Targeted Metabolomic Study Of Acupuncture Along Meridians For Chronic Stable Angina Pectoris

Aim:To provide scientific evidences for elucidating acupoint specificity from the angle of metabolomics, ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) based technology was used to investigate the metabolite regulating effects of acupuncture on points of disease-affected meridian, non-affected meridian and non-acupoints for Chronic Stable Angina Pectoris (CSAP), discuss the discrepancies and rules of metabolomic effects induced by different poiont-selection methods.Methods:1. Clinical observation on the therapeutic effect of CSAP treated by acupuncture with selecting acupoints along channels. Randomized and controlled trial was designed. Under basement treatment,43 patients matched the inclusion criteria were randomly assigned to four groups:Group A, acupuncture on points of disease-affected meridian (PC6, HT5); Group B, acupuncture on points of non-affected meridian (LU9, LU6); Group C, acupuncture on 2 non-acupoints; Group D, received no acupuncture related intervention. Participants in the acupuncture groups received 12 sessions of acupuncture treatment over 4 weeks. Frequency of angina attack in 4 weeks, pain severity of angina (Visual Analogue Scale, VAS), score of Seattle Angina Questionnaire (SAQ),6 Six minutes’ Walk Test (6MWT), self-rating depression scale (SDS), and self-rating anxiety scale (SAS) were selected as outcome measurements. These measurements were evaluated at 1 day before inclusion and the day finished treatment.2. UFLC-MS/MS based metabonomic study on the metabolic changes in the plasma of patients with CSAP treated by acupuncture with selecting acupoints along channels. Plasma samples of CSAP patients from clinical trial were collected at 1 day before inclusion and the day finished treatment. UFLC-MS/MS based metabonomic method was used to test 24 kinds of target metabolites. Analysis methods, such as Partial Least Square Discriminant Analysis (PLS-DA), et al, were applied to process massive metabolomic data, and the discrepancies of metabolomic effects induced by different point-selection methods were discussed.3. The correlation study of CSAP plasma metabolomic data and clinical results of acupuncture with selecting acupoints along channels. Pearson Linear Correlation Analysis was applied to screen metabolites correlated with clinical outcomes, pathway topology analysis and pathway impact analysis were used to analyze related metabolic pathways. Potential biomarkers were confirmed and the discrepancies or rules of metabolomic effects induced by different point-selection methods were discussed.Results:1. Clinical results of CSAP treated by acupuncture with selecting acupoints along channels.(1) After treatment, frequency of angina attack in 4 weeks (P=0.000), pain severity of angina (P=0.006) were remarkably suppressed and SAQ score were improved in Group A, and the efficacy was superior to the other 3 groups. Moreover, scores of SAS (P=0.000) and SDS (P=0.011) were significantly improved in group A.(2) After treatment, subjective improving rate and effective rate in Group A were significant higher than the other 3 groups.2. Plasma metabolomics of patients with CSAP treated by acupuncture with selecting acupoints along channels.(1) Group A:After treatment, plasma levels of L-Isoleucine (P=0.026), L-Leucine (P=0.027), L-Tyrosine (P=0.049) and L-Valine (P=0.045) were significantly decreased. Betaine, Hexanoic acid, myo-Inositol, urea, Oxaloacetic acid, D-Sorbitol, Hypoxanthine, D-Homoserine, L-Isoleucine, L-Tyrosine, L-Phenylalanine, L-Tryptophan, L-Leucine, L-Threonine, L-Valine, D-(+)-Galactose, a-ketoglutaric acid, D-(-)-Fructose and D-(+)-Cellobiose contributed more to the differences before and after acupuncture (VIP value>0.8), were considered as important metabolites.(2) Group B:After treatment, plasma level of metabolites were not significantly changed. Betaine, urea, D-(+)-Galactose, D-(-)-Fructose and Uric acid contributed more to the differences before and after acupuncture (VIP value> 0.8), were considered as important metabolites.(3) Group C:After treatment, plasma level of Glycine (P=0.022) were significantly increased. Urea, D-(+)-Galactose and D-(-)-Fructose contributed more to the differences before and after acupuncture (VIP value>0.8), were considered as important metabolites.