用的比较研究，中国实验方剂学杂志，1998，4(5)：50—3。
2. Dluzen DE, Disshon KA, Mcdermott JL. Estrogen as a modulator of striatal dopaminergic neurotoxicity. In Recent Advances in Neurodegenerative Disorders, Marwah J and Tietelbaum H., Eds. 1998, pp. 149-192, Prominent Press, Scottsdale, AZ.
3. Baldereschi M, Di Carlo A, Rocca WA, Vanni P, Maggi S, Perissinotto E, Grigoletto F, Amaducci L, Inzitari D. For the ILSA Working Group: Parkinson's disease and parkinsonism in a longitudinal study two-fold higher incidence in men. Neurology 2000, 55: 1358-1363.
4. Bower JH, Maraganore DM, McDonnell SK, Rocca WA. Influence of strict, intermediate, and broad diagnostic criteria on the age-and sex-specific incidence of Parkinson's disease. Mov Disord 2000,15,819-825.
5. Saunders-Pullman R, Gordon-Elliott J, Parides M, Fahn S, Saunders HR, Bressman S. The effect of estrogen replacement on early Parkinson's disease. Neurology. 1999 Apr 22;52(7):1417-21.
6．李志龙，乌鸡白凤丸新用，陕西中医，1987，8(6)：264。
7．李祥舒，乌鸡白凤丸治疗中风后痴呆例近期观察，北京中医，1993，12(6)：49。
8．余萌，陈世健等，乌鸡白凤丸的临床应用与药理分析，时珍国药研究，2001(8)：736—7。
9．朱令元，万阜昌，复方乌鸡白凤丸口服液治疗月经不调疗效观察，现代诊断和治疗，1994，5(3)：140—2。
10．沈鸿等，乌鸡白凤丸口服液与丸剂对动物性激素样作用的比较研究，中国实验方剂学杂志，1998，4(5)：50—3。
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