基于~1H-NMR技术对Wilson病模型大鼠及其中、西药干预比较的代谢组学研究
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摘要
目的：Wilson病(Wilson's Disease, WD),又称肝豆状核变性(hepatolenticular degeneration,HLD),是一种与铜代谢障碍有关的神经系统疾病,从本病被报道以来的近一个世纪的时间里,对本病虽有各种各样的研究及发现,但截止到目前为止,对WD的研究尚无法清楚解释其临床表现各异的症状原因和病理。本研究目的如下
(1)基于NMR的代谢组学的研究方法,对WD造模成型前大鼠、WD模型大鼠及其中西药干预疗效比较进行探索。
(2)以NMR和生物信息学等现代技术,通过对血液代谢组的分析,研究WD造模成型前大鼠、WD模型大鼠及其中西药干预后的内源性代谢物的变化,获得相关代谢物指纹图谱探讨血液中有关小分子代谢物变化的原因。
(3)确定与WD发病前、发病后和中西药治疗WD机理密切相关的小分子标志化合物；为WD发病前和发病后,及其中西药疗效比较的系统研究提供科学依据。也试图以此为切入点,运用代谢组学的技术与方法,探索出一种研究WD的新方法、新途径。
方法：选用健康成年Wistar雄性大鼠共66只,体重180±20g, SPF级。以铜负荷法造模WD大鼠模型,随机分为6组：15天模型组(n=11)、15天健康对照组(n=11)、WD模型组(n=11)、健康对照组(n=11)、中药干预组(n=11)和青霉胺干预组(n=11)。以检测尿铜、肝铜为评判造模依据。
在Varian Unityinova 500MHz超导傅里叶变化核磁共振波谱仪上编辑NOEPR-CPMG (NOEPR, Nuclear Overhauser Effect spectroscopy with PResaturation;CPMG,Carr-Purcell-Meiboom-Gill sequence)脉冲序列进行数据采样,其中预饱和模块用于抑制水峰信号。饱和时间为2S,温度300K,谱宽5kHz,信号累加256次。自由感应衰减(FID)信号经过傅里叶变换得到一维NMR谱图,采用MestRe-C 2.3软件及自编软件对谱图进行手动调相、基线校正和谱峰对齐,对0.2-4.6区域按Δδ=0.02等间隔分段积分。将积分数据归一化后构成数据矩阵,并利用PCA方法对数据矩阵进行统计分析。
结果：
1、和15天健康对照组大鼠相比,15天模型组大鼠血清内有以下小分子代谢物的变化：脂质(-CH2-)、乳酸(lactate)、脯氨酸(proline)、丙酮(acetone)含量有所降低,谷氨酸盐(glutamine)、葡萄糖(glucose)、胆碱磷酸(phosphorylcholine)的含量有所升高。
2、和健康对照组大鼠相比,模型组大鼠血清甜菜碱与氧化三甲胺(TAMO+betaine)、低密度脂蛋白(LDL)、极低密度脂蛋白(VLDL)、葡萄糖(glucose)含量明显降低,胆碱(choline)、胆碱磷酸(phosphorylcholine)的含量降低,乳酸(lactate)、谷氨酰胺(glutamine)、糖蛋白(glycoprotein)的含量明显升高,肌氨酸+肌氨酸酐(creatinine),精氨酸(arginine)有所升高。
3、模型组大鼠和15天模型组大鼠相比,其血清代谢物差异存主要在于乳酸(lactate)、葡萄糖(glucose)、精氨酸(arginine)、丙酮(acetone)、酪氨酸(tyrosine)的浓度变化上。
4、相对于模型组大鼠,中药干预组大鼠血清糖蛋白(glycoprotein)、3-羟基丁酸(3-hydroxybutyrate)、脂质(Lipid, mainly VLDL/LDL)、葡萄糖(glucose)、胆碱(choline)、磷酸胆碱(phosphorylcholine)明显升高,亮氨酸(leucine)、异亮氨酸(isoleucine)有所升高。乳酸(lactate)明显下降,谷氨酰胺(glutamine)、肌氨酸+肌氨酸酐(Creatine+ creatinine)、精氨酸(arginine)有所下降。
5、相对于模型组,西药干预组大鼠乳酸(lactate)、糖蛋白(glycoprotein)、谷氨酰胺(glutamine)代谢物含量明显下降,肌氨酸和肌氨酸酐(Creatine+ creatinine)有所下降,葡萄糖(glucose)含量明显升高,甜菜碱(TMAO+ betaine)、脂质(Lipid)、胆碱(choline)、磷酸胆碱(phosphorylcholine)有所升高。
6、比较西药治疗组和中药治疗组,中药组3-羟基丁酸(3-hydroxybutyrate)、亮氨酸(leucine)、异亮氨酸(isoleucine)、缬氨酸(valine)浓度有所下降。
结论及创新点：
1、本实验首次将基于核磁共振的代谢组学方法应用于Wilson病模型大鼠及其中西药干预疗效比较的研究,属于本学科动态前沿,是研究WD方法学的创新。
2、通过对15天模型组和15天健康对照组、模型组和健康对照组的代谢物对比分析,所得出的小分子代谢物能够为进一步为今后对WD发病前、发病后的研究提供一定的参考。其中,谷氨酰胺的结论为研究神经退行性病变中所存在的问题提供了数据参考。通过对所得代谢物的分析,呼应了中医对本病的认识,为对本病的中医现代化研究体供了一条思路。
3、通过对中、西药干预组的研究,为丰富青霉胺对铜的解毒机理提供了一定数据参考。中药治疗组中,一些小分子代谢物的发现初步印证了中药治疗Wilson病的同时具有滋补肝肾,补精填髓功效,为以后深入的从现代化系统医学角度对中药干预Wilson病的研究奠定了基础。
4、通过分析中、西药对治疗本病的差异,从而在整体的、系统的代谢层面探讨了中西药干预本病的异同,并发现中药干预本病的代谢网络比较复杂,呈多靶点,为以后对本病治疗的深入研究提供了一种可行的研究方法。
5、通过实验证明,基于NMR的代谢组学研究方法可以较好地用于WD发病前和发病后与正常对照的区分,及中、西药对WD疗效的对比分析,从而获得具有特征性的代谢物,是寻找WD及其中、西药干预后的小分子特征标志物的有效方法,在未来WD的诊断、病理生理研究以及药物治疗等方面具有广阔的应用前景。
Objectives:Wilson's disease (WD), which is also called hepatolenticular degeneration(HLD), is a kind of neurological disease due to the metabolism disorder of copper. The objective of the research is as the following:
