安颤灵对帕金森病大鼠模型泛素—蛋白酶体系统的影响
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摘要
研究背景帕金森病(Parkinson disease，PD)是一种常见于中老年人的第二大神经退行性疾病，为老年人2大慢性杀手之一。随着老龄化社会的来临，PD患病率正在世界范围内逐年增高，据世界帕金森病协会资料显示，全球现有超过400万帕金森病患者。我国目前约有帕金森病患者172万，居全球第一，而且每年还要新增约10万患者。因此帕金森病已经成为我国乃至世界各国面临的重大医学和社会问题。
研究表明：中脑黑质多巴胺能神经元变性缺失伴胞浆内Lewy小体的形成是帕金森病的主要病理改变。而越来越多的证据显示Lewy小体的形成与泛素蛋白酶体系统(ubiquitin proteasome system，UPS)功能缺损密切相关。目前，在治疗上，人们普遍采用以左旋多巴为主的替代疗法，虽然在缓解PD患者症状上有一定疗效，但同时也会出现一些严重的毒副作用，且不能有效阻止病情的发展。近年来的研究证实：中药治疗PD疗效确切，在延缓疾病的进程，减轻西药治疗后的不良反应，控制PD的一些非运动障碍症状等方面发挥了重要作用。但目前对中药治疗PD的机理探讨上，国内还多局限于从氧化应激、细胞凋亡、兴奋性氨基酸毒性、免疫反应等方面入手，未见对本病关键性病理环节UPS的研究。
本课题所采用的中药复方安颤灵为导师吴正治教授在承担国家十五科技攻关项目“帕金森病中西医结合综合治疗优化方案研究”工作期间，以古今文献相关方药的系统整体为基础，结合多年科研和临床经验，并根据其提出的本病新的中医病机假说——“脾湿困阻、气机不畅、肌肉失主”，以理脾化湿为主，辅以行气活血、柔肝止痉立法组方，经反复筛选优化而成的纯天然中药复方。经过多年临床应用，对早中期帕金森病疗效显著，对晚期和多巴胺制剂失敏的帕金森病患者也有一定疗效。
研究目的本课题采用lactacystin损伤制作的PD体内模型，利用免疫组化，Tunel观察安颤灵在抗凋亡及改善UPS功能等方面的作用机制，旨在为中药安颤灵治疗PD提供实验依据，为PD治疗提供新的思路。
研究方法本研究采用lactacystin立体定向注射入大鼠左侧黑质致密部和黑质纹状体通路，建立PD大鼠模型。经行为测试成功的PD模型大鼠27只，随机选取分为模型组、中药组，每组10只，同时另取设对照组10只。对照组和模型组生理盐水灌胃，中药治疗组同时间等体积安颤灵灌胃，共45d。给药后第1、3、5周及给药结束后分别行APO诱导，观察其旋转行为及其他异常行为并进行记录，分析安颤灵对PD大鼠模型的治疗作用。给药结束后每组随机取2只大鼠行Tunel及免疫组织化学检测，观察各组黑质细胞凋亡的情况，及各组a-突触核蛋白、泛素表达的情况。
研究结果中药组对阿朴吗啡诱导的旋转行为有明显抑制作用，与模型组相比较，其旋转圈数明显减少(P＜0.01)。与模型组相比，中药组黑质细胞凋亡明显减少(P＜0.01)及中药组a-突触核蛋白、泛素阳性细胞数均减少。
研究结论安颤灵可抑制PD大鼠模型黑质细胞的凋亡，并可改善UPS的功能。
Background Parkinson's disease (PD) is the second most common neurodegenerative disorder. With the coming of the old-aged society, the wordwide incidence of PD is rising. According to investigation of Parkinson's Disease Association, there has been up to four millions of PD patients in the world, and our country have 1.72 millions, which occupys the first place. In addition, there is still 100 thousand every year. Therefore, how to deal with PD has become an important medicine and society problem.
