基于超高效液相色谱-质谱联用技术研究婴儿巨细胞病毒肝炎证候实质
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摘要
目的:从尿液代谢组学层面探讨婴儿巨细胞病毒(HCMV)肝炎患儿湿热内蕴证、脾虚湿困证、气滞血瘀证的证候实质。方法:收集HCMV肝炎湿热内蕴证(44例)、脾虚湿困证(35例)、气滞血瘀证(43例)、正常对照组(40名)患儿,进行生化指标统计分析,同时采用超高效液相色谱-质谱联用技术,对患儿尿液样本进行代谢组学检测,经主成分分析寻找各证型的生物标志物。结果:在生化指标分析中,各证型组在丙氨酸氨基转移酶、门冬氨酸氨基转移酶、总胆红素、结合胆红素、总胆汁酸、谷氨酰转肽酶、碱性磷酸酶、乳酸脱氢酶及凝血酶原时间水平有显著差异。在代谢组学研究中,各证型组在正交偏最小二乘法-判别分析图上区分明显。各证型组均涉及氨基酸代谢紊乱。此外,各证型均有特征性生物标志物。结论:HCMV肝炎湿热内蕴证、脾虚湿困证及气滞血瘀证在生化指标和尿液代谢物水平均存在显著差异,表明不同中医证候有其生物学基础。
Objective: To study on the essence of infant cytomegalovirus hepatitis with syndrome of internal retention of damp-heat, syndrome of dampness stagnancy due to spleen deficiency and syndrome of qi stagnation and blood stasis based on urine metabolomics. Methods: Urine samples were collected from patients with syndrome of internal retention of damp-heat(44 cases), syndrome of dampness stagnancy due to spleen deficiency(35 cases), syndrome of qi stagnation and blood stasis(43 cases) and control group(40 cases), and the biochemical results of these patients were collected and analyzed. A non-targeted ultra high performance liquid chromatography-LTQ/orbitrap-mass spectrometry(UHPLC-LTQ/Orbitrap-MS) metabolomics method was used in conjunction with principle component analysis(PCA) to explore the differential metabolites of the three TCM syndromes. Results: In biochemical indicators analysis, three TCM syndromes differ in alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), direct bilirubin(DBIL), total bile acid(TBA), glutamyl transpeptidase(GGT), alkaline phosphatase(ALP), lactate dehydrogenase(LDH) and prothrombin time(PT). In metabolomic study, the orthogonal partial least squares discriminant analysis(OPLS-DA) score plots revealed clear differentiation among the three TCM syndromes. All the three TCM syndromes involve a variety of amino acid metabolism. In addition, every syndrome has its own characteristic biomarker. Conclusion: There are significant differences in biochemical indicators and urinary metabolomic profiles among the three TCM syndromes of infant cytomegalovirus hepatitis, which indicates that different TCM syndromes have their specific biological basis.
Objective: To study on the essence of infant cytomegalovirus hepatitis with syndrome of internal retention of damp-heat, syndrome of dampness stagnancy due to spleen deficiency and syndrome of qi stagnation and blood stasis based on urine metabolomics. Methods: Urine samples were collected from patients with syndrome of internal retention of damp-heat(44 cases), syndrome of dampness stagnancy due to spleen deficiency(35 cases), syndrome of qi stagnation and blood stasis(43 cases) and control group(40 cases), and the biochemical results of these patients were collected and analyzed. A non-targeted ultra high performance liquid chromatography-LTQ/orbitrap-mass spectrometry(UHPLC-LTQ/Orbitrap-MS) metabolomics method was used in conjunction with principle component analysis(PCA) to explore the differential metabolites of the three TCM syndromes. Results: In biochemical indicators analysis, three TCM syndromes differ in alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), direct bilirubin(DBIL), total bile acid(TBA), glutamyl transpeptidase(GGT), alkaline phosphatase(ALP), lactate dehydrogenase(LDH) and prothrombin time(PT). In metabolomic study, the orthogonal partial least squares discriminant analysis(OPLS-DA) score plots revealed clear differentiation among the three TCM syndromes. All the three TCM syndromes involve a variety of amino acid metabolism. In addition, every syndrome has its own characteristic biomarker. Conclusion: There are significant differences in biochemical indicators and urinary metabolomic profiles among the three TCM syndromes of infant cytomegalovirus hepatitis, which indicates that different TCM syndromes have their specific biological basis.
