基于代谢组学研究苦荞蛋白对高脂血症小鼠的干预作用
|
摘要
本实验通过高脂膳食诱导建立高脂血症小鼠模型,利用超高效液相色谱电喷雾电离串联离子阱飞行时间质谱技术获得正常对照小鼠、高脂膳食小鼠和苦荞蛋白干预小鼠血浆样品的代谢指纹图谱,通过模式判别方法结合代谢组网络数据库,初步确定了苦荞蛋白饮食干预高脂血症小鼠潜在的生物标志物及作用通路,从代谢组学角度探究了高脂血症小鼠被干预的代谢通路和苦荞蛋白发挥降血脂功效的作用机制。实验结果表明:高脂血症模型小鼠血浆中存在亚油酸、胞苷二磷酸甘油、磷脂酰乙醇胺、半乳糖神经酰胺、脑苷脂、葡糖神经酰胺、磷脂酰胆碱、葡糖苷酸、前列腺素G2、溶血磷脂酸、溶血磷脂酰胆碱、鞘磷脂12种潜在的生物标志物,苦荞蛋白可通过调节高脂血症小鼠体内12种潜在的生物标志物所对应的6条代谢通路(亚油酸代谢、甘油磷脂代谢、鞘脂类代谢、戊糖-葡萄糖醛酸酯相互转化作用、色氨酸代谢和花生四烯酸代谢),使其向正常状态转变,从而缓解高脂血症小鼠代谢紊乱的症状。
The present study was carried out to investigate the effect of tartary buckwheat protein(TBP) on metabolic pathways in hyperlipidemic mice and to explore its lipid-lowering mechanism from a metabonomic perspective. A hyperlipidemic mouse model was established by feeding mice a high-fat diet. Plasma metabolic ?ngerprinting was performed for normal, hyperlipidemic and TBP-treated mice using ultra-high performance liquid chromatography coupled with electrospray ionization tandem ion trap time-of-?ight mass spectrometry(UPLC/ESI-IT-TOF/MS). The potential biomarkers for the intervention effect of TBP on hyperlipidemic mice and the related metabolic pathways were determined by partial least squares-discriminant analysis(PLS-DA) based on the metabolic network database. The results showed the presence of linoleic acid, cytidine diphosphate glycerol, phosphatidyl ethanolamine, galactosylceramide, cerebroside, glucosylceramide,phosphatidylcholine, glucuronide, prostaglandin G2, lysophosphatidic acid, lysophosphatidyl choline and sphingomyelin as12 potential biomarkers in the plasma of hyperlipidemic mice. Tartary buckwheat protein could regulate 6 metabolic pathways(linoleic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism and pentose-glucuronic acid ester conversion,tryptophan metabolism and four arachidonic acid metabolism) in which 12 potential biomarkers are involved in hyperlipidemic mice, and restore them to normal, thereby ameliorating metabolic disorders in hyperlipidemic mice.
The present study was carried out to investigate the effect of tartary buckwheat protein(TBP) on metabolic pathways in hyperlipidemic mice and to explore its lipid-lowering mechanism from a metabonomic perspective. A hyperlipidemic mouse model was established by feeding mice a high-fat diet. Plasma metabolic ?ngerprinting was performed for normal, hyperlipidemic and TBP-treated mice using ultra-high performance liquid chromatography coupled with electrospray ionization tandem ion trap time-of-?ight mass spectrometry(UPLC/ESI-IT-TOF/MS). The potential biomarkers for the intervention effect of TBP on hyperlipidemic mice and the related metabolic pathways were determined by partial least squares-discriminant analysis(PLS-DA) based on the metabolic network database. The results showed the presence of linoleic acid, cytidine diphosphate glycerol, phosphatidyl ethanolamine, galactosylceramide, cerebroside, glucosylceramide,phosphatidylcholine, glucuronide, prostaglandin G2, lysophosphatidic acid, lysophosphatidyl choline and sphingomyelin as12 potential biomarkers in the plasma of hyperlipidemic mice. Tartary buckwheat protein could regulate 6 metabolic pathways(linoleic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism and pentose-glucuronic acid ester conversion,tryptophan metabolism and four arachidonic acid metabolism) in which 12 potential biomarkers are involved in hyperlipidemic mice, and restore them to normal, thereby ameliorating metabolic disorders in hyperlipidemic mice.