Researchs have previously demonstrated that Parkinson's disease (PD) is characterized by a relatively selective degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) and intracellular proteinaceous inclusions referred to as Lewy bodies. Several studies have suggested that failure of the ubiquitin proteasome system to clear intracellular proteinaceous inclusions may be an important factor in PD. At present, theL-dopamine (L-DA) is wordwide applied to therapy PD. Although it alleviates the symptoms, it can also result in a serises of negative effect, and can't effectively hold back the development of PD. Recently research demonstrates that the effect is significant with Chinese medicine-treated, which not only delays the proccess of PD, but also relieves the negative effect of some chemicals, and can also control other symptoms besides movement disorders. At present, reserchers in our country commomly aimed at the exploration of the anti-oxidation and anti-apoptosis mechanism, However, there is few study on the mechanism of Chinese medicine's therapeutic effect on the ubiquitin proteasome system of PD.
Chinese medicine "AnChanLing" has a beneficial effect on treating PD, which has proved by the many year's clinical application. We established the PD rat model of by injecting lactacystin, and observed the AnChanLing effect on the UPS function and anti-apoptosis by immunohistochemistry and TUNEL. We have intention to provide AnChanLing with some experimental evidence on treating PD, and it would be shed light on the therapic mechanism of PD patients as well.
Objective To Explore AnChanLing effect on UPS function of PD rat models.
Methods we established the rat model of by injecting lactacystin stereotaxically to left side of the mesencephic ventral tegmental area(VTA)and substantian igra pars compactas(SNc) of rats. After 3 weeks, all rats were induced the rotational behavior after injecting apomorphine subcutaneously. 27 successful animals were randomly divided into two groups, model group and Anchanling-treated group. Each group has 10 rats, meanwhile, 10 rats were considered as control group. The control and model groups were treated with physiological saline for 5 weeks, and Anchanling-treated group with Chinese medicine Anchanling decoction. The rotational behavior was evaluated by recording the numer of apomorphine-induced turns once a week. We used tyrosine hydroxylase (TH) immunohistochemical staining to investigate the numbers of dopaminergic neurons in substantia nigra compacta (SNc). TUNEL staining was employed to observe the apoptosis of nerve cells. A-synuclein、ubiquitin positive ceils are applied to elaterate the difference of the UPS function between each group.
Results In the contrary of the group of PD rats, the rotational behavior, apoptosic nigral cells of the group of "AnChanLing" treatment were ameliorated effectively. The TUNEL positive cells in the group of Chinese Medcine were significantly less than the model group(P＜0.01), and a-synuclein、ubiquitin positive cells were also less than that.
Conclusions AnChanLing could obviously inhibit the apoptosis of neuron cell. It was suggested that AnChanLingcould ameliorate effectively the function of UPS.
