基于尿液代谢组学技术分析大川芎方多组分中药制剂干预急性偏头痛大鼠模型的影响
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摘要
目的:考察大川芎方多组分中药制剂对急性偏头痛大鼠尿液中内源性代谢产物的影响,通过尿液代谢组学技术探讨该药可能的作用机制。方法:采用40只SD雄性大鼠随机分为5组,分别为正常组、模型组和大川芎方多组分中药制剂低、中、高剂量组(0. 19,0. 37,0. 74 g·kg~(-1)),每组8只。除正常组外,其余各组大鼠采用皮下注射10 mg·kg~(-1)硝酸甘油方法制备急性偏头痛的模型。造模成功后,大川芎方多组分中药制剂组灌胃给予不同剂量的大川芎方多组分中药制剂。给药0. 5 h后腹主动脉取血,采用酶联免疫吸附测定(ELISA)检测血浆中血清素5-羟色胺(5-HT),5-羟基吲哚乙酸(5-HIAA),一氧化氮(NO),一氧化氮合酶(NOS),降钙素基因相关肽(CGRP)及多巴胺(DA)含量;采用免疫组化对正常组及模型组大鼠下丘脑和脑干部位5-HT和NOS蛋白表达进行分析,并通过平均光密度值进行统计;采用UPLC-TOF/MS,结合主成分分析(PCA)对不同组别大鼠进行尿液代谢组学分析,并发现潜在的生物标记物;通过MetaboAnalyst 3. 0软件对差异代谢物进行代谢通路分析。结果:与正常组比较,模型组大鼠体内的神经递质均有极显著性差异(P <0. 01),说明急性偏头痛大鼠模型已建立;与模型组比较,各给药组体内的神经递质均有显著性差异(P <0. 05,P <0. 01),说明给予大川芎方多组分中药制剂可调节大鼠体内的神经递质含量。根据UPLC-TOF/MS分析,共发现10种潜在的差异代谢物。大川芎方多组分中药制剂给药组可降低模型组中色氨酸、缬氨酸的含量,升高γ-氨基丁酸(GABA),乳酸,β-羟丁酸的含量。根据代谢通路预测研究,发现相关性较大的代谢通路为丙氨酸、天冬氨酸与谷氨酸代谢,丙酸代谢,氮代谢及色氨酸代谢。结论:大川芎方多组分中药制剂对急性偏头痛大鼠模型体内的差异代谢物有一定的回调作用,其作用机制主要与氨基酸代谢有关,特别是犬尿氨酸的代谢。
Objective: To investigate the intervention effect of Dachuanxiong Fang multi-component preparation on acute migraine rats,in order to explore the possible mechanism by using urine metabolomics technology. Method: The forty SD rats were randomly divided into 5 groups( n = 8),control group,model group,low-dose Dachuanxiong Fang multi-component preparation( DCXF) group( 0. 19 g·kg~(-1)),medium-dose DCXF group( 0. 37 g·kg~(-1)),and high-dose DCXF group( 0. 74 g·kg~(-1)). Rats were subcutaneously injected with10 mg·kg~(-1) nitroglycerin for modeling,and Dachuanxiong Fang multi-component preparation was administered through intragastric administration. After half an hour of administration,blood was collected from the abdominal aorta,and the content of 5-hydroxytryptamine( 5-HT),5-hydroxyindole-3-acetic acid( 5-HIAA),nitric oxide( NO),nitric oxide synthase( NOS),calcitonlin gene related peplide( CGRP),dopamine( DA) in plasma were determined by enzyme-linked immuno sorbent assay( ELISA) kits. The contents of 5-HT and NOS in the hypothalamus and brainstem of the control group and the model group were analyzed,and the average optical density value was used for statistical analysis. UPLC-TOF/MS combined with principal component analysis( PCA)analysis was used to analyze different groups of rats and discover differential metabolites. Differential metabolites were analyzed by MetaboAnalyst 3. 0 software for possible metabolic pathways. Result: After modeling,compared with the control group,the content of neurotransmitters in the model group was significantly increased( P < 0. 01).After the intervention with Dachuanxiong Fang multi-component preparation,compared with the model group,the content of neurotransmitters was significantly decreased( P < 0. 05,P < 0. 01). Based on UPLC-TOF/MS analysis,10 potential biomarkers were found. After the intervention with Dachuanxiong multi-component preparation,the contents of tryptophan and proline were down-regulated,while the contents of gamma-aminobutyric acid( GABA),lactic acid and β-hydroxybutyrate showed an upward trend. According to the metabolic pathway prediction study,metabolic pathways with a higher correlation were found to be alanine,aspartic acid and glutamate metabolism,propionic acid metabolism,nitrogen metabolism and tryptophan metabolism. Conclusion: The possible metabolism pathway of Dachuanxiong Fang multi-component preparation is mainly amino acid metabolism in urine,and kynurenine is also the product of tryptophan metabolism pathway. The kynurenine metabolic pathway is also one of the main pathways of tryptophan metabolism.
