达菲干预肾阳虚外感小鼠的血清代谢组学研究
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摘要
目的:通过血清代谢组学的方法,采用LC-MS技术,在正负离子模式下,研究达菲对肾阳虚外感小鼠的干预作用,初步探索达菲在流感治疗过程中调控的代谢通路。方法:36只昆明小鼠随机分为正常组、模型组、达菲组,腹腔注射苯甲酸雌二醇方法复制肾阳虚小鼠模型,然后通过滴鼻感染方法建立病证结合的肾阳虚小鼠外感模型,达菲组以9.75 mg·kg~(-1)·d~(-1)剂量给药干预,每天1次,连续6 d,其他组给予等量的生理盐水;末次给药24 h后取小鼠血清,并进行原始数据采集和相关统计学分析。结果:达菲组小鼠体质量、肛温相对于模型组均有回升趋势;血清样品经过数据分析,最终鉴定了22个存在显著性差异的生物标志物,正离子模式下8个,负离子模式下14个,包括氨基酸类、甘油磷脂类、激素类以及花生四烯酸等物质,以上物质经达菲干预后均出现不同程度的回调。结论:达菲对肾阳虚外感小鼠具有一定干预作用,在干预过程中对甘油磷脂代谢、视黄醇代谢、能量代谢等途径具有一定的调控作用。
Objective:To study the intervention effect of tamiflu on kidney-yang deficiency syndrome mouse model infected with influenza virus A by LC-MS technique in positive and negative ion mode based on serum metabolomics study and explore the metabolic pathways regulated by tamiflu in the course of influenza treatment.Methods:36 Kunming mice were randomly divided into normal group,model group,and tamiflu group.The kidney-yang deficiency syndrome mouse model was established by intraperitoneal injection of estradiol benzoate.The disease-associated kidney-yang deficiency syndrome mouse model was established by nasal infection with influenza virus A.The tamiflu group were intervented with a dose of 9.75 mg·kg~(-1)·d~(-1),once a day for 6 days,other two groups were given the same amount of saline.Serum samples were collected from each mouse of the three groups 24 h after the last administration,and the LC-MS original data were collected and statistical analysis were performed.Results:Compared with the model group,the body weights and rectal temperature of the tamiflu group tended to increase.Twenty-two biomarkers with significant difference were identified in the serum samples.Eight of them were in positive ion mode and fourteen were in negative ion mode.Levels of the substances such as amino acids,glycerophospholipid,hormones and arachidonic acid were regulated with tamiflu intervention.Conclusion:Tamiflu has an intervention effect on kidney-yang deficiency syndrome mouse model infected with influenza virus A and has regulatory effect on glycerophospholipid metabolism,retinol metabolism,energy metabolism during the intervention.
Objective:To study the intervention effect of tamiflu on kidney-yang deficiency syndrome mouse model infected with influenza virus A by LC-MS technique in positive and negative ion mode based on serum metabolomics study and explore the metabolic pathways regulated by tamiflu in the course of influenza treatment.Methods:36 Kunming mice were randomly divided into normal group,model group,and tamiflu group.The kidney-yang deficiency syndrome mouse model was established by intraperitoneal injection of estradiol benzoate.The disease-associated kidney-yang deficiency syndrome mouse model was established by nasal infection with influenza virus A.The tamiflu group were intervented with a dose of 9.75 mg·kg~(-1)·d~(-1),once a day for 6 days,other two groups were given the same amount of saline.Serum samples were collected from each mouse of the three groups 24 h after the last administration,and the LC-MS original data were collected and statistical analysis were performed.Results:Compared with the model group,the body weights and rectal temperature of the tamiflu group tended to increase.Twenty-two biomarkers with significant difference were identified in the serum samples.Eight of them were in positive ion mode and fourteen were in negative ion mode.Levels of the substances such as amino acids,glycerophospholipid,hormones and arachidonic acid were regulated with tamiflu intervention.Conclusion:Tamiflu has an intervention effect on kidney-yang deficiency syndrome mouse model infected with influenza virus A and has regulatory effect on glycerophospholipid metabolism,retinol metabolism,energy metabolism during the intervention.
