基于代谢组学研究熟三七发挥补血作用的机制
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摘要
该文运用高效液相色谱-飞行时间质谱联用(UPLC-Q-TOF-MS)技术研究熟三七(蒸制三七)对乙酰苯肼(APH)致溶血性贫血大鼠血浆中内源性标志物的影响,旨在发现熟三七对溶血性贫血大鼠补血作用的潜在生物标记物及可能的补血作用机制。采用熟三七、药对(熟三七-人参)和复方(三七养血胶囊)分别干预APH诱导的溶血性贫血模型大鼠,测定红细胞(RBC)、血红蛋白(Hb)等血液常规指标以及相关脏器指标。与空白组比较,模型组大鼠RBC,Hb水平显著降低(P<0. 01),肝、脾脏器指数升高(P<0. 05),血常规和脏器指标测定结果表明血虚证大鼠造模成功。而熟三七能显著升高大鼠RBC水平(P<0. 01),各给药组的相关指标均有良好的回调趋势,验证了熟三七的补血作用。对收集的血浆样品应用UPLC-Q-TOF-MS技术,并结合主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA),对正常组、模型组之间的代谢谱进行分析,筛选出26个溶血性贫血的潜在生物标记物。血虚大鼠体内视黄醇、L-缬氨酸、花生四烯酸等9个代谢物下调,原卟啉Ⅸ、烟酰胺等17个代谢物上调。大鼠给予熟三七、药对、复方后可使生物标志物代谢水平向正常状态转归,推测熟三七可能从改善缬氨酸、亮氨酸和异亮氨酸的生物合成、视黄醇代谢、花生四烯酸代谢等8条代谢途径发挥补血的作用。
In this paper,ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOFMS) technique was used to study the effects of steamed notoginseng on endogenous markers in plasma of rats with hemolytic anemia induced by N-acetyl phenyl hydrazine( APH). The aim was to find out the potential biomarkers and possible blood enriching mechanism of steamed notoginseng on hemolytic anemia rats. In the experiment,steamed notoginseng medicine pair( steamed notoginseng-ginseng)and compound medicines( Sanqi Yangxue Capsules) were used respectively to intervene in APH-induced hemolytic anemia model rats.Then blood routine indexes such as red blood cells( RBC),hemoglobin( Hb) and related organ indexes were determined. As compared with the blank group,the RBC and Hb levels in the model group were substantially decreased( P< 0. 01),while the liver and spleen organ indexes were increased( P< 0. 05). The results of blood routine and organ index demonstrated that the blood deficiency model was successfully established. Steamed notoginseng can significantly increase the RBC level of rats( P<0. 01),and the related indicators of each drug group had a trend of returning to normal levels,verifying the blood enriching effect of steamed notoginseng. The UPLC-Q-TOF-MS technique,principal component analysis( PCA) and partial least squares-discrimination analysis( PLS-DA) were used to analyze the metabolic profiles between the normal group and the model group. Twenty-six potential biomarkers for hemolytic anemia were screened in plasma. Nine metabolites such as retinol,L-valine,and arachidonic acid were down-regulated in the blood deficiency rats,and 17 metabolites such as protoporphyrin Ⅸ and niacinamide were up-regulated. The metabolic level of biomarkers could be changed to a normal state after rats were given with steamed notoginseng,drug pairs,and compound prescriptions. It can be speculated that steamed notoginseng may play a role of blood tonifying by improving biosynthesis of valine,leucine and isoleucine,as well as metabolic pathways such as retinol metabolism and arachidonic acid metabolism.