研究表明：中脑黑质多巴胺能神经元变性缺失伴胞浆内Lewy小体的形成是帕金森病的主要病理改变。而越来越多的证据显示Lewy小体的形成与泛素蛋白酶体系统(ubiquitin proteasome system，UPS)功能缺损密切相关。目前，在治疗上，人们普遍采用以左旋多巴为主的替代疗法，虽然在缓解PD患者症状上有一定疗效，但同时也会出现一些严重的毒副作用，且不能有效阻止病情的发展。近年来的研究证实：中药治疗PD疗效确切，在延缓疾病的进程，减轻西药治疗后的不良反应，控制PD的一些非运动障碍症状等方面发挥了重要作用。但目前对中药治疗PD的机理探讨上，国内还多局限于从氧化应激、细胞凋亡、兴奋性氨基酸毒性、免疫反应等方面入手，未见对本病关键性病理环节UPS的研究。
本课题所采用的中药复方安颤灵为导师吴正治教授在承担国家十五科技攻关项目“帕金森病中西医结合综合治疗优化方案研究”工作期间，以古今文献相关方药的系统整体为基础，结合多年科研和临床经验，并根据其提出的本病新的中医病机假说——“脾湿困阻、气机不畅、肌肉失主”，以理脾化湿为主，辅以行气活血、柔肝止痉立法组方，经反复筛选优化而成的纯天然中药复方。经过多年临床应用，对早中期帕金森病疗效显著，对晚期和多巴胺制剂失敏的帕金森病患者也有一定疗效。
研究目的本课题采用lactacystin损伤制作的PD体内模型，利用免疫组化，Tunel观察安颤灵在抗凋亡及改善UPS功能等方面的作用机制，旨在为中药安颤灵治疗PD提供实验依据，为PD治疗提供新的思路。
研究方法本研究采用lactacystin立体定向注射入大鼠左侧黑质致密部和黑质纹状体通路，建立PD大鼠模型。经行为测试成功的PD模型大鼠27只，随机选取分为模型组、中药组，每组10只，同时另取设对照组10只。对照组和模型组生理盐水灌胃，中药治疗组同时间等体积安颤灵灌胃，共45d。给药后第1、3、5周及给药结束后分别行APO诱导，观察其旋转行为及其他异常行为并进行记录，分析安颤灵对PD大鼠模型的治疗作用。给药结束后每组随机取2只大鼠行Tunel及免疫组织化学检测，观察各组黑质细胞凋亡的情况，及各组a-突触核蛋白、泛素表达的情况。
研究结果中药组对阿朴吗啡诱导的旋转行为有明显抑制作用，与模型组相比较，其旋转圈数明显减少(P＜0.01)。与模型组相比，中药组黑质细胞凋亡明显减少(P＜0.01)及中药组a-突触核蛋白、泛素阳性细胞数均减少。
研究结论安颤灵可抑制PD大鼠模型黑质细胞的凋亡，并可改善UPS的功能。
Background Parkinson's disease (PD) is the second most common neurodegenerative disorder. With the coming of the old-aged society, the wordwide incidence of PD is rising. According to investigation of Parkinson's Disease Association, there has been up to four millions of PD patients in the world, and our country have 1.72 millions, which occupys the first place. In addition, there is still 100 thousand every year. Therefore, how to deal with PD has become an important medicine and society problem.
Researchs have previously demonstrated that Parkinson's disease (PD) is characterized by a relatively selective degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) and intracellular proteinaceous inclusions referred to as Lewy bodies. Several studies have suggested that failure of the ubiquitin proteasome system to clear intracellular proteinaceous inclusions may be an important factor in PD. At present, theL-dopamine (L-DA) is wordwide applied to therapy PD. Although it alleviates the symptoms, it can also result in a serises of negative effect, and can't effectively hold back the development of PD. Recently research demonstrates that the effect is significant with Chinese medicine-treated, which not only delays the proccess of PD, but also relieves the negative effect of some chemicals, and can also control other symptoms besides movement disorders. At present, reserchers in our country commomly aimed at the exploration of the anti-oxidation and anti-apoptosis mechanism, However, there is few study on the mechanism of Chinese medicine's therapeutic effect on the ubiquitin proteasome system of PD.
Chinese medicine "AnChanLing" has a beneficial effect on treating PD, which has proved by the many year's clinical application. We established the PD rat model of by injecting lactacystin, and observed the AnChanLing effect on the UPS function and anti-apoptosis by immunohistochemistry and TUNEL. We have intention to provide AnChanLing with some experimental evidence on treating PD, and it would be shed light on the therapic mechanism of PD patients as well.
Objective To Explore AnChanLing effect on UPS function of PD rat models.