引文
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[12]詹同运,石磊,赵树进,等.HPLC-MS/MS法检测人血浆中游离花生四烯酸浓度[J].药物分析杂志,2012,32(3):388ZHAN TY,SHI L,ZHAO SJ,et al.HPLC-MS/MS determination of arachidonic acid in human plasma[J].Chin J Pharm Anal,2012,32(3):388
[13]侯衍豹.麻黄细辛附子汤对肾阳虚外感病证的综合药效评价及调控TLR3-TRIF-NF-κB信号通路的作用[D].济南:山东中医药大学,2016HOU YB.The Comprehensive Evaluation on the Efficacy Kidneyyang Deficiency Mice Model Infected with Influenza Virus and The Intervention Effect on TLR3-TRIF-NF-κB Signaling Pathway of Mahuang Xixin Fuzi Decoction[D].Jinan:Shandong University of Traditional Chinese Medicine,2016
[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):763SUN QH,ZHANG J,LI ZZ,et al.Action mechanism of Mahuang Xixin Fuzi decoction for mice with influenza based on metabolomics information[J].China J Chin Mater Med,2017,42(4):763
[15]MEDEIROS SR,PINHEIRO-ROSA N,LEMOS L,et al.Vitamin A supplementation leads to increases in regulatory CD4+Foxp3+LAP+T cells in mice[J].Nutrition,2015,31(10):1260
[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):1382WEI WF,CHU YT,LIU Y,et al.Serum metabonomics in mice infected with mycoplasma pneumoniae by UPLC-Q-TOF-MS[J].China J Chin Mater Med,2017,42(7):1382
[17]徐海涛.维生素A对感染病毒雄鼠繁殖性能和免疫功能的保护效应[D].成都:四川农业大学,2008XU HT.Protective Effect of Vitamin A on Reproductive Performance and Immune Function of Infected Male Mice[D].Chengdu:Sichuan Agricultural University,2008
[18]孙红暖,杨海明,王志跃,等.精氨酸对动物的营养生理及免疫作用[J].动物营养学报,2014,26(1):54SUN HN,YANG HM,WANG ZY,et al.Nutrition physiological and immune functions of arginine in animals[J].Chin J Anim Nutr,2014,26(1):54
[19]杜丽娜,单进军,谢彤,等.基于液相质谱联用的黄芩水提液抗病毒效应的脂质组学研究[J].中华中医药杂志,2015,30(5):1728DU LN,SHAN JJ,XIE T,et al.Lipidomics study on antiviral effects of baical skullcap root extraction based on liquid chromatographymass spectrography[J].Chin J Tradit Chin Med Pharm,2015,30(5):1728
[20]杜丽娜.基于UPLC-MS技术的呼吸道合胞病毒肺炎痰热闭肺证代谢模式及金欣口服液干预研究[D].南京:南京中医药大学,2015DU LN.Metabolic Pattern of Respiratory Syncytial Virus Pneumonia and Heat-closed Lung Syndrome Based on UPLC-MS Technique and Intervention of Jinxin Oral Liquid[D].Nanjing:Nanjing University of Chinese Medicine,2015
[21]谢彤,杜丽娜,徐建亚,等.基于脂质代谢网络的虎杖抗呼吸道合胞病毒肺炎代谢组学研究[J].世界中医药,2016,11(9):1670XIE T,DU LN,XU JY,et al.Metabonomics study on the effect of Polygonum cuspidatum on anti-respiratory syncytial virus(RSV)basing on lipid metabolism[J].World Chin Med,2016,11(9):1670
[2]符晓四,姚学峰.达菲药物对H1N1病毒作用研究[J].生物信息学,2010,8(4):371FU XS,YAO XF.Effect of tamiflu for curing influenza of H1N1[J].Bioinformatics,2010,8(4):371
[3]窦爱华,李侗曾,刘远志,等.达菲及中药治疗甲型H1N1流感的疗效观察[J].现代中西医结合杂志,2010,19(26):3289DOU AH,LI TZ,LIU YZ,et al.Curative effect observation of tamiflu and traditional Chinese medicine on type A H1N1 influenza[J].Mod J Integr Tradit Chin West Med,2010,19(26):3289
[4]NICHOLSON JK,LINDON JC,HOLMES E.“Metabonomics”:understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data[J].Xenobiotica,1999,29(11):1181
[5]NICHOLSON JK,CONNELLY J,LINDON JC,et al.Metabonomics:a platform for studying drug toxicity and gene function[J].Nat Rev Drug Discov,2002,1(2):153
[6]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):213GUO H,CUI Y,WANG QH,et al.Overview on application of metabonomics in research on Chinese medicine compound[J].Chin J Exp Tradit Med Form,2017,23(1):213