(1)To explore the small mocular metabolites of sub-model, model, TCM treated and penicillamine treated rats of WD, and acquire the related finger-print spectrum and metabolites.
(2)To determine and analyze the typical metabolites and possible mechanisms related closely to the sub-model, model, TCM treated and penicillamine treated rats of WD,in order to get subjective data to support TCM theory on this disease.
(3) To find a new method for researching WD, which can also serve as a new modernization way for TCM.
Methods:66 male Wistar rats (weight=180±20g) were divided into six groups randomly,15-day model group,15-day control group, model group, control group, TCM treated group and penicillamine treated group,with each group containing 11 rats.15-day model group, model group, TCM treated group and penicillamine treated groups were established with excessive copper method. Among them, 15-day model group was administrated with copper food and water for 15 days, while the rest groups for 60 days. Since the 31th day of copper administration, the TCM group was treated with Gandouling tablets by intragastric method, while the penicillamine treated group was treated with penicillamine in the same way. The 15-day control and control groups were naturally taking food and water.
All the rats'serum was taken when the above proceedings were over. The serum was tested with 1H-NMR technology. The spectra were edited with MestRe-C2.3 and self-programmed software and then principal component analysis (PCA) was applied to make differentiation between the 15-day model and 15-day control groups, model and control groups,15-day model and model groups, TCM and model groups, penicillamine and model groups, TCM and penicillamine groups respectively. Then, small modular changes in the serum from the above contrast were analyzed to explore the related mechanisms.
Results:1、Contrasting the small molecular changes between the 15-day control and 15-day model groups, we found lipids, lactate, proline, and acetone concentrations were lower and glutamine, glucose and phosphorylcholine concentrations were higher in the serum of the 15-day model group.
2、Choline and phosphorylcholine concentrations were found to be lower and TAMO+betaine, LDL, VLDL and glucose were significantly lower in the serum of the model group. While creatine+ creatinine and arginine concentrations were found to be higher and lactate, glutamine and glycoprotein were significantly higher in the model group. The small molecular metabolites above may contribute to the discrimination,and serve as references for further research on WD pathogenesis.
3、Contrasting the motabolites of 15-day model and model groups, we found the difference lay in the concentrations of lactate, glucose, arginine, acetone and tyrosine.