引文
[1]解春艳,李丛胜,路艳霞,等.苦杏仁蛋白提取条件优化及加工特性研究[J].食品科技,2015,40(2):294-298.DOI:10.13684/j.cnki.spkj.2015.02.060.
[2]孙青山,王常青,张生万,等.小米油复合饮料降血脂作用的实验研究[J].食品工业科技,2016,37(17):332-334;339.DOI:10.13386/j.issn1002-0306.2016.17.056.
[3]李笑蕊.8种代表性燕麦品种降血脂、抗氧化作用的评价研究[D].天津:天津科技大学,2016:4-20.
[4]SHEN Kuoping,HAO Chilong,YEN H W,et al.Pre-germinated brown rice prevented high fat diet induced hyperlipidemia through ameliorating lipid synthesis and metabolism in C57BL/6J mice[J].Journal of Clinical Biochemistry&Nutrition,2016,59(1):39-44.DOI:10.3164/jcbn.15-117.
[5]聂薇,李再贵.苦荞麦营养成分和保健功能[J].粮油食品科技,2016,24(1):40-45.DOI:10.16210/j.cnki.1007-7561.2016.01.010.
[6]熊德珍,毛金祥.浅析我国苦荞资源的前景[J].农业科技与信息,2016(11):21.DOI:10.15979/j.cnki.cn62-1057/s.2016.11.012.
[7]TOMOTAKE H,SHIMAOKA I,KAYASHITA J,et al.Physicochemical and functional properties of buckwheat protein product[J].Journal of Agricultural and Food Chemistry,2002,50(7):2125-2129.DOI:10.1021/jf011248q.
[8]JAVORNIK B,EGGUM B,KREFT I.Studies on protein fractions and protein quality of buckwheat[J].Genetika,1981,13:115-121.DOI:10.1021/jf011248q.
[9]田斌.荞麦蛋白酶解产物及其体外抗氧化研究[D].西安:陕西科技大学,2008:9-11.
[10]王世霞.甜荞和苦荞降血脂及抗氧化作用的差异化研究[D].天津:天津科技大学,2016:6-8.
[11]KAYASHITA J,SHIMAOKA I,NAKAJYOH M.Hypocholesterolemic effect of buckwheat protein extract in rats fed cholesterol enriched diets[J].Nutrition Research,1995,15(5):691-698.DOI:10.1016/0271-5317(95)00036-I.
[12]张美莉,赵广华,胡小松.荞麦蛋白和类黄酮提取物清除自由基的ESR研究[J].营养学报,2005,27(1):21-24.DOI:10.13325/j.cnki.acta.nutr.sin.2005.01.007.
[13]KAEWKON W,AONSRI C,TIYABOONCHAI W,et al.Sericin consumption suppresses development and progression of colon tumorigenesis in 1,2-dimethylhydrazine-treated rats[J].Biologia,2012,67(5):1007-1012.DOI:10.2478/s11756-012-0093-y.
[14]NICHOLSON J K,LINDON J C,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):1181-1189.DOI:10.1080/004982599238047.
[15]FIEHN O,KOPKA J,D?RMANN P,et al.Metabolite profiling for plant functional genomics[J].Nature Biotechnology,2000,18(11):1157-1161.DOI:10.1038/81137.
[16]罗朵生,李坤平,朴胜华,等.高脂血症大鼠病程进展的尿液代谢组学研究[J].广东药学院学报,2016,32(2):223-227.DOI:10.16809/j.cnki.1006-8783.2016012001.
[17]胡传芹,张雨,王静.代谢组学在个性化功能性食品研究中的应用[J].食品工业科技,2017,38(1):386-390.DOI:10.13386/j.issn1002-0306.2017.01.069.
[18]ROWLAND A,MINERS J O,MACKENZIE P I.The UDP-glucuronosyltransferases:their role in drug metabolism and detoxification[J].International Journal of Biochemistry&Cell Biology,2013,45(6):1121-1132.DOI:10.1016/j.biocel.2013.02.019.
