基于~1HNMR的心肌缺血代谢组学研究及山楂总黄酮的干预作用

Study of1HNMR-based metabolomics in myocardial ischemia and the effects of total flavonoids in hawthorn

目的:基于~1HNMR代谢组学方法研究异丙肾上腺素所致心肌缺血大鼠血浆代谢组学特征,确定血浆生物标志物,探讨心肌缺血相关代谢途径,观察山楂总黄酮的干预作用,分析其可能的作用机制。方法:采用皮下注射异丙肾上腺素建立大鼠心肌缺血模型,以心电图J点偏移绝对值、血液生化学指标和病理组织学评价模型。基于~1HNMR检测正常组、模型组、山楂总黄酮组血浆代谢物信息,采用偏最小二乘判别式法(PLS-DA)进行模式分析,发现潜在生物标志物,通过网络数据库和相关文献进行鉴别,采用单因素方差分析(One-Way ANOVA)检测代谢产物变化的显著性,分析其相关代谢途径。结果:模型组心电图J点偏移显著;血浆ET、MDA、LDH和CK明显增高,NO和SOD明显降低;心肌出现明显病理改变。三组大鼠~1HNMR血浆代谢指纹谱实现明显分离。发现10个特征性代谢产物,分别为低密度脂蛋白/极低密度脂蛋白(LDL/VLDL)、乳酸(Lactate)、N-乙酰糖蛋白(NAC)、乙酰乙酸(Acetoacetate)、不饱和脂肪酸(Unsaturated fatty acids)、丙氨酸(Alanine)、组氨酸(Histidine)、甲酸盐(Formate)、柠檬酸(Citrate)和肌酸酐(Creatinine);与正常组比,模型组血浆低密度脂蛋白/极低密度脂蛋白、乳酸、N-乙酰糖蛋白、乙酰乙酸和不饱和脂肪酸显著增加;丙氨酸、组氨酸、甲酸、柠檬酸和肌酸酐显著降低;0.276 g/kg山楂总黄酮干预后LDL/VLDL、乳酸、N-乙酰糖蛋白、乙酰乙酸和不饱和脂肪酸水平有所下降,丙氨酸、组氨酸、甲酸、柠檬酸和肌酸酐的含量均有所上升。结论:模型组与正常组~1HNMR血浆代谢轮廓存在明显差异。10个特征性代谢产物构成心肌缺血代谢水平的物质基础,有助于阐释心肌缺血的本质。0.276 g/kg山楂总黄酮使心肌缺血代谢状态向正常代谢状态回归,可能通过调节脂代谢、糖代谢改善心肌能量代谢,此外,可能调控ROS诱导的氧化应激以及炎症反应。上述机制的探讨为进一步深入阐释山楂总黄酮抗心肌缺血作用提供了参考。

Objective:To study the ~1HNMR based serum metabolomics characteristics of myocardial ischemia rats induced by isoproterenol(ISO),discover the serum biomarkers and explore the metabolic pathways closely related to myocardial ischemia.Also observe the effects of hawthorn flavonoids and analyze its possible mechanism.Methods:The myocardial ischemia rat model was established by ISO sc.The absolute value of J-point offset,blood biochemical parameters and histopathological evaluation were adopted to confirm the model.The serum metabolites of three groups,normal,model and hawthorn flavonoids were detected by ~1HNMR,and PLS-DA was used for pattern analysis.The characteristic metabolites were identified by network database and related literature.One-Way ANOVA was used to detect the significance.Results:For model group,the J-point deviation of electrocardiogram was significant,the plasma level of ET,MDA,LDH and CK was significantly increased,and NO,SOD was distinctly decreased(P<0.05 or P<0.01),also the ischemia pathological changes were observed.Rats serum metabolic fingerprinting of three groups was obviously separated by PLS-DA analysis.lipoprotein,lactate,N-acetylglucosamine,acetoacetate,unsaturated fatty acids,alanine,histidine,formate,citrate and creatinine were found as characteristic metabolites.Compared with the normal group,serum lipoprotein,lactate,N-acetylglucosamine,acetoacetate and unsaturated fat in the model group was significantly increased(P<0.05 or P<0.01),while alanine,histidine,creatinine was significantly decreased(P<0.05 or P<0.01).LDL/VLDL,lactate,N-acetylglucosamine,acetoacetate,unsaturated fatty acids was decreased and alanine,citrate,creatinine,histadine and formate was increased under the dose of 0.276 g/kg Hawthorn flavonoids.Conclusion:Significant difference in serum metabolic profile was detected in myocardial ischemia rats and normal rats.Ten characteristic metabolites were found as material basis of myocardial ischemia,which might elucidate the nature of myocardial ischemia.Regression from myocardial metabolic status to normal status was observed on dose of 0.276 g/kg Hawthorn flavonoids,which probably improved energy metabolism through regulation of lipid and glucose metabolism.In addition,regulated oxidative stress induced by ROS,and inflammatory response was observed.