(4) Group D:After treatment, plasma level of L-Threonine (P=0.014) were significantly decreased. D-(+)-Galactose and D-(-)-Fructose contributed more to the differences before and after treatment (VIP value>0.8), were considered as important metabolites.(5) Compared with the other 3 groups, plasma level of Urea was highest, D-(+)-Galactose and D-(-)-Fructose were lowest in Group A, suggesting this 3 metabolites may be biomarkers of therapeutic effects of acupuncture with selecting acupoints along channels compared with other acupoint selection methods.3. The results of correlation study of CSAP plasma metabolomic data and clinical results of acupuncture with selecting acupoints along channels.(1) The Pearson correlation analysis of baseline CSAP plasma metabolomic data and clinical manifestations showed that plasma level of Citrulline positively correlated with pain severity of angina, L-Phenylalanine, L-Leucine and L-Valine positively correlated with 6MWT, Hexanoic acid positively correlated with SAS and SDS.(2) Group A:The Pearson correlation analysis of CSAP plasma metabolic changes and clinical manifestation alterations showed that plasma levels of L-Isoleucine, L-Leucine, L-Threonine, L-Valine, D-(+)-Galactose, D-(-)-Fructose, Hexanoic acid, L-Phenylalanine and Citrulline positively correlated with frequency of angina attack in 4 weeks, D-(+)-Cellobiose, urea, myo-Inositol, D-Sorbitol, Pimelic acid, Hypoxanthine and a-ketoglutaric acid negatively correlated with frequency of angina attack in 4 weeks; Hexanoic acid positively correlated with SDS, Uric acid and Glycine negatively correlated with SDS.(3) Group B:The Pearson correlation analysis of CSAP plasma metabolic changes and clinical manifestation alterations showed that plasma levels of Hypoxanthine positively correlated with frequency of angina attack in 4 weeks, Glycine negatively correlated with frequency of angina attack in 4 weeks; L-Tyrosine positively correlated with 6MWT; L-Tyrosine, L-Tryptophan and Glycine negatively correlated with SAS; Glycine negatively correlated with SDS.(4) Group C:The Pearson correlation analysis showed that none of CSAP plasma metabolic changes correlated with clinical manifestation alterations.(5) Group D:The Pearson correlation analysis of CSAP plasma metabolic changes and clinical manifestation alterations showed that plasma level of a-ketoglutaric acid negatively correlated with frequency of angina attack in 4 weeks,6MWT and SDS.(6) Metabolic pathway analysis showed that acupuncture on points of disease-affected meridian involved in amino acid, purine and energy metabolic pathways; acupuncture on points of non-affected meridian involved in aromatic amino acid and purine metabolic pathway; acupuncture on non-acupoints involved in purine metabolic pathway.Conclusions:1. Clinical efficacy of acupuncturing are better at the acupoints on disease-affected meridian (Group A) for CSAP than at the acupoints on non-affected meridian (Group B), at the non-acupoints and basement treatment. Moreover, acupuncture on points of disease-affected meridian can ameliorate anxiety and depression state of CSAP patients.2. All of the 4 kinds of treatments can regulate CSAP plasma metabolites to a certain extent, but in separated direction. Acupuncture on points of disease-affected meridian can affect most metabolites and wider range of metabolic pathways, suggesting significant difference compared with other treatments.3. After acupuncture at points on disease-affected meridian and non-affected meridian, changes of some plasma metabolites correlated with clinical manifestation alterations, but the metabolomic effect of former treatment involved in more cardiovascular biomarkers, correspond better to clinical improvements; metabolomic changes after acupuncture on non-acupoints or basement treatment were not obviously correlated with clinical alteration.4. Amino acid metabolism, especially branched chain amino acids (BCAAs) metabolic pathway has closest relation to metabolomic effect and clinical efficacy of acupuncture at points on disease-affected meridian, amino acids are key metabolites differ from other interventions, main metabolomic mechanism of acupuncture at points on disease-affected meridian for CSAP may involve in amino acid metabolic pathway.