[19]缪璐欢,杜静芳,白凤翎,等.代谢组学在发酵食品有毒代谢产物分析中的研究进展[J].食品工业科技,2016,37(5):388-393.DOI:10.13386/j.issn1002-0306.2016.05.071.
[20]任向楠,梁琼麟.基于质谱分析的代谢组学研究进展[J].分析测试学报,2017,36(2):161-169.DOI:10.3969/j.issn.1004-4957.2017.02.002.
[21]马红.基于代谢组学技术的新疆哈萨克族食管鳞癌血浆特征代谢物及临床意义研究[D].乌鲁木齐:新疆医科大学,2014:22-23.
[22]申国安,段礼新,漆小泉.植物代谢组学数据分析和数据库[J].生命科学,2015,27(8):995-999.DOI:10.13376/j.cbls/2015138.
[23]贾金萍,张福生,李震宇,等.基于1H-NMR代谢组学技术研究“杀青”对连翘化学成分的影响[J].中草药,2016,47(8):1375-1381.DOI:10.7501/j.issn.0253-2670.2016.08.022.
[24]赵小梅,刘歆颖,续畅,等.基于LC-MS代谢组学的雷公藤多苷致肝毒性生物标志物的初步筛查[J].中国中药杂志,2015,40(19):3851-3858.DOI:10.4268/cjcmm20151927.
[25]王昕泰,杜丽娜,汪受传.基于代谢组学的小儿腺病毒肺炎痰热闭肺证生物标志物研究思路[J].世界科学技术:中医药现代化,2015,17(10):2177-2180.DOI:10.11842/wst.2015.12.040.
[26]董宇,俞忠明,李洪玉,等.基于超高效液相色谱-高分辨质谱联用技术的桑黄干预后大鼠尿液代谢组学分析[J].色谱,2016,34(8):811-816.DOI:10.3724/SP.J.1123.2016.04009.
[27]李慧,刘其南,高叶,等.二至丸干预大鼠急性酒精肝损伤的尿液代谢组学研究[J].中国医院药学杂志,2016,36(19):1665-1670.DOI:10.13286/j.cnki.chinhosppharmacyj.2016.19.12.
[28]闫翠环,王亚利,王鑫国,等.卫气虚相关血浆代谢标志物的季节性变化研究[J].中国中医药信息杂志,2017,24(1):66-70.DOI:10.3969/j.issn.1005-5304.2017.01.017.
[29]ZHU L L,GE G B,LIU Y,et al.Characterization of UDP-glucuronosyltransferases involved in glucuron i d a t i o n o f diethylstilbestrol in human liver and intestine[J].Chemical Research in Toxicology,2012,25(12):2663-2669.DOI:10.1021/tx300310k.
[30]LI X L,CHEN Y N,LIU J P,et al.Serum metabolic variables associated with impaired glucose tolerance induced by high-fathigh-cholesterol diet in Macaca mulatta[J].Experimental Biology&Medicine,2012,237(11):1310-1321.DOI:10.1258/ebm.2012.012157.
[31]周传波.多烯磷脂酰胆碱对酒精性脂肪肝患者的临床疗效研究[J].国际医药卫生导报,2016,22(4):532-534.DOI:10.3760/cma.j.issn.1007-1245.2016.04.030.
[32]SCHERTZER J W,BROWN E D.Use of CDP-glycerol as an alternate acceptor for the teichoic acid polymerase reveals that membrane association regulates polymer length[J].Journal of Bacteriology,2008,190(21):6940-6947.DOI:10.1128/JB.00851-08.
[33]JIN Shuna,SONG Chengwu,LI Sen,et al.Preventive effects of turmeric on the high-fat diet-induced hyperlipidaemia in mice associated with a targeted metabolomic approach for the analysis of serum lysophosphatidylcholine using LC-MS/MS[J].Journal of Functional Foods,2014,11:130-141.DOI:10.1016/j.jff.2014.09.016.