[7]吕圭源,苏洁,陈素红,等.相同性味不同归经中药对雌二醇致肾阳虚小鼠的影响[J].上海中医药杂志,2011,45(10):70LU GY,SU J,CHEN SH,et al.Effects of herbs of same nature and flavor but different meridian tropism on estradiol-induced kidneyyang deficiency in mice[J].Shanghai J Tradit Chin Med,2011,45(10):70
[8]郭元吉,程小雯.流行性感冒病毒及其实验技术[M].北京:中国三峡出版社,1997:249GUO YJ,CHENG XW.Influenza Virus and Its Experimental Techniques[M].Beijing:China Three Gorges Publishing House,1997:249
[9]孙启慧,相美容,王朋展,等.麻黄细辛附子汤对正常小鼠外感及肾阳虚模型小鼠外感影响的比较[J].中国中医急症,2015,24(12):2 072SUN QH,XIANG MR,WANG PZ,et al.Intervention comparison of Mahuang Xixin Fuzi decoction on influenza virus in normal mice and kidney-yang deficiency mice model[J].J Emerg Tradit Chin Med,2015,24(12):2072
[10]蒋海强,聂磊,李运伦,等.清热降压胶囊干预SHR海马区组织代谢组学研究[J].中国中药杂志,2014,39(1):134JIANG HQ,NIE L,LI YL,et al.Effect of qingrejiangya capsule on histologic metabolomics of SHR hippocampus[J].China J Chin Mater Med,2014,39(1):134
[11]王一红,冯家力,潘振球,等.液相色谱-质谱/质谱联用技术分析18种游离氨基酸[J].中国卫生检验杂志,2006,16(2):161WANG YH,FENG JL,PAN ZQ,et al.The analysis of 18 free amino acids by liquid chromatography-mass spectrometry/mass spectrometry[J].Chin J Health Lab Technol,2006,16(2):161
[12]詹同运,石磊,赵树进,等.HPLC-MS/MS法检测人血浆中游离花生四烯酸浓度[J].药物分析杂志,2012,32(3):388ZHAN TY,SHI L,ZHAO SJ,et al.HPLC-MS/MS determination of arachidonic acid in human plasma[J].Chin J Pharm Anal,2012,32(3):388
[13]侯衍豹.麻黄细辛附子汤对肾阳虚外感病证的综合药效评价及调控TLR3-TRIF-NF-κB信号通路的作用[D].济南:山东中医药大学,2016HOU YB.The Comprehensive Evaluation on the Efficacy Kidneyyang Deficiency Mice Model Infected with Influenza Virus and The Intervention Effect on TLR3-TRIF-NF-κB Signaling Pathway of Mahuang Xixin Fuzi Decoction[D].Jinan:Shandong University of Traditional Chinese Medicine,2016
[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):763SUN QH,ZHANG J,LI ZZ,et al.Action mechanism of Mahuang Xixin Fuzi decoction for mice with influenza based on metabolomics information[J].China J Chin Mater Med,2017,42(4):763
[15]MEDEIROS SR,PINHEIRO-ROSA N,LEMOS L,et al.Vitamin A supplementation leads to increases in regulatory CD4+Foxp3+LAP+T cells in mice[J].Nutrition,2015,31(10):1260
[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):1382WEI WF,CHU YT,LIU Y,et al.Serum metabonomics in mice infected with mycoplasma pneumoniae by UPLC-Q-TOF-MS[J].China J Chin Mater Med,2017,42(7):1382
[17]徐海涛.维生素A对感染病毒雄鼠繁殖性能和免疫功能的保护效应[D].成都:四川农业大学,2008XU HT.Protective Effect of Vitamin A on Reproductive Performance and Immune Function of Infected Male Mice[D].Chengdu:Sichuan Agricultural University,2008
[18]孙红暖,杨海明,王志跃,等.精氨酸对动物的营养生理及免疫作用[J].动物营养学报,2014,26(1):54SUN HN,YANG HM,WANG ZY,et al.Nutrition physiological and immune functions of arginine in animals[J].Chin J Anim Nutr,2014,26(1):54
[19]杜丽娜,单进军,谢彤,等.基于液相质谱联用的黄芩水提液抗病毒效应的脂质组学研究[J].中华中医药杂志,2015,30(5):1728DU LN,SHAN JJ,XIE T,et al.Lipidomics study on antiviral effects of baical skullcap root extraction based on liquid chromatographymass spectrography[J].Chin J Tradit Chin Med Pharm,2015,30(5):1728
[20]杜丽娜.基于UPLC-MS技术的呼吸道合胞病毒肺炎痰热闭肺证代谢模式及金欣口服液干预研究[D].南京:南京中医药大学,2015DU LN.Metabolic Pattern of Respiratory Syncytial Virus Pneumonia and Heat-closed Lung Syndrome Based on UPLC-MS Technique and Intervention of Jinxin Oral Liquid[D].Nanjing:Nanjing University of Chinese Medicine,2015
[21]谢彤,杜丽娜,徐建亚,等.基于脂质代谢网络的虎杖抗呼吸道合胞病毒肺炎代谢组学研究[J].世界中医药,2016,11(9):1670XIE T,DU LN,XU JY,et al.Metabonomics study on the effect of Polygonum cuspidatum on anti-respiratory syncytial virus(RSV)basing on lipid metabolism[J].World Chin Med,2016,11(9):1670