In this paper,ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOFMS) technique was used to study the effects of steamed notoginseng on endogenous markers in plasma of rats with hemolytic anemia induced by N-acetyl phenyl hydrazine( APH). The aim was to find out the potential biomarkers and possible blood enriching mechanism of steamed notoginseng on hemolytic anemia rats. In the experiment,steamed notoginseng medicine pair( steamed notoginseng-ginseng)and compound medicines( Sanqi Yangxue Capsules) were used respectively to intervene in APH-induced hemolytic anemia model rats.Then blood routine indexes such as red blood cells( RBC),hemoglobin( Hb) and related organ indexes were determined. As compared with the blank group,the RBC and Hb levels in the model group were substantially decreased( P< 0. 01),while the liver and spleen organ indexes were increased( P< 0. 05). The results of blood routine and organ index demonstrated that the blood deficiency model was successfully established. Steamed notoginseng can significantly increase the RBC level of rats( P<0. 01),and the related indicators of each drug group had a trend of returning to normal levels,verifying the blood enriching effect of steamed notoginseng. The UPLC-Q-TOF-MS technique,principal component analysis( PCA) and partial least squares-discrimination analysis( PLS-DA) were used to analyze the metabolic profiles between the normal group and the model group. Twenty-six potential biomarkers for hemolytic anemia were screened in plasma. Nine metabolites such as retinol,L-valine,and arachidonic acid were down-regulated in the blood deficiency rats,and 17 metabolites such as protoporphyrin Ⅸ and niacinamide were up-regulated. The metabolic level of biomarkers could be changed to a normal state after rats were given with steamed notoginseng,drug pairs,and compound prescriptions. It can be speculated that steamed notoginseng may play a role of blood tonifying by improving biosynthesis of valine,leucine and isoleucine,as well as metabolic pathways such as retinol metabolism and arachidonic acid metabolism.
引文
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[20]罗年翠.当归-白芍药对配伍效应与物质基础研究[D].南京:南京中医药大学,2013.
[21]周永峰,牛明,房吉祥,等.基于UPLC-Q-TOF-MS的五味子保肝作用代谢组学研究[J].中国中药杂志,2018,43(18):3756.
[22]曲婷丽,王二兵,李震宇,等.黄芪注射液乙酸乙酯萃取物对小鼠白细胞减少症作用的代谢组学[J].中成药,2017,39(3):455.
[23]郑惠婷,李克宁,陈驰,等.基于1H-NMR人参煎治疗2型糖尿病大鼠的胰腺代谢组学研究[J].中国中药杂志,2018,43(14):3012.
[24]张淑丽,王怡萍,杜振霞,等.基于UPC2-/TOF-MS技术的果糖诱导高尿酸血症大鼠血清脂质代谢组学研究[J].中国中药杂志,2016,41(6):1135.
[25]曹慧婷,朱华旭,张启春,等.基于代谢组学研究黄连-黄芩药对治疗脑缺血大鼠的作用机制[J].中国中药杂志,2017,42(11):2159.
[26] Winkelman J W,Collins G H. Neurotoxicity of tetraphenylporphinesulfonate TPPS4 and its relation to photodynamic therapy[J]. Photochem Photobiol,2010,46(5):801.
[27] Storbeck K H,Swart P,Swart A C. Cytochrome P450 side-chain cleavage:insights gained from homology modeling[J]. Mol Cell Endocrinol,2007,265/266(4):65.
[2]陈斌,贾晓斌.三七的炮制研究进展与研究思路[J].中草药,2013,44(4):482.
[3]王蕊,贾波,余常,等.生、熟三七配伍规律与功效[J].实用中医内科杂志,2015(9):167.
[4]国家药品监督管理局.国家中成药标准汇编(内科气血津液分册)[M].北京:化学工业出版社,2002:359.
[5]钱宏梁,潘志强.中医血虚证及其动物模型制备方法评析[J].广州中医药大学学报,2018(1):176.
[6]黄波,李静,王小中,等.溶血性贫血大鼠模型的建立及实验评价[J].重庆医科大学学报,2010,35(5):703.
[7]陈斌,蔡涛,贾晓斌. UPLC同时测定蒸制三七中的10种人参皂苷类活性成分的含量[J].中国中药杂志,2014,39(9):1614.