[34]LI Sen,JIN Shuna,SONG Chengwu,et al.The metabolic change of serum lysophosphatidylcholines involved in the lipid lowering effect of triterpenes from Alismatis rhizoma on high-fat diet induced hyperlipidemia mice[J].Journal of Ethnopharmacology,2016,177:10-18.DOI:10.1016/j.jep.2015.11.017.
[35]ZHAO Y Y,MIAO H,CHENG X L,et al.Lipidomics:novel insight into the biochemical mechanism of lipid metabolism and dysregulation-associated disease[J].Chemico-Biological Interactions,2015,240:220-238.DOI:10.1016/j.cbi.2015.09.005.
[36]OBACH R S.Pharmacologically active drug metabolites:impact on drug discovery and pharmacotherapy[J].Pharmacological Reviews,2013,65(2):578-640.DOI:10.1124/pr.111.005439.
[37]金姝娜.基于组合LC-MS/MS技术的中药姜黄调节脂代谢紊乱活性及其成分姜黄素类化合物的全面表征和分离[D].武汉:华中科技大学,2015:32-34.
[38]CHOI S,SNIDER A J.Sphingolipids in high fat diet and obesityrelated diseases[J].Mediators of Inflammation,2015,2015:1-12.DOI:10.1155/2015/520618.
[39]易吉平,曾明,何兴轩.鞘磷脂类信号通路在肺纤维化发病机制中的作用[J].中国药理学与毒理学杂志,2016,30(2):158-164.DOI:10.3867/j.issn.1000-3002.2016.02.011.
[40]王少媛,张金兰,张丹,等.鞘脂在肝脏疾病中的研究进展[J].药学学报,2015,50(12):1551-1558.DOI:10.16438/j.0513-4870.2015.12.014.
[41]WORGALL T S.Lipid metabolism in cystic fibrosis[J].Current Opinion in Clinical Nutrition and Metabolic Care,2009,12(2):105-109.DOI:10.1097/MCO.0b013e32832595b7.
[42]SCHMITZ G,ORSóE.Intracellular cholesterol and phospholipid trafficking:comparable mechanisms in macrophages and neuronal cells[J].Neurochemical Research,2001,26(8/9):1045-1068.DOI:10.1023/A:1012357106398.
[43]DUMAS M E,BARTON R H,TOYE A,et al.Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(33):12511-12516.DOI:10.1073/pnas.0601056103.
[2]孙青山,王常青,张生万,等.小米油复合饮料降血脂作用的实验研究[J].食品工业科技,2016,37(17):332-334;339.DOI:10.13386/j.issn1002-0306.2016.17.056.
[3]李笑蕊.8种代表性燕麦品种降血脂、抗氧化作用的评价研究[D].天津:天津科技大学,2016:4-20.
[4]SHEN Kuoping,HAO Chilong,YEN H W,et al.Pre-germinated brown rice prevented high fat diet induced hyperlipidemia through ameliorating lipid synthesis and metabolism in C57BL/6J mice[J].Journal of Clinical Biochemistry&Nutrition,2016,59(1):39-44.DOI:10.3164/jcbn.15-117.
[5]聂薇,李再贵.苦荞麦营养成分和保健功能[J].粮油食品科技,2016,24(1):40-45.DOI:10.16210/j.cnki.1007-7561.2016.01.010.
[6]熊德珍,毛金祥.浅析我国苦荞资源的前景[J].农业科技与信息,2016(11):21.DOI:10.15979/j.cnki.cn62-1057/s.2016.11.012.
[7]TOMOTAKE H,SHIMAOKA I,KAYASHITA J,et al.Physicochemical and functional properties of buckwheat protein product[J].Journal of Agricultural and Food Chemistry,2002,50(7):2125-2129.DOI:10.1021/jf011248q.
[8]JAVORNIK B,EGGUM B,KREFT I.Studies on protein fractions and protein quality of buckwheat[J].Genetika,1981,13:115-121.DOI:10.1021/jf011248q.
[9]田斌.荞麦蛋白酶解产物及其体外抗氧化研究[D].西安:陕西科技大学,2008:9-11.
[10]王世霞.甜荞和苦荞降血脂及抗氧化作用的差异化研究[D].天津:天津科技大学,2016:6-8.
