电针“内关”预处理对心肌缺血再灌注损伤大鼠血清代谢物的影响
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摘要
目的:基于核磁共振氢谱技术(1 H NMR)观察电针"内关"穴预处理对心肌缺血再灌注损伤(MIRI)大鼠血清代谢物及其代谢通路的影响,从代谢组学角度探讨电针预处理对MIRI大鼠产生保护作用的机制。方法:雄性SD大鼠48只随机分为正常组、模型组、电针内关组(内关组)、电针合谷组(合谷组),每组12只。采用结扎冠状动脉左前降支40min后恢复血流1h制备MIRI大鼠模型。两电针组造模前每天予电针相应穴位30min,连续7d。模型制备成功后,收集各组大鼠血清,利用~1 H NMR进行代谢物检测,利用最小二乘分析、正交偏最小二乘分析进行模式识别。结果:在造模后,模型组、电针组大鼠心电图均表现出T波高耸,R波与T波间无明显ST段。再灌注后T波下降超过0.2mV,无明显ST段。与正常组比较,模型组大鼠血清代谢模式发生明显改变,乙酰乙酸、乳酸、肌酸、丙三醇、葡萄糖升高,丙氨酸、谷氨酰胺、甘油磷酸胆碱、磷酸胆碱降低。与模型组比较,内关组大鼠血清代谢模式发生明显改变,表现为葡萄糖上升,亮氨酸、异亮氨酸、缬氨酸、三羟基丁酸、乳酸、醋酸盐、丙酮、乙酰乙酸、丙酮酸、谷氨酰胺、肌酸、丙三醇下降;合谷组与模型组代谢模式无显著差异。结论:电针"内关"预处理明显改变了MIRI大鼠糖代谢、丙酮酸代谢、氨基酸代谢、酮体代谢、能量代谢的模式,电针"内关"可能通过调节丙酮酸、氨基酸、酮体、能量等对MIRI大鼠起到一定的预保护作用。
Objective To observe the effect of"Neiguan"(PC6)-electroacupuncture(EA)preconditioning on serum metabolites in myocardial ischemia-reperfusion injury(MIRI)rats,so as to reveal its mechanism underlying improvement of ischemic myocardium from metabonomics.Methods A total of 48 male SD rats were randomly divided into control,model,EA"Neiguan"(PC6)and EA"Hegu"(LI4)groups(n=12 rats/group).Rats of the control group were just banded on animal boards for 30 min,once daily for 7 days.The MIRI model was established by occlusion of the left anterior descending branch of the left coronary artery for 40 min,followed by reperfusion for 1 h,and rats of the model group were also banded as those in the control group.Before modeling,EA(10 Hz/50 Hz,1 mA)was applied to bilateral"Neiguan"(PC6)and"Hegu"(LI4)for 30 min,once daily for 7 successive days.After the treatment,serum samples were collected to be analyzed by proton nuclear magnetic resonance(1 H NMR)spectroscopy.The orthogonal partial least squares discriminate analysis(PLS-DA)was employed to distinguish the serum differential metabolic profile of rats in different groups and identify potential biomarkers.Results After modeling,the ECG of model group and electroacupuncture groups showed T wave towering,and there was no obvious ST segment between R wave and T wave.The T wave decreased more than 0.2 mV after reperfusion,and there was no obvious ST segment.Compared with the control group,MIRI induced significant changes of metabolites in the serum including increase of acetoacetate acid,lectic acid,creatine,glycerol and glucose,and decrease of alanine,glutamine,glycerophosphoryl choline and phosphorylcholine.In comparison with the model group,PC6-EA preconditioning induced significant changes,including an increase of glucose,and a decrease of leucine,isoleucine,valine,3-hydroxybutyric acid,lactate,acetate,acetone,acetoacetate acid,pyruvic acid,glutamine,creatine and glycerol.There is no significant difference in metabolic patterns between"Hegu"group and model group.Metabolic pathway enrichment analysis indicated that the protective effect of PC6-EA pretreatment was realized mainly by regulating pathways of glycolysis,gluconeogenesis,citric acid metabolism,pyruvate metabolism,ketone body metabolism,etc.Conclusion PC6-EA pretreatment has a role in regulating gluconeogenesis,pyruvate metabolism,amino metabolism,ketone body metabolism and energy metabolism in rats with MIRI,which maybe contribute to its protective effect on ischemic myocardium,but the specific metabolic pathways and mechanisms need being studied further.