Objective: To investigate the intervention effect of Dachuanxiong Fang multi-component preparation on acute migraine rats,in order to explore the possible mechanism by using urine metabolomics technology. Method: The forty SD rats were randomly divided into 5 groups( n = 8),control group,model group,low-dose Dachuanxiong Fang multi-component preparation( DCXF) group( 0. 19 g·kg~(-1)),medium-dose DCXF group( 0. 37 g·kg~(-1)),and high-dose DCXF group( 0. 74 g·kg~(-1)). Rats were subcutaneously injected with10 mg·kg~(-1) nitroglycerin for modeling,and Dachuanxiong Fang multi-component preparation was administered through intragastric administration. After half an hour of administration,blood was collected from the abdominal aorta,and the content of 5-hydroxytryptamine( 5-HT),5-hydroxyindole-3-acetic acid( 5-HIAA),nitric oxide( NO),nitric oxide synthase( NOS),calcitonlin gene related peplide( CGRP),dopamine( DA) in plasma were determined by enzyme-linked immuno sorbent assay( ELISA) kits. The contents of 5-HT and NOS in the hypothalamus and brainstem of the control group and the model group were analyzed,and the average optical density value was used for statistical analysis. UPLC-TOF/MS combined with principal component analysis( PCA)analysis was used to analyze different groups of rats and discover differential metabolites. Differential metabolites were analyzed by MetaboAnalyst 3. 0 software for possible metabolic pathways. Result: After modeling,compared with the control group,the content of neurotransmitters in the model group was significantly increased( P < 0. 01).After the intervention with Dachuanxiong Fang multi-component preparation,compared with the model group,the content of neurotransmitters was significantly decreased( P < 0. 05,P < 0. 01). Based on UPLC-TOF/MS analysis,10 potential biomarkers were found. After the intervention with Dachuanxiong multi-component preparation,the contents of tryptophan and proline were down-regulated,while the contents of gamma-aminobutyric acid( GABA),lactic acid and β-hydroxybutyrate showed an upward trend. According to the metabolic pathway prediction study,metabolic pathways with a higher correlation were found to be alanine,aspartic acid and glutamate metabolism,propionic acid metabolism,nitrogen metabolism and tryptophan metabolism. Conclusion: The possible metabolism pathway of Dachuanxiong Fang multi-component preparation is mainly amino acid metabolism in urine,and kynurenine is also the product of tryptophan metabolism pathway. The kynurenine metabolic pathway is also one of the main pathways of tryptophan metabolism.
引文
[1] Pradhan A,Mcguire B,Charles A. Characterization of a novel model of chronic migraine[J]. PAIN,2014,155(2):269-274.
[2] Lipton R B,Bigal M E,Diamond M,et al. Migraine prevalence,disease burden,and the need for preventive therapy[J]. Neurology,2007,68(5):343-349.
[3]郭慧,崔扬,王秋红,等.基于代谢组学技术的中药复方研究近况[J].中国实验方剂学杂志,2017,23(1):213-219.
[4] LEI Y,LI D,DENG J,et al. Metabolomic profiling of three brain regions from a postnatal infected Borna disease virus Hu-H1 rat model[J]. Metabonomics,2014,10(3):10484-10495.
[5] MA S Y,SHEN L,CHEN M W,et al. The study of metabonomics combined with diversity of intestinal flora in LDP intervention in kidney-Yin deficiency hyperthyroid rats[J]. RSC Adv,2015,5(71):57975-57983.
[6]陈勤.少阳经穴针刺治疗偏头痛的代谢组学研究[D].四川:成都中医药大学,2009.
[7]高骏.针刺治疗偏头痛经穴效应特异性的1HNMR代谢组学基础研究[D].四川:成都中医药大学,2011.
[8]周本宏,周静,吴丽宁,等.天麻-川芎对偏头痛模型大鼠尿液内源性物质代谢的影响[J].中国实验方剂学杂志,2014,20(3):104-108.
[9]李慧,吴艳华,陈宝田,等.正天丸对偏头痛大鼠的血浆代谢组学研究[J].浙江中医药大学学报,2014,38(9):1046-1049.
[10] WANG Q, SHEN L, MA S Y, et al. Effects of ligusticum Chuanxiong and gastrodia elata on blood-brain barrier permeability in migraine rats[J]. Pharmazie,2015,70(6):421-426.
