基于UPLC-MS技术的针刺合募配穴治疗功能性消化不良的代谢组学研究

发布时间：2019-02-15 10:04

【摘要】：目的采用基于超高效液相色谱串联四级杆轨道阱质谱(UPLC-Q- Exactive Orbitrap-MS)的技术和方法研究针刺合募配穴对功能性消化不良(Functional Dyspepsia, FD)患者血浆中内源性小分子(分子量1000)代谢物的影响,探讨针刺合募配穴对效应物质基础含量变化的影响及其规律,为揭示针刺合募配穴治疗FD的代谢组学机制提供新的生物信息学依据和线索。方法1.FD患者的血浆代谢组学研究：采用UPLC-Q- Exactive Orbitrap-MS代谢组学技术,通过与健康组比较,找出FD的血浆潜在生物标志物,利用MetPA平台分析潜在生物标志物的代谢途径。2.合募配穴治疗FD的代谢组学研究：分别采用针刺合募配穴、下合穴、募穴治疗FD,运用UPLC-Q- Exactive Orbitrap-MS代谢组学技术,通过与各组治疗前比较,找出各组FD患者在针刺治疗2个疗程(10天)、4个疗程(20天)后血浆中差异具有统计学意义的生物标志物。3.各组血浆中差异标志物的含量变化分析：以健康组的血浆标志物的均值含量为参考标准,对针刺合募配穴、下合穴、募穴治疗FD的治疗前、治疗2个疗程、治疗4个疗程后血浆中的差异标志物的均值含量做整体比较,观察各标志物在各组中的均值含量变化趋势及变化规律,探讨针刺合募配穴对FD患者血浆代谢物的影响。结果1.与健康组相比,FD患者血浆中苯乙胺、(3R,7R)-1,3,7-Octanetriol含量明显下降(P0.01);亚硝酸盐、2-苯乙酰胺、L-别异亮氨酸含量明显升高(P0.01);2-己基甘油、4-羟基-L-谷氨酸含量明显下降(P0.05);1-二氢吡咯—2—羧酸酸、D-脯氨酸含量明显升高(P0.05),是FD的潜在生物标志物。与FD疾病相关的代谢通路是苯丙氨酸代谢、精氨酸和脯氨酸代谢。2.从针刺合募配穴(足三里、中脘)治疗FD患者2个疗程和4个疗程后的血浆中,共筛选出15个具有统计学差异的标志物(P0.05),并成功鉴定10个,其中亚硝酸盐、2-苯乙酰胺、D-脯氨酸、L-别异亮氨酸在针刺合募配穴治疗后的血浆中均呈下调趋势；2-己基甘油、氨基丙二酸、1-吡咯啉-2-羧酸、4-羟基-L-谷氨酸、3-甲基组氨酸、12酮脱氧胆酸在针刺合募配穴治疗后的血浆中均呈上调趋势。针刺合募配穴治疗FD的相关调节通路是苯丙氨酸代谢、精氨酸和脯氨酸代谢。3.从针刺下合穴(足三里)治疗FD患者2个疗程和4个疗程后的血浆中,共筛选出11个具有统计学差异的生物标志物(P0.05),并成功鉴定9个,其中亚硝酸盐、2-苯乙酰胺、D-脯氨酸、(3R,7R)-1,3,7-Octanetriol.L-异亮氨酸在针刺足三里穴治疗后的血浆中均呈下调趋势；2-己基甘油、4-羟基-L-谷氨酸、12酮脱氧胆酸、烟酰胺在针刺足三里穴治疗后的血浆中均呈上调趋势。针刺下合穴治疗FD的相关调节通路是苯丙氨酸代谢。4.从针刺募穴(中脘)治疗FD患者2个疗程和4个疗程后的血浆中,共筛选出11个具有统计学差异的标志物(P0.05),并成功鉴定10个,其中亚硝酸盐、2-苯乙酰胺、D-脯氨酸在针刺中脘穴治疗后的血浆中均呈下调趋势；2-己基甘油、氨基丙二酸、1-吡咯啉-2-羧酸、4-羟基-L-谷氨酸、3-甲基组氨酸、12酮脱氧胆酸、L-别苏氨酸在针刺中脘穴治疗后的血浆中均呈上调趋势。针刺募穴治疗FD的相关调节通路是苯丙氨酸代谢、精氨酸和脯氨酸代谢。结论1.一系列代谢物的含量变化异常是FD患者血浆代谢物的共同特点,其中,血浆苯乙胺、亚硝酸盐、2-苯乙酰胺、2-己基甘油、4-羟基-L-谷氨酸、1-吡咯啉-2-羧酸、D-脯氨酸、(3R,7R)-1,3,7-Octanetriol.L-别异亮氨酸,是FD潜在的生物标志物。2.与FD疾病相关的潜在靶标通路可能是苯丙氨酸代谢、精氨酸和脯氨酸代谢。3.针刺合募配穴治疗FD的代谢组学机制可能与调控苯丙氨酸代谢、精氨酸和脯氨酸代谢有关。4.针刺合募配穴、下合穴、募穴对FD疾病相关代谢标志物都有一定的良性调节作用,合募配穴的总体调节作用优于下合穴和募穴。
[Abstract]:Objective To study the effect of acupuncture on the endogenous small molecule (molecular weight 1000) of functional dyspepsia (FD) in patients with functional dyspepsia (FD) by means of ultra-high performance liquid chromatography tandem quadrupole mass spectrometry (UPLC-Q-active Orbitrap-MS). To study the effect of acupuncture on the change of the material base content and the law, to provide a new bioinformatics basis and a clue for revealing the mechanism of the metabolic group of the acupuncture combined point treatment FD. Methods The plasma metabolic group of FD patients was studied by means of UPLC-Q-Exactive Orbitrap-MS, and the potential biomarkers of FD were found by comparison with the healthy group. The metabolic pathway of potential biomarkers was analyzed by the MetPA platform. In the study of the metabolic group of the treatment FD of the combined point, the treatment FD was treated by means of acupuncture combined with the points, the lower and the lower, and the point of raising, and by means of UPLC-Q-Exactive Orbitrap-MS, the group of FD patients were found to be treated with acupuncture for 2 courses of treatment (10 days). There was a statistically significant biomarker in plasma after 4 courses of treatment (20 days). the content of the difference marker in each group of plasma is analyzed, the mean content of the plasma mark in the healthy group is taken as the reference standard, the acupuncture combined point, the lower cavity and the point raising point are used for treating the treatment of the FD, and the two treatment courses are treated, The mean content of the difference markers in the plasma after 4 treatment courses was compared and the change of the mean content of the markers in each group was observed, and the effect of the acupuncture on the plasma metabolites in the FD patients was discussed. Results 1. Compared