Objective To observe the effect of"Neiguan"(PC6)-electroacupuncture(EA)preconditioning on serum metabolites in myocardial ischemia-reperfusion injury(MIRI)rats,so as to reveal its mechanism underlying improvement of ischemic myocardium from metabonomics.Methods A total of 48 male SD rats were randomly divided into control,model,EA"Neiguan"(PC6)and EA"Hegu"(LI4)groups(n=12 rats/group).Rats of the control group were just banded on animal boards for 30 min,once daily for 7 days.The MIRI model was established by occlusion of the left anterior descending branch of the left coronary artery for 40 min,followed by reperfusion for 1 h,and rats of the model group were also banded as those in the control group.Before modeling,EA(10 Hz/50 Hz,1 mA)was applied to bilateral"Neiguan"(PC6)and"Hegu"(LI4)for 30 min,once daily for 7 successive days.After the treatment,serum samples were collected to be analyzed by proton nuclear magnetic resonance(1 H NMR)spectroscopy.The orthogonal partial least squares discriminate analysis(PLS-DA)was employed to distinguish the serum differential metabolic profile of rats in different groups and identify potential biomarkers.Results After modeling,the ECG of model group and electroacupuncture groups showed T wave towering,and there was no obvious ST segment between R wave and T wave.The T wave decreased more than 0.2 mV after reperfusion,and there was no obvious ST segment.Compared with the control group,MIRI induced significant changes of metabolites in the serum including increase of acetoacetate acid,lectic acid,creatine,glycerol and glucose,and decrease of alanine,glutamine,glycerophosphoryl choline and phosphorylcholine.In comparison with the model group,PC6-EA preconditioning induced significant changes,including an increase of glucose,and a decrease of leucine,isoleucine,valine,3-hydroxybutyric acid,lactate,acetate,acetone,acetoacetate acid,pyruvic acid,glutamine,creatine and glycerol.There is no significant difference in metabolic patterns between"Hegu"group and model group.Metabolic pathway enrichment analysis indicated that the protective effect of PC6-EA pretreatment was realized mainly by regulating pathways of glycolysis,gluconeogenesis,citric acid metabolism,pyruvate metabolism,ketone body metabolism,etc.Conclusion PC6-EA pretreatment has a role in regulating gluconeogenesis,pyruvate metabolism,amino metabolism,ketone body metabolism and energy metabolism in rats with MIRI,which maybe contribute to its protective effect on ischemic myocardium,but the specific metabolic pathways and mechanisms need being studied further.
引文
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[6]鹿小燕,顾焕,徐浩,等.基于气相色谱质谱联用对冠心病“痰”“瘀”证候血清代谢组学的研究[J].中西医结合心脑血管病杂志,2016,14(9):929-932.
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[8]李杰,胡超,许龙龙,等.参麦方对于大鼠心肌损伤保护作用的代谢组学研究[J].中国药理学通报,2016,32(11):1559-1565.
[9]金文敏,穆殿平.芪参益气滴丸用于大鼠急性心肌梗死的代谢组学研究[J].天津药学,2015,27(6):9-13.
[10]王超,田岳凤,周丹,等.电针“内关”穴对心肌缺血再灌注大鼠心肌组织一氧化氮、一氧化氮合酶和细胞内钙的影响[J].针刺研究,2010,35(2):113-117.
[11]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:314-319.
[12]ABDURRACHIM D,LUIKEN J J,NICOLAY K,et al.Good and bad consequences of altered fatty acid metabolism in heart failure:evidence from mouse models[J].Cardiovasc Res,2015,106(2):194-205.
[13]周国鹏,赵志杰.心肌缺血与糖代谢[J].中华老年心脑血管病杂志,2001,3(3):204-207.
