利用LC-MS/MS技术分析蜂毒对大鼠内源性代谢的影响
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摘要
为了探究蜂毒对Sprague-Dawley(SD)大鼠机体内源性代谢的影响,采用LC-MS/MS技术分析了蜂毒对大鼠血清中代谢物及可能涉及的代谢通路的影响。选取年龄、性别、身体状况相同的SD大鼠,设置为蛰刺组(A组)、创伤组(B组)和对照组(C组)。对A、B两组大鼠分别进行蜜蜂蛰刺和针蛰刺处理,处理后10 min取血清进行LC-MS/MS分析。正负离子模式下,采用偏最小二乘判别分析(PLS-DA)方法进行数学建模,并验证其稳定性。结果表明,蜇刺组、创伤组、对照组的两两之间受影响代谢物种类及数量均有显著差异,其中71种物质与蜂蛰相关,39种物质与蜂毒相关,24种物质与创伤相关。经进一步筛选,共得到与蜂毒相关的9个比较主要差异代谢物,分别为肉毒碱、溶血磷酯酸、溶血磷脂酰胆碱、乙酰肉毒碱、白介素20、亚油酸、溶血磷酯酰乙醇胺、鞘氨醇、溶血磷脂酰丝氨酸;以及4个受影响代谢通路,包括亚油酸代谢、神经鞘脂类代谢、甘油磷脂代谢、丙氨酸和天门冬氨酸代谢。蛰刺、创伤和蜂毒均对大鼠的内源性代谢产生一定影响,其中,与创伤相比,蜂毒影响相对更大。根据受影响代谢通路的生理作用,推测蜂毒对机体内源性代谢的影响主要涉及脂肪酸的代谢过程,具有重要的生理学作用。
To explore the influence of bee venom on endogenous metabolism,this study used liquid chromatographymass spectrometry( LC-MS/MS) to analyze the effect of bee venom on the metabolites and possibly involved metabolism pathways in rats serum. Sprague Dawley( SD) rats with the same age,the same gender and the same physical condition were chosen and divided into 3 groups including bee sting group( group A),stabbing group( group B) and the control group( group C) at random. Rats in group A and B were single stung by honeybees and needle,respectively. 10 min after sting,serum specimens from these 3 groups would be analyzed by LC-MS/MS. In positive and negative ion mode,partial least squares-discriminate analysis was adopted to establish mathematical model,and their stability would be tested later. The results indicated that there were significant differences in types and quantities of influenced metabolites among different groups. The number of differential metabolite related to bee venom,sting and stabbing were 71,39 and 24,respectively. After further screening,9 potential biomarkers were found showing relation with bee venom,including carnitine,acetylcarnitine,Lyso PC,Lyso-PA,lyso-PS,lyso-PE,IL-20,linoleic,and sphinganine. And they were mainly involved in 4 metabolic pathways,including linoleic acidmetabolism,sphingolipids catabolism, phospholipid catabolism, Alanine and Aspartate anabolism. The results indicated that sting,stabbing and bee venom all influence endogenous metabolites of SD rats. Comparing with stabbing,bee venom showed relatively stronger effect. According to the physiological function of affected metabolic pathways,it was speculated that the effect of bee venom on endogenous metabolisms was mainly involved in the metabolic process of fatty-acid,and exploration about this process had significant meaning for physiology.
To explore the influence of bee venom on endogenous metabolism,this study used liquid chromatographymass spectrometry( LC-MS/MS) to analyze the effect of bee venom on the metabolites and possibly involved metabolism pathways in rats serum. Sprague Dawley( SD) rats with the same age,the same gender and the same physical condition were chosen and divided into 3 groups including bee sting group( group A),stabbing group( group B) and the control group( group C) at random. Rats in group A and B were single stung by honeybees and needle,respectively. 10 min after sting,serum specimens from these 3 groups would be analyzed by LC-MS/MS. In positive and negative ion mode,partial least squares-discriminate analysis was adopted to establish mathematical model,and their stability would be tested later. The results indicated that there were significant differences in types and quantities of influenced metabolites among different groups. The number of differential metabolite related to bee venom,sting and stabbing were 71,39 and 24,respectively. After further screening,9 potential biomarkers were found showing relation with bee venom,including carnitine,acetylcarnitine,Lyso PC,Lyso-PA,lyso-PS,lyso-PE,IL-20,linoleic,and sphinganine. And they were mainly involved in 4 metabolic pathways,including linoleic acidmetabolism,sphingolipids catabolism, phospholipid catabolism, Alanine and Aspartate anabolism. The results indicated that sting,stabbing and bee venom all influence endogenous metabolites of SD rats. Comparing with stabbing,bee venom showed relatively stronger effect. According to the physiological function of affected metabolic pathways,it was speculated that the effect of bee venom on endogenous metabolisms was mainly involved in the metabolic process of fatty-acid,and exploration about this process had significant meaning for physiology.
引文
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[13]Lopaschuk G D. Fatty acid oxidation and its relation with insulin resistance and associated disorders[J]. Ann. Nutr.Metab.,2016,68(Suppl. 3):15-20.