4、Contrasting the small molecular changes between the TCM and model groups, we found glycoprotein、3-hydroxybutyrate、Lipid, glucose、choline、phosphorylcholine concentrations were significantly higher, and leucine, isoleucine concentrations were higher, while lactate concentration was significantly lower, and glutamine Creatine+ creatinine, arginine concentrations were lower in the serum of TCM group.
5、Contrasting the small molecular changes between the penicillamine and model groups, we found lactate, glycoprotein, glutamine concentrations were significantly lower, Creatine, creatinine concentrations were lower, glucose concentration was significant higher, and betaine, lipid, choline and phosphorylcholine concentrations were higher in the serum of penicillamine group.
6、Contrasting the small molecular changes between the penicillamine and TCM groups, we found 3-hydroxybutyrate, leucine, isoleucine and valine concentrations were higher in the serum of TCM group.
Conclusions and highlights:
1、It is the first time for NMR based metabonomics to be applied to the research of WD model rats and the comparison of the motabolites after its TCM and western medicine treatment. This research is on the leading of this subject, and is a methodological innovation.
2、The small molecular metabolites from the contrast and analysis of the 15-day model and 15-day control,model and control, 15-day model and model groups can be a possible references for the future research on the disease. Among them, the result of glutamine can be possible data reference for nrurological degeneration diseases. Through the analysis of the metabolites, we found the results could coordinate with the TCM interpretation of this disease, which could put forth a new idea for modernization of TCM.
3、Through the research on TCM and penicillamine groups, some data could be taken into consideration in the future research on mechanism of penicillamine chelating copper. The detection of some motabolites in the TCM group proved TCM beared the function of nourishing and stenghening liver and kidney.This laid the foundation for researching the curative effect of TCM on WD from a modernized medical perception.
4、We explored the difference of TCM and western medicine treating this disease from a systematic facet, through analyzing the difference of the metabolites. As a result, we found the TCM treating WD beared a complex metabolic net and manifested multi-target spot. Which showed metabonomics provided a feasible way for advanced research in the future.
5、By experiment, we proved NMR based metabonomics method could be applied to discriminate the sub-model, model rats and control rats, and to discriminate the metabolites after the TCM and penicillamine groups. And through this method, we could get the typical metabolites, which proved this method had a broad application prospect in the future TCM and western medical research of diagnisis, pathophysiology and treatment on WD.
(1)基于NMR的代谢组学的研究方法,对WD造模成型前大鼠、WD模型大鼠及其中西药干预疗效比较进行探索。