[14]EBERLI F R,WEINBERG E O,GRICE W N,et al.Protective effect of increased glycolytic substrate against systolic and diastolic dysfunction and increased coronary resistance from prolonged global underperfusion and reperfusion in isolated rabbit hearts perfused with erythrocyte suspensions[J].Circ Res,1991,68(2):466-481.
[15]HAIST J V,HIRST C N,KARMAZYN M.Effective protection by NHE-1inhibition in ischemic and reperfused heart under preconditioning blockade[J].Am J Physiol Heart Circ Physiol,2003,284(3):H798-H803.
[16]任江华,姜黎,曹茂银.丙酮酸对顿抑心肌功能及能量代谢的作用[J].中国老年学杂志,2004,24(8):720-722.
[17]HOLECEK M.Relation between glutamine,branched-chain amino acids,and protein metabolism[J].Nutrition,2002,18(2):130-133.
[18]李爱玲,高兰兴.支链氨基酸对心肌缺血大鼠线粒体损伤的保护作用[J].中国应用生理学杂志,1999,15(4):326-328.
[19]王镜岩,朱圣庚,徐长法.生物化学[M].3版,北京:高等教育出版社,2002.
[20]王超,谢文娟,刘密,等.针灸预处理对不同时间心肌缺血再灌注损伤兔血浆内皮素、血清肌酸激酶和心肌组织热休克蛋白70表达的影响[J].针刺研究,2014,39(5):372-376.
[21]王超,张佳丽,刘昭,等.电针内关穴预处理对兔心肌缺血再灌注CK及心肌细胞活性的影响[J].上海针灸杂志,2015,34(1):67-69.
[22]林海波,严洁,常小荣,等.针灸内关穴预处理对兔缺血再灌注损伤心肌细胞死亡受体通路Fas/FasL蛋白表达的影响[J].中华中医药杂志,2013,28(5):1286-1290.
[23]成泽东,陈以国,李晓梅,等.电针“内关”穴对急性心肌缺血小鼠心肌组织氯离子通道蛋白表达的影响[J].针刺研究,2016,41(5):423-428.
[24]白增华,吴兆利,苏妆,等.电针“内关”穴对心肌缺血大鼠心肌氯离子通道相关基因表达的影响[J].针刺研究,2015,40(6):439-443.
[25]谭成富,严洁,王超,等.针灸预处理对心肌缺血再灌注损伤兔不同时间热休克蛋白27、70、90表达的影响[J].针刺研究,2017,42(1):31-38.
[26]宋瑾,王超,阳仁达,等.电针内关穴预处理对心肌缺血再灌注损伤大鼠NO、NOS及腺苷A1受体的影响[J].中国中医急症,2017,26(4):565-567.
[27]PECHLIVANIS A,KOSTIDIS S,SARASLANIDIS P,et al.1 H NMR study on the short-and long-term impact of two training programs of sprint running on the metabolic fingerprint of human serum[J].J Proteome Res,2013,12(1):470-480.
[28]钟欢,佘畅,呙安林,等.基于1 H-NMR技术探讨艾灸与针刺对血清代谢物的调节作用[J].世界科学技术-中医药现代化,2016,18(3):402-409.
[2]魏星,万玲,刘石蜜,等.冠心病血瘀证血浆代谢组学研究[J].中医杂志,2013,54(7):587-591.
[3]吴倩怡,阎江洪,梁小雪,等.急性ST段抬高型心肌梗死患者的血清代谢组学研究[J].重庆医科大学学报,2016,41(9):875-882.
[4]郑景辉,陈建军,简维雄,等.冠心病心血瘀阻证患者血浆代谢组学的通路分析[J].时珍国医国药,2015,26(2):485-487.
[5]陈夏,郑景辉,梁健,等.冠心病心血瘀阻证患者尿液代谢组学的通路分析[J].中国中医基础医学杂志,2015,21(1):69-71.
[6]鹿小燕,顾焕,徐浩,等.基于气相色谱质谱联用对冠心病“痰”“瘀”证候血清代谢组学的研究[J].中西医结合心脑血管病杂志,2016,14(9):929-932.
[7]牟菲,段佳林,边海旭,等.降香水提物和挥发油对心肌缺血/再灌注损伤大鼠预防作用的代谢组学研究[J].中国药理学通报,2016,32(10):1377-1382.