[14]Wang X Y,Luo J P,Chen R,et al.. Dendrobium huoshanense polysaccharide prevents ethanol-induced liver injury in mice by metabolomic analysis[J]. Int. J. Biol. Macromol.,2015,78:354-362.
[15]Badr G,Hozzein W N, Badr B M, et al.. Bee venom accelerates wound healing in diabetic mice by suppressing activating transcription factor-3(ATF-3)and inducible nitric oxide synthase(iN OS)-mediated oxidative stress and recruiting bone marrow-derived endothelial progenitor cells[J]. J. Cell.Physiol.,2016,231(10):2159-2171.
[16]钟珊,周智,赵有蓉,等.蜂毒致亚急性肝衰竭1例[J].中华肝脏病杂志,2005,13(11):827-827.Zhong S,Zhou Z,Zhao YR,et al.. A case of subacute liver failure resulted from bee venom[J]. Chin. J. Hepatol.,2001,58(3):477-484.
[17]邹香国,高雅丽,李红伟,等.血液透析、血液滤过抢救急性蜂毒中毒疗效比较[J].中国实用医刊,2007,34(13):72-72.Zou X G,Gao Y L,Li H W,et al.. Compare the curative effect of hemodialysis and hemofiltration to rescue acute bee venom[J].Central Plains Med. J.,2007,34(13):72-72.
[18]Yamamoto J, Omura M, Tuchiya K, et al.. Preferable existence of polyunsaturated lysophosphatidic acids in human follicular fluid from phosphatidic acidtients programmed with in vitro fertilization[J]. Prostag. Oth. Lipid M.,2016,126:16-23.
[19]杨林,夏敬彪,彭爱民,等.大剂量甲泼尼龙联合连续性血液净化治疗蜂毒所致多脏器功能障碍[J].中国急救医学,2007,27(11):1029-1032.Yang L, Xia J B, Peng A M, et al.. Large-dose methylprednisolone with continuous blood purification(CBP)for the treatment of multiple organ dysfunction syndrome(MODS)induced by bee venom[J]. Chin. J. Crit. Care Med.,2007,27(11):1029-1032.
[20]Hurali P B,Ghaisas M M. Evaluation of anti-inflammatory,anti-nociceptive,and anti-arthritic activities of Indian Apis dorsata bee venom in exphosphatidyl ethanolaminerimental animals:biochemical,histological,and radiological assessment[J]. Immunopharm. Immunot.,2015,37(2):171-184.
[21]Al-Tamimi J,Alhazza I M,Al-Khalifa M,et al.. Potential effects of samsum ant,Brachyponera sennaarensis,venom on TNF-α/NF-κB mediated inflammation in CCL4-toxicity in vivo[J]. Lipids Health Dis.,2016,15(1):198-209.
[22]Kang S Y,Roh D H,Choi J W,et al.. Repetitive treatment with diluted bee venom attenuates the induction of below-level neuropathic pain behaviors in a rat spinal cord injury model[J]. Toxins,2015,7(7):2571-2585.
[23]Cruz-Gonzalez I, Martín Moreiras J, García E. Thrombus formation after left atrial appendage exclusion using an amplatzer cardiac plug device[J]. Catheter. Cardio. Int.,2011,78(6):970-973.
[24]Pavkova G M,Nemec P,Lipkova J,et al.. Relation of IL-6,IL-13 and IL-15 gene polymorphisms to the rheumatoid factors,anti-CCP and other measures of rheumatoid arthritis activity[J]. Int. J. Immun.,2014,41(1):34-40.
[2]Nicholson J K,Wilson I D. Understanding “global”systems biology:metabonomics and the continuum of metabolism[J].Nat. Rev. Drug Discov.,2003,2(8):668-676
[3]骆建新,郑崛村,马用信,等.人类基因组计划与后基因组时代[J].中国生物工程杂志,2003,23(11):87-94.Luo J X,Zheng J C,Ma Y M,et al.. Human genome project and the post genome era[J]. Prog. Biotechnol.,2003,23(11):87-94.
[4]贾伟.医学代谢组学[M].上海:上海科学技术出版社,2011.Jia W. Medical metabonomics[M]. Shanghai:Shanghai Science and Technology Press,2011.
[5]高丽娇,吴杰.蜜蜂蜂毒主要成分与功能研究进展[J].基因组学与应用生物学,2013,32(2):246-253.Gao L J,Wu J. Advance on the main compositions and the functions of honeybee venom[J]. Genomics Appl. Biol.,2013,32(2):246-253.
[6]赵燕,张帆,曹灵红.蜂蜇伤蜂毒免疫治疗的研究进展[J].医学综述,2016,22(13):2582-2585.Zhao Y,Zhang F,Cao L H. Research progress of bee sting bee venom immunotherapy[J]. Med. Rev.,2016,22(13):2582-2585.
[7]Sanchonlo B, Everett J R. New methodology for known metabolite identification in metabonomics/metabolomics:Topological metabolite identification carbon efficiency(tM ICE)[J]. J. Proteome Res.,2016,15(9):3405-3419.
