Metabolic Response of Basal Ganglia in Copper-laden Rat to Gandouling Based on ~1H NMR Spectroscopy

采用核磁共振氢谱(1H NMR)技术与多元统计分析相结合的方法,对肝豆灵对大鼠基底节铜损伤的调节机制进行研究。24只雄性Wistar大鼠随机分为正常组、模型组和肝豆灵组,每组8只,以铜负荷法造模。从造模第7周开始,肝豆灵组大鼠以肝豆灵灌胃。数据分析显示:与正常组比较,模型组的基底节细胞凋亡指数显著增高(p<0.01);基底节组织中的谷氨酰胺、尿苷、苏氨酸含量升高(p<0.05),甘露醇、腺苷、N-乙酰天冬氨酸、谷氨酸、N-乙酰谷氨酸(NAG)、天冬酰胺、乙酸、天冬氨酸、肌醇、三磷酸腺苷(ATP)含量降低(p<0.05);与模型组相比,肝豆灵组的基底节细胞凋亡指数显著降低(p<0.01);基底节组织中的谷氨酰胺、尿苷、乙酸、天冬氨酸含量降低(p<0.05),甘露醇、腺苷、苏氨酸、N-乙酰天冬氨酸、谷氨酸、N-乙酰谷氨酸、天冬酰胺、肌醇、ATP含量升高(p<0.05)。研究结果表明,肝豆灵可影响铜负荷大鼠基底节的代谢,对铜损伤具有一定的修复作用,其机制可能是通过调节氨的解毒和兴奋性氨基酸类神经递质的代谢,干预能量代谢,恢复神经元和神经胶质细胞功能。1H NMR spectroscopy combined with multivariate statistical method was introduced in this paper to explore the regulation effect of Gandouling on basal ganglia injury in copper-laden rats. 24 male Wistar rats were randomly divided into control group,model group and Gandouling group,with8 rats in each group. The rat model was established by copper-laden method. From the 7th week of modeling,Gandouling group was given Gandouling formula via gavage. Compared with the control group,the index of apoptosis cells in basal ganglia of model group increased significantly( p < 0. 01),and glutamine,uridine and threonine in basal ganglia were increased( p < 0. 05) while mannitol,adenosine,N-acetylaspartate, glutamate, N-acetylglutamine asparagine, acetate, aspartate, myoinositol and ATP were decreased( p < 0. 05). Compared with the model group,the index of apoptosis cells in basal ganglia of Gandouling group was significantly decreased( p < 0. 01),and glutamine,uridine,acetate and aspartate were reduced( p < 0. 05) while mannitol, adenosine, threonine,N-acetylaspartate,glutamate,N-acetylglutamine,asparagine,myo-inositol and ATP were elevated( p < 0. 05). The results showed that Gandouling could influence the metabolism of basal ganglia in copper-laden rats,which has a certain therapeutic effect on damage caused by copper. The mechanism may be restoration of the functions of neurons and glial cells,through adjusting the detoxification of ammonia and metabolism of excitatory amino acid neurotransmitter and interfering the energy metab-olism.国家自然科学基金(81202691,81201143,81371639);; 福建省自然科学基金(2015Y0032

Comparison of mechanism for copper-excretion between Gandouling Tablets and penicillamine by metabonomics