Methods we established the rat model of by injecting lactacystin stereotaxically to left side of the mesencephic ventral tegmental area(VTA)and substantian igra pars compactas(SNc) of rats. After 3 weeks, all rats were induced the rotational behavior after injecting apomorphine subcutaneously. 27 successful animals were randomly divided into two groups, model group and Anchanling-treated group. Each group has 10 rats, meanwhile, 10 rats were considered as control group. The control and model groups were treated with physiological saline for 5 weeks, and Anchanling-treated group with Chinese medicine Anchanling decoction. The rotational behavior was evaluated by recording the numer of apomorphine-induced turns once a week. We used tyrosine hydroxylase (TH) immunohistochemical staining to investigate the numbers of dopaminergic neurons in substantia nigra compacta (SNc). TUNEL staining was employed to observe the apoptosis of nerve cells. A-synuclein、ubiquitin positive ceils are applied to elaterate the difference of the UPS function between each group.
Results In the contrary of the group of PD rats, the rotational behavior, apoptosic nigral cells of the group of "AnChanLing" treatment were ameliorated effectively. The TUNEL positive cells in the group of Chinese Medcine were significantly less than the model group(P＜0.01), and a-synuclein、ubiquitin positive cells were also less than that.
Conclusions AnChanLing could obviously inhibit the apoptosis of neuron cell. It was suggested that AnChanLingcould ameliorate effectively the function of UPS.
引文
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[1] 孙红梅，张军，白丽敏，等．银杏平颤方对帕金森病鼠黑质半胱氨酸天冬氨酸蛋白酶和Bcl-2蛋白表达的影响[J]．中国临床康复，2005，9(17)：137。
[2] 陈建宗，黄晨，李晓苗，等．培补肝肾法治疗帕金森病的研究思路及实践[J]．中国医药学报，2004，19(11)：688。
[3] 何建成，袁灿兴，卫洪昌，等．滋补肝肾通络解毒中药对PD大鼠细胞凋亡的影响[J]．中国老年学杂志，2003，22(5)：217。
[4] 黄艳，朱汉卿，郑鸿燕，等。白藜芦醇抗帕金森病黑质细胞凋亡的实验性研究[J]．江苏药学与临床研究，2005，13(2)：7．
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[7] 祝维峰，罗荣敬，周丽萍，等．震颤宁治疗帕金森病的实验研究[J]．广州中医药大学学报，2004，21(4)：284。
[8] 张军，孙红梅，白丽敏，等．PD小鼠脑内SOD、MDA和GSH-Px含量的变化和银杏平颤方及其拆方的影响[J1．中医药学刊，2004，22(5)：911。
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[11] 田季雨，陈晓莉，陈建宗．培补肝肾中药预处理对帕金森病小鼠模型氧化应激反应的影响[J]．辽宁中医杂志，2000，30(11)：936．
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[1] 孙红梅，张军，白丽敏，等．银杏平颤方对帕金森病鼠黑质半胱氨酸天冬氨酸蛋白酶和Bcl-2蛋白表达的影响[J]．中国临床康复，2005，9(17)：137。
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[3] 何建成，袁灿兴，卫洪昌，等．滋补肝肾通络解毒中药对PD大鼠细胞凋亡的影响[J]．中国老年学杂志，2003，22(5)：217。
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[6] 蒲小平，李晓蓉，李蔗浓，等．肉苁蓉成分camoneoside Ⅱ对神经毒素MPP+诱发细胞凋亡的保护作用[J]．北京大学学报(医学版)，2002，37(5)：339．
[7] 祝维峰，罗荣敬，周丽萍，等．震颤宁治疗帕金森病的实验研究[J]．广州中医药大学学报，2004，21(4)：284。
[8] 张军，孙红梅，白丽敏，等．PD小鼠脑内SOD、MDA和GSH-Px含量的变化和银杏平颤方及其拆方的影响[J1．中医药学刊，2004，22(5)：911。
[9] 何建成，袁灿兴，李亚明，等．滋补肝肾通络解毒中药对帕金森病大鼠模型 氧化应激反应的影响[J]．中国新药与临床杂志，2003，22(3)：160．
[10] 李如奎，屠英梅，赵虹，等．平颤汤及其拆方对帕金森病模型大鼠自由基损伤保护作用的实验研究[J]．中国中医药科技，2000，8(6)：346．
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