(2)以NMR和生物信息学等现代技术,通过对血液代谢组的分析,研究WD造模成型前大鼠、WD模型大鼠及其中西药干预后的内源性代谢物的变化,获得相关代谢物指纹图谱探讨血液中有关小分子代谢物变化的原因。
(3)确定与WD发病前、发病后和中西药治疗WD机理密切相关的小分子标志化合物；为WD发病前和发病后,及其中西药疗效比较的系统研究提供科学依据。也试图以此为切入点,运用代谢组学的技术与方法,探索出一种研究WD的新方法、新途径。
方法：选用健康成年Wistar雄性大鼠共66只,体重180±20g, SPF级。以铜负荷法造模WD大鼠模型,随机分为6组：15天模型组(n=11)、15天健康对照组(n=11)、WD模型组(n=11)、健康对照组(n=11)、中药干预组(n=11)和青霉胺干预组(n=11)。以检测尿铜、肝铜为评判造模依据。
在Varian Unityinova 500MHz超导傅里叶变化核磁共振波谱仪上编辑NOEPR-CPMG (NOEPR, Nuclear Overhauser Effect spectroscopy with PResaturation;CPMG,Carr-Purcell-Meiboom-Gill sequence)脉冲序列进行数据采样,其中预饱和模块用于抑制水峰信号。饱和时间为2S,温度300K,谱宽5kHz,信号累加256次。自由感应衰减(FID)信号经过傅里叶变换得到一维NMR谱图,采用MestRe-C 2.3软件及自编软件对谱图进行手动调相、基线校正和谱峰对齐,对0.2-4.6区域按Δδ=0.02等间隔分段积分。将积分数据归一化后构成数据矩阵,并利用PCA方法对数据矩阵进行统计分析。
结果：
1、和15天健康对照组大鼠相比,15天模型组大鼠血清内有以下小分子代谢物的变化：脂质(-CH2-)、乳酸(lactate)、脯氨酸(proline)、丙酮(acetone)含量有所降低,谷氨酸盐(glutamine)、葡萄糖(glucose)、胆碱磷酸(phosphorylcholine)的含量有所升高。
2、和健康对照组大鼠相比,模型组大鼠血清甜菜碱与氧化三甲胺(TAMO+betaine)、低密度脂蛋白(LDL)、极低密度脂蛋白(VLDL)、葡萄糖(glucose)含量明显降低,胆碱(choline)、胆碱磷酸(phosphorylcholine)的含量降低,乳酸(lactate)、谷氨酰胺(glutamine)、糖蛋白(glycoprotein)的含量明显升高,肌氨酸+肌氨酸酐(creatinine),精氨酸(arginine)有所升高。
3、模型组大鼠和15天模型组大鼠相比,其血清代谢物差异存主要在于乳酸(lactate)、葡萄糖(glucose)、精氨酸(arginine)、丙酮(acetone)、酪氨酸(tyrosine)的浓度变化上。
4、相对于模型组大鼠,中药干预组大鼠血清糖蛋白(glycoprotein)、3-羟基丁酸(3-hydroxybutyrate)、脂质(Lipid, mainly VLDL/LDL)、葡萄糖(glucose)、胆碱(choline)、磷酸胆碱(phosphorylcholine)明显升高,亮氨酸(leucine)、异亮氨酸(isoleucine)有所升高。乳酸(lactate)明显下降,谷氨酰胺(glutamine)、肌氨酸+肌氨酸酐(Creatine+ creatinine)、精氨酸(arginine)有所下降。
5、相对于模型组,西药干预组大鼠乳酸(lactate)、糖蛋白(glycoprotein)、谷氨酰胺(glutamine)代谢物含量明显下降,肌氨酸和肌氨酸酐(Creatine+ creatinine)有所下降,葡萄糖(glucose)含量明显升高,甜菜碱(TMAO+ betaine)、脂质(Lipid)、胆碱(choline)、磷酸胆碱(phosphorylcholine)有所升高。
6、比较西药治疗组和中药治疗组,中药组3-羟基丁酸(3-hydroxybutyrate)、亮氨酸(leucine)、异亮氨酸(isoleucine)、缬氨酸(valine)浓度有所下降。
结论及创新点：
1、本实验首次将基于核磁共振的代谢组学方法应用于Wilson病模型大鼠及其中西药干预疗效比较的研究,属于本学科动态前沿,是研究WD方法学的创新。
2、通过对15天模型组和15天健康对照组、模型组和健康对照组的代谢物对比分析,所得出的小分子代谢物能够为进一步为今后对WD发病前、发病后的研究提供一定的参考。其中,谷氨酰胺的结论为研究神经退行性病变中所存在的问题提供了数据参考。通过对所得代谢物的分析,呼应了中医对本病的认识,为对本病的中医现代化研究体供了一条思路。
3、通过对中、西药干预组的研究,为丰富青霉胺对铜的解毒机理提供了一定数据参考。中药治疗组中,一些小分子代谢物的发现初步印证了中药治疗Wilson病的同时具有滋补肝肾,补精填髓功效,为以后深入的从现代化系统医学角度对中药干预Wilson病的研究奠定了基础。
4、通过分析中、西药对治疗本病的差异,从而在整体的、系统的代谢层面探讨了中西药干预本病的异同,并发现中药干预本病的代谢网络比较复杂,呈多靶点,为以后对本病治疗的深入研究提供了一种可行的研究方法。
5、通过实验证明,基于NMR的代谢组学研究方法可以较好地用于WD发病前和发病后与正常对照的区分,及中、西药对WD疗效的对比分析,从而获得具有特征性的代谢物,是寻找WD及其中、西药干预后的小分子特征标志物的有效方法,在未来WD的诊断、病理生理研究以及药物治疗等方面具有广阔的应用前景。
Objectives:Wilson's disease (WD), which is also called hepatolenticular degeneration(HLD), is a kind of neurological disease due to the metabolism disorder of copper. The objective of the research is as the following:
(1)To explore the small mocular metabolites of sub-model, model, TCM treated and penicillamine treated rats of WD, and acquire the related finger-print spectrum and metabolites.
(2)To determine and analyze the typical metabolites and possible mechanisms related closely to the sub-model, model, TCM treated and penicillamine treated rats of WD,in order to get subjective data to support TCM theory on this disease.