目的探讨肝豆灵片(大黄、黄芩、黄连、半枝莲等)和青霉胺对大鼠的代谢物,以比较两者排铜机制异同。方法适应性饲养1周后,大鼠被随机分为对照组、模型组、肝豆灵组、青霉胺组。造模结束后,取各组大鼠血清样品分析其核磁共振谱峰。结果模型组大鼠血清乳酸、糖蛋白、肌酸、肌酐、谷氨酰胺、精氨酸的量升高,葡萄糖、甜菜碱、胆碱、磷酸胆碱、脂质的量降低;青霉胺组大鼠血清葡萄糖、甜菜碱、胆碱、胆碱磷酸、脂质的量升高,糖蛋白、乳酸、肌酸、肌酐、谷氨酰胺的量降低;肝豆灵组大鼠血清3-羟基丁酸、亮氨酸、异亮氨酸、缬氨酸、胆碱、胆碱磷酸、葡萄糖、脂质的量升高,谷氨酰胺、乳酸、肌酸、肌酐、精氨酸的量降低。结论青霉胺与肝豆灵均可明显调节铜负荷大鼠的葡萄糖、乳酸、肌酸、肌酐、胆碱、磷酸胆碱、谷氨酰胺和脂质向正常范围回归的趋势。而肝豆灵片还可调节鸟氨酸循环、支链氨基酸、3-羟基丁酸及氨的代谢。AIM To apply metabonomics to the exploration of differences in mechanism for copper-excretion between Gandouling Tablets( Rheum officinale Baill.,Scutellaria baicalensis Georgi,Coptis chinensis Franch.,Scutellaria barbata D. Don,etc.) and penicillamine. METHODS After adaptive breeding for one week,the tested rats were randomly divided into four groups, including control, model, Gandouling Tablets and penicillamine group. After modeling,the sera of all groups were analyzed by1H-NMR spectroscopy. RESULTS The model group showed increased levels of lactate, glutamine, glycoprotein, creatine, creatinine and arginine,together with decreased levels of glucose,betaine,choline,phosphocholine and lipid. Penicillamine group had increased levels of glucose,betaine,choline,phosphocholine and lipid,together with decreased levels of glutamine,glycoprotein,lactate,creatine and creatinine. While Gandouling Tablets increased 3-hydroxybutyrate,leucine, isoleucine, valine, choline, phosphocholine, glucose and lipid, but decreased glutamine,lactate,creatine,creatinine and arginine. CONCLUSION There is a similarity of mechanism for copper excretion between Gandouling Tablets and penicillamine, mainly in the metabolic regulation of glutamine, lactate,creatine,creatinine, glucose, choline, phosphocholine and lipid returning to normal. Moreover,Gandouling Tablets can regulate the metabolisms of ornithine cycle,3- hydroxybutyric acid and branched chain amino acids.国家自然科学基金青年基金资助(81202691);; 安徽省高校自然科学基金重点项目(KJ2012Z228);; 安徽省高校博士后岗位项目(2013年

~1H-NMR Spectroscopy-based Metabonomic Research on Urine of Model Rats of Wilson's Disease

目的:以基于核磁共振(nuClEAr MAgnETIC rESOnAnCE,nMr)的代谢组学方法对WIlSOn病(WIlSOn'S dISEASE,Wd)铜负荷模型大鼠及正常对照组大鼠的尿液进行研究,分析模型大鼠尿液中代谢物的变化,继而从小分子层面探讨铜过量对机体的损伤机制,以更加清楚的认识本病。方法:28只雄性WISTAr大鼠,体重(180±20)g,随机被分为模型组(n=14)和健康对照组(n=14)。采用铜负荷法制作WIlSOn病大鼠模型,以nMr技术对大鼠尿液进行检测。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。对样品进行分段积分,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:相对于正常对照组,模型组大鼠尿液醋酸盐(ACETATE)含量有显著升高,柠檬酸盐(CITrATE)、苯乙酰甘氨酸(PAg)、琥珀酸盐(SuCCInATE)、甲胺(METHylAMInE)、肌氨酸+肌氨酸酐(CrEATInE/CrEATInInE)、丙酮酸盐(PyruVATE)、二甲基甘氨酸(dMg)、丙氨酸(AlAnInE)含量有所升高,胆碱(CHOlInE)、牛磺酸(TAurInE)含量有所降低。这些发生改变的代谢物可能是潜在的Wd铜负荷小分子代谢标志物,可为进一步研究Wd的铜过量代谢机制提供参考。Objective :Applying 1H nuclear magnetic resonance(1H-NMR)based metabonomics to study the changes of small molecular metabolites in the urine of the model rats of Wilson's disease.To explore the pathogenesis of Wilson's disease in small molecular aspect.Methods :28 male Wistar rats[weight=(180±20) g] were divided into two groups randomly,the model group(n=14)and the control group(n=14),with the models established by copper-loaded method.Urine of the rats was tested with 1H-NMR technology.The spectra was edited with MestRe-C2.3 and self-programmed software and then principal component analysis(PCA)was applied to differentiate the two groups.Results :Acetate concentration was found to be significantly higher in the urine of the model group;citrate,PAG,succinate,methylamine,creatine/creatinine,pyruvate,DMG,and alanine were higher,and choline and taurine were lower in the urine of the model group.The small molecular metabolites mentioned above may contribute to the discrimination of the two groups,and provide references for further researches on the pathogenesis of WD.安徽省高校自然科学基金重点项目(KJ2012Z228); 安徽中医学院自然科学基金项目(2011ZR008B

