Serum metabolomics strategy for understanding the therapeutic effects of Yin-Chen-Hao-Tang against Yanghuang syndrome

Abstract

Yin-Chen-Hao-Tang (YCHT), a classic Chinese herbal formula, is characterized by its strong therapeutic effects of liver regulation and relief of jaundice, especially Yanghuang syndrome (YHS). YHS is a type of jaundice with damp-heat pathogenesis, and it is considered a complicated Chinese medicine syndrome (CMS). The accurate mechanism for healing YHS has not yet been completely reported. The purpose of the current research is to investigate the expression of endogenous biomarkers in YHS mice and evaluate the clinical therapeutic effect of YCHT. Serum samples were analyzed using UPLC-Q/TOF-MS techniques in order to determine differential metabolites to elucidate the functional mechanism of YCHT on YHS through metabolite profiling combined with multivariate analysis. Simultaneously, the exact diversification of YHS mice was elucidated using blood biochemistry indexes and histopathological examination, and the results indicated that YHS is markedly improved by YCHT. Unsupervised principal component analysis (PCA) patterns were constructed to dissect the variances of metabolic profiling. Overall, 22 potential biomarkers were identified using a metabolomics approach based on an accurate MS/MS approach, clustering and distinguishing analysis. The present work demonstrates that the effectiveness of YCHT against YHS prompts distinct discrepancies in metabolic profiles by adjusting biomarkers and regulating metabolic disorders. A total of 15 metabolic pathways were involved in biological disturbance. This demonstrates that metabolomic techniques are powerful means to explore the pathogenesis of CMS and the therapeutic effects of traditional Chinese formulae.

Fig. 1. Biochemical analysis of the therapeutic effects of YCHT against Yanghuang syndrome. All of the results are expressed as mean ± SD. () Control, () YHS and () YCHT. The YHS group compared with the control group: *p < 0.05, **p < 0.01; and the YCHT group compared with the YHS group: #p < 0.05, ##p < 0.01. The specific values and units are shown in ESI Table 1.

Fig. 2. H & E staining in the therapeutic study of YCHT against Yanghuang syndrome. Control group (A), YHS group (B) and YCHT group (C) (magnification ×100). The structure of the hepatic lobule in the control group is complete, the hepatocytes are arranged radially in a central vein, there is dyeing uniformity, and the shape of hepatocytes is normal. But hepatocytes in the YHS group showed significant changes according to the histopathological observation. In particular, a large area of focal necrosis, ballooning degeneration and inflammatory cell infiltration are detected, and the liver tissue shows homogeneous powder staining in the YHS group compared with the control group. The shape of the hepatocytes is normal and the outline of the sinus hepaticus is clear in YCHT group mice compared with the YHS group.

Fig. 3. Multivariate data analysis of the serum metabolite data. 2D score plot of PCA showing the control group (), YHS group () and QC samples () in the positive mode (A) and negative mode (B). 3D score plot of PCA in the positive mode (C) and negative mode (D).

Fig. 4. Relative signal intensities of serum metabolic biomarkers identified using UPLC/MS. Data are expressed as mean ± SD. The YHS group compared with the control group: *p < 0.05, **p < 0.01; and the YCHT group compared with the YHS group: #p < 0.05, ##p < 0.01.

Fig. 5. Metabolic pathway analysis of serum biomarkers. (1) Glycerophospholipid metabolism; (2) phenylalanine metabolism; (3) pentose and glucuronate interconversions; (4) ascorbic acid metabolism; (5) glycerolipid metabolism; (6) tryptophan metabolism; (7) linoleic acid metabolism, (8) biosynthesis of phenylalanine, tyrosine and tryptophan; (9) alpha-linolenic acid metabolism; (10) glutathione metabolism; (11) phosphoinositol metabolism; (12) starch and sucrose metabolism; (13) arachidonic acid metabolism; (14) tyrosine metabolism; (15) amino sugar and nucleotide glucose metabolism.

Fig. 6. Pattern recognition analysis of the 3D score plot of PCA of YHS after YCHT treatment in the positive mode (A) and negative mode (B). () Control group, () YHS group, () YCHT group, and () QC samples.

Fig. 7. Heat map analysis of potential biomarkers among the control, YHS and YCHT groups. The degree of change is marked by different colors: red denotes upregulation and blue denotes downregulation. Each column represents an individual sample, and each row represents a biomarker.

Fig. 8. Correlation networks of all of the potential biomarkers on KEGG. The red font represents the biomarkers detected in this experiment.