with the healthy group, the content of phenethylamine, (3R, 7R) -1, 3, 7-Octanetriol in the plasma of FD patients decreased significantly (P0.01), and the content of nitrite, 2-phenethyl amine and L-isoleucine increased significantly (P0.01), and the content of 4-hydroxy-L-glutamic acid in the 2-hexanyl glycerol, 4-hydroxy-L-glutamic acid decreased significantly (P0.05). The contents of D-proline and D-proline increased significantly (P0.05). The metabolic pathway associated with FD is phenylalanine metabolism, arginine and proline metabolism. A total of 15 markers of statistical difference (P0.05) were selected and 10 were successfully identified, including nitrite, 2-phenethyl amine and D-proline. L-isoleucine is down-regulated in plasma after acupuncture combined with point-in-point treatment; 2-hexyl glycerol, amino malonic acid, 1-methyl-2-coumaric acid, 4-hydroxy-L-glutamic acid, 3-methylhistidine and 12-ketodesodeoxycholic acid were up-regulated in plasma after acupuncture combined with the point-in-point treatment. The related regulatory pathway of the treatment of FD with the acupuncture combined point is phenylalanine metabolism, arginine and proline metabolism. A total of 11 biomarkers (P0.05) were selected and 9 were successfully identified, including nitrite, 2-phenethamine, D-proline, (3R, (R) -1, 3, 7-Octanetriol. L-isoleucine was down-regulated in the plasma after the treatment of the acupuncture and the third-point acupuncture point; 2-hexyl glycerol, 4-hydroxy-L-glutamic acid, and 12-one deoxycholic acid, and the smoke and the amine were all up-regulated in the plasma treated by the acupuncture and the three-point acupuncture point. The relevant regulatory pathway of the treatment of FD with acupuncture at the lower point of acupuncture is phenylalanine metabolism. A total of 11 markers of statistical difference (P0.05) were selected from the treatment of 2 treatment courses and 4 treatment courses of the FD patients from the acupuncture point-raising point (Zhongzi), and 10 were successfully identified, of which the nitrite, 2-phenylidenamine, D-proline is down-regulated in plasma after acupuncture, and 2-hexyl glycerol, amino malonic acid, 1-methyl-2-coumaric acid, 4-hydroxy-L-glutamic acid, 3-methylhistidine, and 12-one deoxycholic acid, L-non-threonine was up-regulated in plasma after acupuncture. The related regulatory pathway of the acupuncture point-raising treatment FD is the metabolism of phenylalanine, arginine and proline. Conclusion 1. The variation in the content of a series of metabolites is a common characteristic of the plasma metabolite of the FD patient, in which the plasma phenethylamine, the nitrite, the 2-phenylglyoxyamine, the 2-hexanyl glycerol, the 4-hydroxy-L-glutamic acid, the 1-ethanone-2-coumaric acid, the D-proline, (3R, 7R) -1,3,7-Octanetriol. L-Inisoleucine is a potential biomarker for FD. The potential target pathway associated with the FD disease may be phenylalanine metabolism, arginine and proline metabolism. The mechanism of the metabolic group in the treatment of FD by acupuncture combined with the acupoint may be related to the regulation of the metabolism of phenylalanine, arginine and proline. Acupuncture combined with the points of distribution, the lower combined point and the point-raising point have a certain benign regulation effect on the metabolic markers of the FD-related metabolic markers, and the overall regulation effect of the combined points is better than that of the lower and the point-raising points.
【学位授予单位】：成都中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R246.1

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