[8]李杰,胡超,许龙龙,等.参麦方对于大鼠心肌损伤保护作用的代谢组学研究[J].中国药理学通报,2016,32(11):1559-1565.
[9]金文敏,穆殿平.芪参益气滴丸用于大鼠急性心肌梗死的代谢组学研究[J].天津药学,2015,27(6):9-13.
[10]王超,田岳凤,周丹,等.电针“内关”穴对心肌缺血再灌注大鼠心肌组织一氧化氮、一氧化氮合酶和细胞内钙的影响[J].针刺研究,2010,35(2):113-117.
[11]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:314-319.
[12]ABDURRACHIM D,LUIKEN J J,NICOLAY K,et al.Good and bad consequences of altered fatty acid metabolism in heart failure:evidence from mouse models[J].Cardiovasc Res,2015,106(2):194-205.
[13]周国鹏,赵志杰.心肌缺血与糖代谢[J].中华老年心脑血管病杂志,2001,3(3):204-207.
[14]EBERLI F R,WEINBERG E O,GRICE W N,et al.Protective effect of increased glycolytic substrate against systolic and diastolic dysfunction and increased coronary resistance from prolonged global underperfusion and reperfusion in isolated rabbit hearts perfused with erythrocyte suspensions[J].Circ Res,1991,68(2):466-481.
[15]HAIST J V,HIRST C N,KARMAZYN M.Effective protection by NHE-1inhibition in ischemic and reperfused heart under preconditioning blockade[J].Am J Physiol Heart Circ Physiol,2003,284(3):H798-H803.
[16]任江华,姜黎,曹茂银.丙酮酸对顿抑心肌功能及能量代谢的作用[J].中国老年学杂志,2004,24(8):720-722.
[17]HOLECEK M.Relation between glutamine,branched-chain amino acids,and protein metabolism[J].Nutrition,2002,18(2):130-133.
[18]李爱玲,高兰兴.支链氨基酸对心肌缺血大鼠线粒体损伤的保护作用[J].中国应用生理学杂志,1999,15(4):326-328.
[19]王镜岩,朱圣庚,徐长法.生物化学[M].3版,北京:高等教育出版社,2002.
[20]王超,谢文娟,刘密,等.针灸预处理对不同时间心肌缺血再灌注损伤兔血浆内皮素、血清肌酸激酶和心肌组织热休克蛋白70表达的影响[J].针刺研究,2014,39(5):372-376.
[21]王超,张佳丽,刘昭,等.电针内关穴预处理对兔心肌缺血再灌注CK及心肌细胞活性的影响[J].上海针灸杂志,2015,34(1):67-69.
[22]林海波,严洁,常小荣,等.针灸内关穴预处理对兔缺血再灌注损伤心肌细胞死亡受体通路Fas/FasL蛋白表达的影响[J].中华中医药杂志,2013,28(5):1286-1290.
[23]成泽东,陈以国,李晓梅,等.电针“内关”穴对急性心肌缺血小鼠心肌组织氯离子通道蛋白表达的影响[J].针刺研究,2016,41(5):423-428.
[24]白增华,吴兆利,苏妆,等.电针“内关”穴对心肌缺血大鼠心肌氯离子通道相关基因表达的影响[J].针刺研究,2015,40(6):439-443.
[25]谭成富,严洁,王超,等.针灸预处理对心肌缺血再灌注损伤兔不同时间热休克蛋白27、70、90表达的影响[J].针刺研究,2017,42(1):31-38.
[26]宋瑾,王超,阳仁达,等.电针内关穴预处理对心肌缺血再灌注损伤大鼠NO、NOS及腺苷A1受体的影响[J].中国中医急症,2017,26(4):565-567.
[27]PECHLIVANIS A,KOSTIDIS S,SARASLANIDIS P,et al.1 H NMR study on the short-and long-term impact of two training programs of sprint running on the metabolic fingerprint of human serum[J].J Proteome Res,2013,12(1):470-480.
[28]钟欢,佘畅,呙安林,等.基于1 H-NMR技术探讨艾灸与针刺对血清代谢物的调节作用[J].世界科学技术-中医药现代化,2016,18(3):402-409.