[11]KAYASHITA J,SHIMAOKA I,NAKAJYOH M.Hypocholesterolemic effect of buckwheat protein extract in rats fed cholesterol enriched diets[J].Nutrition Research,1995,15(5):691-698.DOI:10.1016/0271-5317(95)00036-I.
[12]张美莉,赵广华,胡小松.荞麦蛋白和类黄酮提取物清除自由基的ESR研究[J].营养学报,2005,27(1):21-24.DOI:10.13325/j.cnki.acta.nutr.sin.2005.01.007.
[13]KAEWKON W,AONSRI C,TIYABOONCHAI W,et al.Sericin consumption suppresses development and progression of colon tumorigenesis in 1,2-dimethylhydrazine-treated rats[J].Biologia,2012,67(5):1007-1012.DOI:10.2478/s11756-012-0093-y.
[14]NICHOLSON J K,LINDON J C,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):1181-1189.DOI:10.1080/004982599238047.
[15]FIEHN O,KOPKA J,D?RMANN P,et al.Metabolite profiling for plant functional genomics[J].Nature Biotechnology,2000,18(11):1157-1161.DOI:10.1038/81137.
[16]罗朵生,李坤平,朴胜华,等.高脂血症大鼠病程进展的尿液代谢组学研究[J].广东药学院学报,2016,32(2):223-227.DOI:10.16809/j.cnki.1006-8783.2016012001.
[17]胡传芹,张雨,王静.代谢组学在个性化功能性食品研究中的应用[J].食品工业科技,2017,38(1):386-390.DOI:10.13386/j.issn1002-0306.2017.01.069.
[18]ROWLAND A,MINERS J O,MACKENZIE P I.The UDP-glucuronosyltransferases:their role in drug metabolism and detoxification[J].International Journal of Biochemistry&Cell Biology,2013,45(6):1121-1132.DOI:10.1016/j.biocel.2013.02.019.
[19]缪璐欢,杜静芳,白凤翎,等.代谢组学在发酵食品有毒代谢产物分析中的研究进展[J].食品工业科技,2016,37(5):388-393.DOI:10.13386/j.issn1002-0306.2016.05.071.
[20]任向楠,梁琼麟.基于质谱分析的代谢组学研究进展[J].分析测试学报,2017,36(2):161-169.DOI:10.3969/j.issn.1004-4957.2017.02.002.
[21]马红.基于代谢组学技术的新疆哈萨克族食管鳞癌血浆特征代谢物及临床意义研究[D].乌鲁木齐:新疆医科大学,2014:22-23.
[22]申国安,段礼新,漆小泉.植物代谢组学数据分析和数据库[J].生命科学,2015,27(8):995-999.DOI:10.13376/j.cbls/2015138.
[23]贾金萍,张福生,李震宇,等.基于1H-NMR代谢组学技术研究“杀青”对连翘化学成分的影响[J].中草药,2016,47(8):1375-1381.DOI:10.7501/j.issn.0253-2670.2016.08.022.
[24]赵小梅,刘歆颖,续畅,等.基于LC-MS代谢组学的雷公藤多苷致肝毒性生物标志物的初步筛查[J].中国中药杂志,2015,40(19):3851-3858.DOI:10.4268/cjcmm20151927.
[25]王昕泰,杜丽娜,汪受传.基于代谢组学的小儿腺病毒肺炎痰热闭肺证生物标志物研究思路[J].世界科学技术:中医药现代化,2015,17(10):2177-2180.DOI:10.11842/wst.2015.12.040.
[26]董宇,俞忠明,李洪玉,等.基于超高效液相色谱-高分辨质谱联用技术的桑黄干预后大鼠尿液代谢组学分析[J].色谱,2016,34(8):811-816.DOI:10.3724/SP.J.1123.2016.04009.
[27]李慧,刘其南,高叶,等.二至丸干预大鼠急性酒精肝损伤的尿液代谢组学研究[J].中国医院药学杂志,2016,36(19):1665-1670.DOI:10.13286/j.cnki.chinhosppharmacyj.2016.19.12.
