Therapeutic Effect and Mechanism of Si-Miao-Yong-An-Tang on Thromboangiitis Obliterans Based on the Urine Metabolomics Approach

Abstract

Si-Miao-Yong-An-Tang (SMYAT) is a classic prescription for the treatment of thromboangiitis obliterans (TAO). However, the effect and mechanism are still unclear. This experiment aims to evaluate the therapeutic effect and mechanism of SMYAT on sodium laurate solution induced thromboangiitis obliterans model rats using urine metabolomics. The therapeutic effect of SMYAT was evaluated by histopathology, hemorheology and other indexes. The urine metabolomic method, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for clustering group and discriminant analysis to screen urine differential metabolic biomarkers, and explore new insight into pathophysiological mechanisms of SMYAT in the treatment of TAO. SMYAT has significant antithrombotic and anti-inflammatory effects, according to the results of urine metabolomic analysis, and regulate the metabolic profile of TAO rats, and its return profile is close to the state of control group. Through metabolomics technology, a total of 35 urine biomarkers of TAO model were characterized. Among them, SMYAT treatment can regulate 22 core biomarkers, such as normetanephrine and 4-pyridoxic acid. It is found that the therapeutic effect of SMYAT is closely related to the tyrosine metabolism, vitamin B6 metabolism and cysteine and methionine metabolism. It preliminarily explored the therapeutic mechanism of SMYAT, and provided a scientific basis for the application of SMYAT.

Keywords: biomarker; metabolites; metabolomics; pathway; therapeutic effect and mechanism.

FIGURE 1

Metabolomic profiling of TAO. PCA model results for TAO model group in positive mode (A); OPLS-DA model results for TAO model group in positive mode (B); Black spot represents the control group and red spot represents the model group.

FIGURE 2

Change trend of biomarker content in control group and model group. *Significant difference between the control group and model group (p < 0.05). **Very significant difference between the control group and model group (p < 0.01).

FIGURE 3

Systems analysis of metabolomic alterations of the model and control samples with MetaboAnalyst’s data annotation tools: (A) PLS-plot revealed differences between the two groups. (B) Correlation analysis of the 10 differential metabolites are marked on the plot. Three clusters were identified representing the different groups of metabolites. Top significant features of the metabolite markers based the VIP projection (B). (C) Hierarchical clustering of the differential metabolites. (D) Heatmap visualization constructed based on the differential metabolites of importance for the urine of model. The heatmaps were constructed based on the potential candidates of importance, which were extracted with OPLS-DA analysis. Rows: metabolites; columns: samples; Variable differences are revealed between the control and model groups.

FIGURE 4

Main metabolic pathways of potential biomarkers. (1) beta-Alanine metabolism; (2) Phenylalanine metabolism; (3) Histidine metabolism; (4) Cysteine and methionine metabolism; (5) Citrate cycle (TCA cycle); (6) Steroid hormone biosynthesis; (7) Pantothenate and CoA biosynthesis; (8) Glyoxylate and dicarboxylate metabolism; (9) Tyrosine metabolism; (10) Tryptophan metabolism; (11) Vitamin B6 metabolism; (12) Ascorbate and aldarate metabolism; (13) Propanoate metabolism; (14) Glutathione metabolism; (15) Arginine and proline metabolism; (16) Pyrimidine metabolism; (17) Aminoacyl-tRNA biosynthesis.

FIGURE 5

(A) Effects of administration on plasma viscosity and whole blood viscosity of rats in each group; (B) Effects of administration on related indexes of red blood cells of rats in each group; *p < 0.05, **p < 0.01 compared with control group; ^# p < 0.05, ^## p < 0.01 compared with model group.

FIGURE 6

Femoral artery sections of rats in each group on the 25th day of the experiment (×400); (A): control group; (B): model group; (C): SMYAT group; (D): MLN group.

FIGURE 7

The PCA score plot of control, model, SMYAT, and MLN groups in urine metabolism profile. (A) Positive ion mode and (B) negative ion mode. Black spot represents the control group, red spot represents the model group, green spot represents the SMYAT group and blue spot represents the MLN group.

FIGURE 8

*Significant difference between the control group and model group (p < 0.05). **Very significant difference between the control group and model group (p < 0.01). ^# Significant difference between SMYAT and model groups, p < 0.05. ^## Very significant difference compared between SMYAT and model groups, p < 0.01.

FIGURE 9

Correlation networks of the potential biomarkers based on the KEGG.