引文
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[19]白冠锋,郝一鸣,钱鹏,等.基于气-质联用技术的慢性肾功能衰竭湿证患者尿液代谢组学研究.中华中医药杂志,2018,33(4):1337-1340
[20]宋红,郑小伟,鲍晓东,等.基于核磁共振技术的模式识别方法在肾气虚哮喘大鼠血清代谢组学中的应用.中华中医药杂志,2017,32(1):259-262
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[22]Chen Y,Feng J,Guo J,et al.Dysbiosis of small intestinal microbiota in liver cirrhosis and its association with etiology.Scientific Reports,2016,6:1-9
[23]Ma X,Chi Y H,Niu M,et al.Metabolomics coupled with multivariate data and pathway analysis on potential biomarkers in cholestasis and intervention effect of paeonia lactiflora Pall.Frontiers in Pharmacology,2016,7(155):1-12
[24]Li W W,Shan J J,Lin L L,et al.Disturbance in plasma metabolic profile in different types of human cytomegalovirus-induced liver injury in infants.Scientific Reports,2017,7(1):1-10
[25]Li W W,Yang Y,Dai Q G, et al.Non-invasive urinary metabolomic profiles discriminate biliary atresia from infantile hepatitis syndrome.Metabolomics,2018,14(7):89-90
[26]Wlodarska M,Luo C,Kolde R,et al.Indoleacrylic acid produced by commensal peptostreptococcus species suppresses inflammation.Cell Host&Microbe,2017,22(1):25-37
[27]Gao H,Lu Q,Liu X,et al.Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.Cancer Science,2010,100(4):782-785
[28]Munshi S U,Taneja S,Bhavesh N S,et al.Metabonomic analysis of hepatitis E patients shows deregulated metabolic cycles and abnormalities in amino acid metabolism.Journal of Viral Hepatitis,2011,18(10):591-602
[29]Seidel A,Brunner S,Seidel P,et al.Modified nucleosides:an accurate tumour marker for clinical diagnosis of cancer,early detection and therapy control.British Journal of Cancer,2006,94(11):1726-1733
[30]郭晓红,储明星,周忠孝.视黄醇结合蛋白及其基因的分子生物学.遗传,2004,26(2):257-262
[31]龚兰,卢业成,陈伟辉,等.病毒性肝炎患者血清视黄醇结合蛋白检测的临床意义.临床肝胆病杂志,2006,22(2):103-104
[32]何佳,宁会彬,曾艳丽,等.血清中β2微球蛋白、视黄醇结合蛋白、胱抑素C在替诺福韦或恩替卡韦单药治疗慢性乙型肝炎早期肾功能变化中的意义.中华肝脏病杂志,2016,24(9):643-646
[33]邵珺,李平,于乐成,等.血清视黄醇结合蛋白在肝损伤中的意义.肝脏,2015(1):11-13
[34]杜蕊.学龄前反复呼吸道感染患儿中医体质类型与维生素A相关性分析.沈阳:辽宁中医药大学,2011
[35]张淑莲.高血压、冠心病、血脂谱异常与内源性维生素A、E及中医证型变化特点的研究.北京:北京中医药大学,2013
[36]魏军平,林兰.气阴两虚型糖尿病与血清维生素AEC含量关系的探讨-附103的临床观察.辽宁中医杂志,1994(5):201-202
[37]李维薇,杨燕,汪受传.组学技术在肝脏疾病证本质领域的研究进展.中华中医药杂志,2019,34(2):139-142
[2]Shibata Y,Kitajima N,Kawada J,et al.Association of cytomegalovirus with infantile hepatitis.Microbiology&Immunology,2013,49(8):771-777
[3]Ozkan T B,Mistik R,Dikici B,et al.Antiviral therapy in neonatal cholestatic cytomegalovirus hepatitis.BMC Gastroenterology,2007,7(1):1-5
[4]Bilavsky E,Schwarz M,Bar-Sever Z,et al.Hepatic involvement in congenital cytomegalovirus infection-infrequent yet significant.Journal of Viral Hepatitis,2015,22(9):763-768
[5]胡亚美,江载芳,申昆玲,等.诸福棠实用儿科学.8版.北京:人民卫生出版社,2015:910-915
[6]李维薇,杨燕,贺丽丽,等.婴儿巨细胞病毒肝炎的中医药研究进展.辽宁中医杂志,2015(12):2458-2461
[7]舒静,闫慧敏,杨燕.婴儿肝炎综合征辨证分型临床观察.北京中医药,2010,29(11):813-816
[8]赵骞,杨燕,舒静.婴儿肝炎综合征中医证候学研究.北京中医药,2010,29(8):575-577
[9]赵骞,杨燕.161例婴儿肝炎综合征症状与舌象指纹聚类分析.辽宁中医药大学学报,2013(6):154-156
[10]郭蕾,王永炎,张志斌.关于证候概念的诠释.北京中医药大学学报,2003,26(2):5-8
[11]王永炎.完善中医辨证方法体系的建议.中医杂志,2004,45(10):729-731
[12]Zhang X,Fang A,Riley C P,et al.Multi-dimensional liquid chromatography in proteomics:A review.Analytica Chimica Acta,2010,664(2):101-113