--1H-NMR Spectroscopy-based Metabonomic Research on Serum of Model Rats of Wilson's Disease

目的:以基于核磁共振(nMr)的代谢组学方法对WIlSOn病大鼠模型及正常对照组大鼠血清进行研究,分析血清中小分子代谢物的变化,从小分子代谢物层面上探讨WIlSOn病的内在机制,以更加清楚的认识本病。方法:22只雄性WISTAr大鼠,体重(180±20)g,随机被分为模型组(n=11)和健康对照组(n=11),采用铜负荷法制作WIlSOn病大鼠模型,以核磁共振(nMr)技术对大鼠血清进行检测。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。对样品进行分段积分,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:相对于正常对照组,模型组大鼠血清甜菜碱(bETAInE)、氧化三甲胺(TAMO)、低密度脂蛋白(ldl)、极低密度脂蛋白(Vldl)、葡萄糖(gluCOSE)含量有显著降低,胆碱(CHOlInE)、胆碱磷酸(PHOSPHOrylCHOlInE)的含量有所降低,乳酸(lACTATE)、谷氨酰胺(gluTAMInE)、糖蛋白(glyCOPrOTEIn)有显著升高,肌氨酸+肌氨酸酐(CrEATInE+CrEATInInE),精氨酸(ArgInInE)有所升高。这些发生改变的代谢物可以作为Wd的小分子代谢标志物,为进一步研究Wd的内在代谢机制提供参考。Objective:Applying 1H nuclear magnetic resonance(1H-NMR)spectroscopy-based metabonomic approach to investigate the changes of small molecular metabolites in the serum from the rats of the model group of Wilson's disease contrasted with those of the control group.Exploring the pathogenesis of Wilson's disease from small molecular aspect.Methods:22 male Wistar rats[weight=(180±20)g]were divided into two groups randomly,the model group(n=11)and the control group(n=11),with the models established with excessive copper method.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 differentiate the two groups.Results: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 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.“十一五”国家科技支撑计划分课题重大疑难疾病中医防治研究项目(2006BA104A02

常幅、程序和随机三种不同载荷下的平台用钢的表面裂纹扩展规律及比较

本文使用导管架平台所用的材料 A537研究表面裂纹在常幅、程序和随机三种不同载荷下的疲劳裂纹扩展规律、以及对它们的影响,并把它们的寿命进行比较,找出裂纹形状变化的规律,探索把常幅加载疲劳裂纹扩展数据用于平台疲劳寿命估算的可能性

Metabonomic Research on Curative Mechanism of Gandouling Treating Model Rats of Wilson's Disease