(3) To find a new method for researching WD, which can also serve as a new modernization way for TCM.
Methods:66 male Wistar rats (weight=180±20g) were divided into six groups randomly,15-day model group,15-day control group, model group, control group, TCM treated group and penicillamine treated group,with each group containing 11 rats.15-day model group, model group, TCM treated group and penicillamine treated groups were established with excessive copper method. Among them, 15-day model group was administrated with copper food and water for 15 days, while the rest groups for 60 days. Since the 31th day of copper administration, the TCM group was treated with Gandouling tablets by intragastric method, while the penicillamine treated group was treated with penicillamine in the same way. The 15-day control and control groups were naturally taking food and water.
All the rats'serum was taken when the above proceedings were over. The serum was tested with 1H-NMR technology. The spectra were edited with MestRe-C2.3 and self-programmed software and then principal component analysis (PCA) was applied to make differentiation between the 15-day model and 15-day control groups, model and control groups,15-day model and model groups, TCM and model groups, penicillamine and model groups, TCM and penicillamine groups respectively. Then, small modular changes in the serum from the above contrast were analyzed to explore the related mechanisms.
Results:1、Contrasting the small molecular changes between the 15-day control and 15-day model groups, we found lipids, lactate, proline, and acetone concentrations were lower and glutamine, glucose and phosphorylcholine concentrations were higher in the serum of the 15-day model group.
2、Choline and phosphorylcholine concentrations were found to be lower and TAMO+betaine, LDL, VLDL and glucose were significantly lower in the serum of the model group. While creatine+ creatinine and arginine concentrations were found to be higher and lactate, glutamine and glycoprotein were significantly higher in the model group. The small molecular metabolites above may contribute to the discrimination,and serve as references for further research on WD pathogenesis.
3、Contrasting the motabolites of 15-day model and model groups, we found the difference lay in the concentrations of lactate, glucose, arginine, acetone and tyrosine.
4、Contrasting the small molecular changes between the TCM and model groups, we found glycoprotein、3-hydroxybutyrate、Lipid, glucose、choline、phosphorylcholine concentrations were significantly higher, and leucine, isoleucine concentrations were higher, while lactate concentration was significantly lower, and glutamine Creatine+ creatinine, arginine concentrations were lower in the serum of TCM group.
5、Contrasting the small molecular changes between the penicillamine and model groups, we found lactate, glycoprotein, glutamine concentrations were significantly lower, Creatine, creatinine concentrations were lower, glucose concentration was significant higher, and betaine, lipid, choline and phosphorylcholine concentrations were higher in the serum of penicillamine group.
6、Contrasting the small molecular changes between the penicillamine and TCM groups, we found 3-hydroxybutyrate, leucine, isoleucine and valine concentrations were higher in the serum of TCM group.
Conclusions and highlights:
1、It is the first time for NMR based metabonomics to be applied to the research of WD model rats and the comparison of the motabolites after its TCM and western medicine treatment. This research is on the leading of this subject, and is a methodological innovation.
2、The small molecular metabolites from the contrast and analysis of the 15-day model and 15-day control,model and control, 15-day model and model groups can be a possible references for the future research on the disease. Among them, the result of glutamine can be possible data reference for nrurological degeneration diseases. Through the analysis of the metabolites, we found the results could coordinate with the TCM interpretation of this disease, which could put forth a new idea for modernization of TCM.
3、Through the research on TCM and penicillamine groups, some data could be taken into consideration in the future research on mechanism of penicillamine chelating copper. The detection of some motabolites in the TCM group proved TCM beared the function of nourishing and stenghening liver and kidney.This laid the foundation for researching the curative effect of TCM on WD from a modernized medical perception.
4、We explored the difference of TCM and western medicine treating this disease from a systematic facet, through analyzing the difference of the metabolites. As a result, we found the TCM treating WD beared a complex metabolic net and manifested multi-target spot. Which showed metabonomics provided a feasible way for advanced research in the future.
5、By experiment, we proved NMR based metabonomics method could be applied to discriminate the sub-model, model rats and control rats, and to discriminate the metabolites after the TCM and penicillamine groups. And through this method, we could get the typical metabolites, which proved this method had a broad application prospect in the future TCM and western medical research of diagnisis, pathophysiology and treatment on WD.
引文
[1]Dedoussis GV, Genechel J, Sialvera TE, et al. Wilson disease: high prevalence in mountainous area of Crete [J]. Ann Hum Genet, 2005,69 (Pt3):268-274.
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