[13]Wang X,Zhang A,Han Y,et al.Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease.Molecular&Cellular Proteomics,2012,11(8):370-371
[14]Gou X,Tao Q,Feng Q,et al.Urinary metabonomics characterization of liver fibrosis induced by CCl4 in rats and intervention effects of Xia Yu Xue Decoction.Journal of Pharmaceutical&Biomedical Analysis,2013,74(4):62-65
[15]中华中医药学会.中医儿科常见病诊疗指南.北京:中国中医药出版社,2012:163-166
[16]Tsugawa H,Cajka T,Kind T,et al.MS-DIAL:Data-independent MS/MS deconvolution for comprehensive metabolome analysis.Nature Methods,2015,12(6):523-526
[17]Xia J,Sinelnikov I V,Han B,et al.MetaboAnalyst 3.0-making metabolomics more meaningful.Nucleic Acids Research,2015,43(1):1-7
[18]马欣,顾宏图,赵瑜,等.不同证候的非酒精性脂肪肝合并肝损伤患者的血清代谢组学分析.中华中医药杂志,2017,32(3):1246-1250
[19]白冠锋,郝一鸣,钱鹏,等.基于气-质联用技术的慢性肾功能衰竭湿证患者尿液代谢组学研究.中华中医药杂志,2018,33(4):1337-1340
[20]宋红,郑小伟,鲍晓东,等.基于核磁共振技术的模式识别方法在肾气虚哮喘大鼠血清代谢组学中的应用.中华中医药杂志,2017,32(1):259-262
[21]Xie Q,Fan F,Wei W,et al.Multi-omics analyses reveal metabolic alterations regulated by hepatitis B virus core protein in hepatocellular carcinoma cells.Scientific Reports,2017,7:1-12
[22]Chen Y,Feng J,Guo J,et al.Dysbiosis of small intestinal microbiota in liver cirrhosis and its association with etiology.Scientific Reports,2016,6:1-9
[23]Ma X,Chi Y H,Niu M,et al.Metabolomics coupled with multivariate data and pathway analysis on potential biomarkers in cholestasis and intervention effect of paeonia lactiflora Pall.Frontiers in Pharmacology,2016,7(155):1-12
[24]Li W W,Shan J J,Lin L L,et al.Disturbance in plasma metabolic profile in different types of human cytomegalovirus-induced liver injury in infants.Scientific Reports,2017,7(1):1-10
[25]Li W W,Yang Y,Dai Q G, et al.Non-invasive urinary metabolomic profiles discriminate biliary atresia from infantile hepatitis syndrome.Metabolomics,2018,14(7):89-90
[26]Wlodarska M,Luo C,Kolde R,et al.Indoleacrylic acid produced by commensal peptostreptococcus species suppresses inflammation.Cell Host&Microbe,2017,22(1):25-37
[27]Gao H,Lu Q,Liu X,et al.Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.Cancer Science,2010,100(4):782-785
[28]Munshi S U,Taneja S,Bhavesh N S,et al.Metabonomic analysis of hepatitis E patients shows deregulated metabolic cycles and abnormalities in amino acid metabolism.Journal of Viral Hepatitis,2011,18(10):591-602
[29]Seidel A,Brunner S,Seidel P,et al.Modified nucleosides:an accurate tumour marker for clinical diagnosis of cancer,early detection and therapy control.British Journal of Cancer,2006,94(11):1726-1733
[30]郭晓红,储明星,周忠孝.视黄醇结合蛋白及其基因的分子生物学.遗传,2004,26(2):257-262
[31]龚兰,卢业成,陈伟辉,等.病毒性肝炎患者血清视黄醇结合蛋白检测的临床意义.临床肝胆病杂志,2006,22(2):103-104
[32]何佳,宁会彬,曾艳丽,等.血清中β2微球蛋白、视黄醇结合蛋白、胱抑素C在替诺福韦或恩替卡韦单药治疗慢性乙型肝炎早期肾功能变化中的意义.中华肝脏病杂志,2016,24(9):643-646
[33]邵珺,李平,于乐成,等.血清视黄醇结合蛋白在肝损伤中的意义.肝脏,2015(1):11-13
[34]杜蕊.学龄前反复呼吸道感染患儿中医体质类型与维生素A相关性分析.沈阳:辽宁中医药大学,2011
[35]张淑莲.高血压、冠心病、血脂谱异常与内源性维生素A、E及中医证型变化特点的研究.北京:北京中医药大学,2013
[36]魏军平,林兰.气阴两虚型糖尿病与血清维生素AEC含量关系的探讨-附103的临床观察.辽宁中医杂志,1994(5):201-202
[37]李维薇,杨燕,汪受传.组学技术在肝脏疾病证本质领域的研究进展.中华中医药杂志,2019,34(2):139-142
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