[28]闫翠环,王亚利,王鑫国,等.卫气虚相关血浆代谢标志物的季节性变化研究[J].中国中医药信息杂志,2017,24(1):66-70.DOI:10.3969/j.issn.1005-5304.2017.01.017.
[29]ZHU L L,GE G B,LIU Y,et al.Characterization of UDP-glucuronosyltransferases involved in glucuron i d a t i o n o f diethylstilbestrol in human liver and intestine[J].Chemical Research in Toxicology,2012,25(12):2663-2669.DOI:10.1021/tx300310k.
[30]LI X L,CHEN Y N,LIU J P,et al.Serum metabolic variables associated with impaired glucose tolerance induced by high-fathigh-cholesterol diet in Macaca mulatta[J].Experimental Biology&Medicine,2012,237(11):1310-1321.DOI:10.1258/ebm.2012.012157.
[31]周传波.多烯磷脂酰胆碱对酒精性脂肪肝患者的临床疗效研究[J].国际医药卫生导报,2016,22(4):532-534.DOI:10.3760/cma.j.issn.1007-1245.2016.04.030.
[32]SCHERTZER J W,BROWN E D.Use of CDP-glycerol as an alternate acceptor for the teichoic acid polymerase reveals that membrane association regulates polymer length[J].Journal of Bacteriology,2008,190(21):6940-6947.DOI:10.1128/JB.00851-08.
[33]JIN Shuna,SONG Chengwu,LI Sen,et al.Preventive effects of turmeric on the high-fat diet-induced hyperlipidaemia in mice associated with a targeted metabolomic approach for the analysis of serum lysophosphatidylcholine using LC-MS/MS[J].Journal of Functional Foods,2014,11:130-141.DOI:10.1016/j.jff.2014.09.016.
[34]LI Sen,JIN Shuna,SONG Chengwu,et al.The metabolic change of serum lysophosphatidylcholines involved in the lipid lowering effect of triterpenes from Alismatis rhizoma on high-fat diet induced hyperlipidemia mice[J].Journal of Ethnopharmacology,2016,177:10-18.DOI:10.1016/j.jep.2015.11.017.
[35]ZHAO Y Y,MIAO H,CHENG X L,et al.Lipidomics:novel insight into the biochemical mechanism of lipid metabolism and dysregulation-associated disease[J].Chemico-Biological Interactions,2015,240:220-238.DOI:10.1016/j.cbi.2015.09.005.
[36]OBACH R S.Pharmacologically active drug metabolites:impact on drug discovery and pharmacotherapy[J].Pharmacological Reviews,2013,65(2):578-640.DOI:10.1124/pr.111.005439.
[37]金姝娜.基于组合LC-MS/MS技术的中药姜黄调节脂代谢紊乱活性及其成分姜黄素类化合物的全面表征和分离[D].武汉:华中科技大学,2015:32-34.
[38]CHOI S,SNIDER A J.Sphingolipids in high fat diet and obesityrelated diseases[J].Mediators of Inflammation,2015,2015:1-12.DOI:10.1155/2015/520618.
[39]易吉平,曾明,何兴轩.鞘磷脂类信号通路在肺纤维化发病机制中的作用[J].中国药理学与毒理学杂志,2016,30(2):158-164.DOI:10.3867/j.issn.1000-3002.2016.02.011.
[40]王少媛,张金兰,张丹,等.鞘脂在肝脏疾病中的研究进展[J].药学学报,2015,50(12):1551-1558.DOI:10.16438/j.0513-4870.2015.12.014.
[41]WORGALL T S.Lipid metabolism in cystic fibrosis[J].Current Opinion in Clinical Nutrition and Metabolic Care,2009,12(2):105-109.DOI:10.1097/MCO.0b013e32832595b7.
[42]SCHMITZ G,ORSóE.Intracellular cholesterol and phospholipid trafficking:comparable mechanisms in macrophages and neuronal cells[J].Neurochemical Research,2001,26(8/9):1045-1068.DOI:10.1023/A:1012357106398.
[43]DUMAS M E,BARTON R H,TOYE A,et al.Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(33):12511-12516.DOI:10.1073/pnas.0601056103.