[11] HONG Y L,FENG Y,XU D S,et al. Study on extraction and purification of active parts from Da Chuan Xiong Fang for treatment of migraine[J]. J Chin Med Mater,2007,10(6):721-723.
[12]王强.基于体内过程的大川芎方中风药川芎“引药上行”的配伍原理研究[D].上海:上海中医药大学,2014.
[13]马诗瑜,沈岚,王清清,等.大川芎方多组分制剂释药特性的评价[J].中成药,2017,39(5):939-945.
[14]王高玉,刘红宁,戈淑超,等.铁皮石斛水提物对胃癌前病变作用的尿液代谢组学分析[J].中国实验方剂学杂志,2018,24(21):77-85.
[15]杨瑞,唐思,董晓茜,等.基于UPLC-Q-TOF-MS分析雷公藤配伍甘草治疗肾病综合征大鼠的尿液代谢组学[J].中国实验方剂学杂志,2018,24(12):150-156.
[16]王晓丹. D-犬尿氨酸中枢神经系统的代谢及神经化学作用[D].天津:天津医科大学,2012.
[17]何磊,包永睿,孟宪生,等.基于活血化瘀功效的川芎酚酸组分治疗大鼠偏头痛作用机制研究[J].中南药学,2018,16(1):45-49.
[18]于雪,胡文忠,姜爱丽,等.天麻的活性成分及功能性研究进展[J].食品工业科技,2016,37(8):392-395,399.
[19]田慧,张丹参.天麻素神经保护机制的研究进展[J].神经药理学报,2013,3(5):58-64.
[2] Lipton R B,Bigal M E,Diamond M,et al. Migraine prevalence,disease burden,and the need for preventive therapy[J]. Neurology,2007,68(5):343-349.
[3]郭慧,崔扬,王秋红,等.基于代谢组学技术的中药复方研究近况[J].中国实验方剂学杂志,2017,23(1):213-219.
[4] LEI Y,LI D,DENG J,et al. Metabolomic profiling of three brain regions from a postnatal infected Borna disease virus Hu-H1 rat model[J]. Metabonomics,2014,10(3):10484-10495.
[5] MA S Y,SHEN L,CHEN M W,et al. The study of metabonomics combined with diversity of intestinal flora in LDP intervention in kidney-Yin deficiency hyperthyroid rats[J]. RSC Adv,2015,5(71):57975-57983.
[6]陈勤.少阳经穴针刺治疗偏头痛的代谢组学研究[D].四川:成都中医药大学,2009.
[7]高骏.针刺治疗偏头痛经穴效应特异性的1HNMR代谢组学基础研究[D].四川:成都中医药大学,2011.
[8]周本宏,周静,吴丽宁,等.天麻-川芎对偏头痛模型大鼠尿液内源性物质代谢的影响[J].中国实验方剂学杂志,2014,20(3):104-108.
[9]李慧,吴艳华,陈宝田,等.正天丸对偏头痛大鼠的血浆代谢组学研究[J].浙江中医药大学学报,2014,38(9):1046-1049.
[10] WANG Q, SHEN L, MA S Y, et al. Effects of ligusticum Chuanxiong and gastrodia elata on blood-brain barrier permeability in migraine rats[J]. Pharmazie,2015,70(6):421-426.
[11] HONG Y L,FENG Y,XU D S,et al. Study on extraction and purification of active parts from Da Chuan Xiong Fang for treatment of migraine[J]. J Chin Med Mater,2007,10(6):721-723.
[12]王强.基于体内过程的大川芎方中风药川芎“引药上行”的配伍原理研究[D].上海:上海中医药大学,2014.
[13]马诗瑜,沈岚,王清清,等.大川芎方多组分制剂释药特性的评价[J].中成药,2017,39(5):939-945.
[14]王高玉,刘红宁,戈淑超,等.铁皮石斛水提物对胃癌前病变作用的尿液代谢组学分析[J].中国实验方剂学杂志,2018,24(21):77-85.
[15]杨瑞,唐思,董晓茜,等.基于UPLC-Q-TOF-MS分析雷公藤配伍甘草治疗肾病综合征大鼠的尿液代谢组学[J].中国实验方剂学杂志,2018,24(12):150-156.
[16]王晓丹. D-犬尿氨酸中枢神经系统的代谢及神经化学作用[D].天津:天津医科大学,2012.
[17]何磊,包永睿,孟宪生,等.基于活血化瘀功效的川芎酚酸组分治疗大鼠偏头痛作用机制研究[J].中南药学,2018,16(1):45-49.
[18]于雪,胡文忠,姜爱丽,等.天麻的活性成分及功能性研究进展[J].食品工业科技,2016,37(8):392-395,399.
[19]田慧,张丹参.天麻素神经保护机制的研究进展[J].神经药理学报,2013,3(5):58-64.