目的:以基于核磁共振(nuClEAr MAgnETIC rESOnAnCE,nMr)的代谢组学方法对肝豆灵干预WIlSOn病(WIlSOn'S dISEASE,Wd)模型大鼠进行研究,分析肝豆灵干预后,Wd模型大鼠尿液中代谢物的变化,从小分子层面探讨肝豆灵对Wd的治疗作用机制。方法:42只雄性WISTAr大鼠,体重(180±20)g,随机被分为健康对照组(n=14)、模型组(n=14)和肝豆灵组(n=14)。采用铜负荷法制作WIlSOn病大鼠模型,以nMr技术对大鼠尿液进行检测,经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。对样品进行分段积分,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:相对于模型组,肝豆灵组大鼠尿液色氨酸(TryPTOPHAn)、马尿酸盐(HIPPurATE)、苯基丙氨酸(PHEnylAlAnInE)、甘氨酸(glyCInE)、苯乙酰甘氨酸(PAg)含量有显著升高;乳酸(lACTATE)、甜菜碱(TMAO/bETAInE)、甲酸盐(fOrMATE)、二甲基甘氨酸(dMg)含量有所升高;柠檬酸盐(CITrATE)、肌氨酸酐(CrEATInInE)、谷氨酰胺(gluTAMInE)含量显著降低;丙酮酸盐(PyruVATE)甲基胍(METHylguAnIdInE)、丙酮(ACETOnE)、牛磺酸(TAurInE)含量有所降低。这些发生改变的代谢物可为进一步研究肝豆灵干预Wd的作用机制提供参考。Objective:Applying 1H nuclear magnetic resonance(1H-NMR)based metabonomics to study the changes of small molecular metabolites in the urine of model rats of WD treated with Gandouling.To explore the curative mechanism of Gandouling on the model rats in small molecular aspect.Methods:42 male Wistar rats[weight=(180±20)g]were divided into three groups randomly,the model group(n=14),the control group(n=14),and the Gandouling group(n=14),with the models established with copper-loaded method.Urine of the rats 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 differentiate the three groups.Results:Compared with those of the model group,tryptophan,hippurate,phenylalanine,glycine and PAG concentration were found to be significantly higher;lactate,TMAO/betaine,formate and DMG were higher;citrate,creatinine and glutamine were significantly lower;pyruvate,methylguanidine,acetone and taurine were lower in the urine of the Gandouling group.The small molecular metabolites mentioned above may contribute to the curative mechanism of Gandouling,and provide references for further researches on the treatment mechanism of WD.安徽省高校自然科学基金重点项目(KJ2012Z228); 安徽中医学院自然科学基金项目(2011ZR008B

Research on metabolomics of liver injury rats with Wilson's disease due to copper overload

目的利用代谢组学技术研究铜负荷大鼠肝组织的小分子变化,探讨铜过量对肝脏小分子代谢的影响。方法 16只Wistar大鼠随机分为正常组和模型组,以铜负荷法造模,通过核磁共振氢谱技术采集大鼠肝组织的代谢轮廓,以PLS-DA方法分析铜中毒后,大鼠肝组织代谢物的变化。结果与正常组对比,模型组大鼠肝组织中尿素囊、牛磺酸、肌醇、赖氨酸、尼克酰胺、乙醇胺、乙酸、谷氨酸、酪氨酸、尿苷、甲硫氨酸、苏氨酸、异亮氨酸、3-羟基丁酸、缬氨酸、乳酸、亮氨酸、苯丙氨酸、N-乙酰天冬氨酸、延胡索酸和腺苷/肌苷的含量升高(P<0.05),肌酸、天冬酰胺、天冬氨酸、磷酸胆碱和甘露醇的含量降低(P<0.05)。结论铜对大鼠肝组织的损伤可能涉及鸟氨酸和三羧酸循环、磷脂、氨基酸、能量、核苷酸和糖的代谢。Objective To explore the changes of small molecules in liver tissues of rats with copper overload and the influences of copper overload on metabolism of small molecules in liver tissues. Methods Sixteen Wistar rats were randomly divided into normal control group and model group. Copper overload method was used to establish the model rats.1H-NMR was used to acquire the metabolic profile of rat liver tissues,and PLS-DA was used to analyze the changes of metabolites in rat liver tissues after copper poisoning. Results Compared with normal control group,the contents of allantoin,taurine,myoinositol,lysine,nicotinamide,ethanolamine,acetate,glutamate,tyrosine,uridine,methionine,threonine,isoleucine,3-hydroxybutyrate,valine,lactate,leucine,phenylalanine,Nacetylaspartate,fumarate,and adenosine / inosine were increased( P < 0. 05),while the contents of creatine,asparagine,aspartate,phosphorylcholine and mannitol were decreased in model group( P < 0. 05). Conclusion Copper damage in rat liver tissues may be involved in the metabolism of ornithine and Krebs cycles,lecithin,amino acid,energy,nucleotides and glucose.国家自然科学基金青年基金资助项目(81202691);; 安徽省高校自然科学基金重点基金资助项目(KJ2012Z228);; 安徽中医学院自然科学基金资助项目(2011ZR008B);; 安徽省高校博士后岗位项目基金资助项目;; 安徽省高校优秀青年骨干教师国外访问研修重点项目(gxfxZ D2016121