[8]杨秀娟,邓毅,吴国霞,等.基于UPLC-Q-TOF/MS的归身和归尾补血机制的代谢组学初步研究[J].天然产物研究与开发,2018(2):176.
[9] Qiu S B,Zhang H X,Fei Q Q,et al. Urine and plasma metabolomics study on potential hepatoxic biomarkers identification in rats induced by Gynura segetum[J]. J Ethnopharmacol,2018,216:202.
[10]方晓艳,苗明三.当归补血汤粗多糖补气生血作用研究和组方合理性探讨[J].时珍国医国药,2010,21(1):93.
[11]邓毅,张明,张艳萍.甘肃当归对乙酰苯肼溶血性血虚小鼠模型的干预作用[J].西部中医药,2015,28(10):5.
[12] Zhang Z Z,Fan M L,Hao X,et al. Integrative drug efficacy assessment of Danggui and European Danggui using NMR-based metabolomics[J]. J Pharm Biomed Anal,2016,120:1.
[13] Campos-Ferraz P L,Bozza T,Nicastro H,et al. Distinct effects of leucine or a mixture of the branched-chain amino acids(leucine,isoleucine,and valine)supplementation on resistance to fatigue,and muscle and liver-glycogen degradation,in trained rats[J]. Nutrition,2013,29(11/12):1388.
[14]孙启慧,张静,李壮壮,等.基于代谢组学的麻黄细辛附子汤治疗流感小鼠的作用机制研究[J].中国中药杂志,2017,42(4):763.
[15]李灿,孙启慧,付业佩,等.基于粪便代谢物组学分析麻黄细辛附子汤干预H1N1流感病毒感染小鼠的作用机制[J].中国实验方剂学杂志,2017,23(9):74.
[16]魏文峰,褚衍涛,刘烨,等.基于超高效液相色谱-飞行时间质谱技术的肺炎支原体感染小鼠血清代谢组学研究[J].中国中药杂志,2017,42(7):178.
[17]亢泽峰,庄曾渊,万素君,等.血虚证大鼠视网膜病理改变的研究[J].中国中医基础医学杂志,2001,7(3):43.
[18]李达.烟酰胺与代谢综合征关系的研究[D].沈阳:中国医科大学,2013.
[19]王颖,邹丽红,侯丽辉,等.基于超高液相色谱-质谱分析代谢组学在芪苓温肾消囊方治疗痰湿型多囊卵巢综合征的应用[J].时珍国医国药,2018,29(7):1591.
[20]罗年翠.当归-白芍药对配伍效应与物质基础研究[D].南京:南京中医药大学,2013.
[21]周永峰,牛明,房吉祥,等.基于UPLC-Q-TOF-MS的五味子保肝作用代谢组学研究[J].中国中药杂志,2018,43(18):3756.
[22]曲婷丽,王二兵,李震宇,等.黄芪注射液乙酸乙酯萃取物对小鼠白细胞减少症作用的代谢组学[J].中成药,2017,39(3):455.
[23]郑惠婷,李克宁,陈驰,等.基于1H-NMR人参煎治疗2型糖尿病大鼠的胰腺代谢组学研究[J].中国中药杂志,2018,43(14):3012.
[24]张淑丽,王怡萍,杜振霞,等.基于UPC2-/TOF-MS技术的果糖诱导高尿酸血症大鼠血清脂质代谢组学研究[J].中国中药杂志,2016,41(6):1135.
[25]曹慧婷,朱华旭,张启春,等.基于代谢组学研究黄连-黄芩药对治疗脑缺血大鼠的作用机制[J].中国中药杂志,2017,42(11):2159.
[26] Winkelman J W,Collins G H. Neurotoxicity of tetraphenylporphinesulfonate TPPS4 and its relation to photodynamic therapy[J]. Photochem Photobiol,2010,46(5):801.
[27] Storbeck K H,Swart P,Swart A C. Cytochrome P450 side-chain cleavage:insights gained from homology modeling[J]. Mol Cell Endocrinol,2007,265/266(4):65.
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