[8]李田乐.心功能不全合并糖尿病大鼠模型建立及代谢组学研究[D].天津:天津医科大学,硕士学位论文,2016.Li T L. Heart function insufficiency Tatara model with diabetes mellitus rats and metabolomics studies[D]. Tianjin:Medical University of Tianjin,Master Dissertation,2016.
[9]Wang X, Zhang A, Han Y, et al.. Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease[J]. Mol. Cell.Proteomics,2012,11(8):370-380.
[10]Liang Q, Liu H, Xing H, et al.. Urinary UPLC-MS metabolomics dissecting the underlying mechanisms of huaxian capsule protects against sepsis[J]. RSC Adv.,2016,6(46):40436-40441.
[11]Clark-Matott J,Saleem A,Dai Y,et al.. Metabolomic analysis of exercise effects in the POLG mitochondrial DNA mutator mouse brain[J]. Neurobiol. Aging,2015,36(11):2972-2983.
[12]刘军,查英,陈灶萍,等. 2型糖尿病患者丙氨酸氨基转移酶、天冬氨酸氨基转移酶水平与代谢紊乱、胰岛素抵抗之间的关系[J].临床内科杂志,2006,23(4):262-265.Liu J,Zha Y,Chen Z P,et al.. The relationship between serum Alanine aminotransferase, Aspartate aminotransferase and metabolic disorder,insulin resistance in type 2 diabetes[J]. J. Clin. Intern. Med.,2006,23(4):262-265.
[13]Lopaschuk G D. Fatty acid oxidation and its relation with insulin resistance and associated disorders[J]. Ann. Nutr.Metab.,2016,68(Suppl. 3):15-20.
[14]Wang X Y,Luo J P,Chen R,et al.. Dendrobium huoshanense polysaccharide prevents ethanol-induced liver injury in mice by metabolomic analysis[J]. Int. J. Biol. Macromol.,2015,78:354-362.
[15]Badr G,Hozzein W N, Badr B M, et al.. Bee venom accelerates wound healing in diabetic mice by suppressing activating transcription factor-3(ATF-3)and inducible nitric oxide synthase(iN OS)-mediated oxidative stress and recruiting bone marrow-derived endothelial progenitor cells[J]. J. Cell.Physiol.,2016,231(10):2159-2171.
[16]钟珊,周智,赵有蓉,等.蜂毒致亚急性肝衰竭1例[J].中华肝脏病杂志,2005,13(11):827-827.Zhong S,Zhou Z,Zhao YR,et al.. A case of subacute liver failure resulted from bee venom[J]. Chin. J. Hepatol.,2001,58(3):477-484.
[17]邹香国,高雅丽,李红伟,等.血液透析、血液滤过抢救急性蜂毒中毒疗效比较[J].中国实用医刊,2007,34(13):72-72.Zou X G,Gao Y L,Li H W,et al.. Compare the curative effect of hemodialysis and hemofiltration to rescue acute bee venom[J].Central Plains Med. J.,2007,34(13):72-72.
[18]Yamamoto J, Omura M, Tuchiya K, et al.. Preferable existence of polyunsaturated lysophosphatidic acids in human follicular fluid from phosphatidic acidtients programmed with in vitro fertilization[J]. Prostag. Oth. Lipid M.,2016,126:16-23.
[19]杨林,夏敬彪,彭爱民,等.大剂量甲泼尼龙联合连续性血液净化治疗蜂毒所致多脏器功能障碍[J].中国急救医学,2007,27(11):1029-1032.Yang L, Xia J B, Peng A M, et al.. Large-dose methylprednisolone with continuous blood purification(CBP)for the treatment of multiple organ dysfunction syndrome(MODS)induced by bee venom[J]. Chin. J. Crit. Care Med.,2007,27(11):1029-1032.
[20]Hurali P B,Ghaisas M M. Evaluation of anti-inflammatory,anti-nociceptive,and anti-arthritic activities of Indian Apis dorsata bee venom in exphosphatidyl ethanolaminerimental animals:biochemical,histological,and radiological assessment[J]. Immunopharm. Immunot.,2015,37(2):171-184.
[21]Al-Tamimi J,Alhazza I M,Al-Khalifa M,et al.. Potential effects of samsum ant,Brachyponera sennaarensis,venom on TNF-α/NF-κB mediated inflammation in CCL4-toxicity in vivo[J]. Lipids Health Dis.,2016,15(1):198-209.
[22]Kang S Y,Roh D H,Choi J W,et al.. Repetitive treatment with diluted bee venom attenuates the induction of below-level neuropathic pain behaviors in a rat spinal cord injury model[J]. Toxins,2015,7(7):2571-2585.
[23]Cruz-Gonzalez I, Martín Moreiras J, García E. Thrombus formation after left atrial appendage exclusion using an amplatzer cardiac plug device[J]. Catheter. Cardio. Int.,2011,78(6):970-973.
[24]Pavkova G M,Nemec P,Lipkova J,et al.. Relation of IL-6,IL-13 and IL-15 gene polymorphisms to the rheumatoid factors,anti-CCP and other measures of rheumatoid arthritis activity[J]. Int. J. Immun.,2014,41(1):34-40.