Exploration of the anti-hyperuricemia effect of TongFengTangSan (TFTS) by UPLC-Q-TOF/MS-based non-targeted metabonomics

Abstract

Background: TongFengTangSan (TFTS) is a commonly used Tibetan prescription for gout treatment. Previously, TFTS (CF) was confirmed to have a significant uric acid-lowering effect. However, the anti-hyperuricemia mechanisms and the main active fractions remain unclear. The current study aimed to investigate the anti-hyperuricemia mechanism using metabolomics and confirm the active CF fraction.

Methods: The hyperuricemia model was established through intraperitoneal injection containing 100 mg/kg potassium oxonate and 150 mg/kg hypoxanthine by gavage. We used serum uric acid (sUA), creatinine (CRE), blood urea nitrogen (BUN), xanthine oxidase (XOD) activity, interleukin-6 (IL-6) and interleukin-1β (IL-1β) as indicators to evaluate the efficacy of CF and the four fractions (SX, CF30, CF60, and CF90). The anti-hyperuricemia mechanism of CF was considered through non-targeted metabolomics depending on the UPLC-Q-TOF-MS technology. Principle component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) helped explore the potential biomarkers in hyperuricemia. Moreover, the differential metabolites and metabolic pathways regulated by CF and four fractions were also assessed.

Results: CF revealed a significant anti-hyperuricemia effect by down-regulating the level of sUA, sCRE, sIL-1β, and XOD. SX, CF30, CF60, and CF90 differed in the anti-hyperuricemia effect. Only CF60 significantly lowered the sUA level among the four fractions, and it could be the main efficacy fraction of TFTS. Forty-three differential metabolites were identified in hyperuricemia rats from plasma and kidney. Pathway analysis demonstrated that seven pathways were disrupted among hyperuricemia rats. CF reversed 19 metabolites in hyperuricemia rats and exerted an anti-hyperuricemia effect by regulating purine metabolism. CF60 was the main active fraction of TFTS and exerted a similar effect of CF by regulating purine metabolism.

Conclusions: CF and CF60 could exert an anti-hyperuricemia effect by regulating the abnormal purine metabolism because of hyperuricemia while improving intestinal and renal function. CF60 could be the main active fraction of TFTS.

Keywords: Hyperuricemia; Mechanism; Metabolomics; TFTS.

Fig.1

Anti-hyperuricemia effect of CF and four fractions. Compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with model group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001

Fig.2

Histopathological study of kidney (× 40 and × 200).The renal tubules in kidney exhibited expansion (black arrow) and vacuolar degeneration (blue arrow)

Fig.3

TIC of differential metabolites in plasma and kidney samples obtained in positive and negative modes

Fig.4

Multivariate statistical analysis of metabolomics. A PCA score plot. B OPLS-DA score plot. C Permutation Plot. D S-plot

Fig.5

Heatmap of the differential metabolites between CF and four fractions. *p < 0.05, **p < 0.01. Red: up-regulation; Blue: down-regulation

Fig.6

Overlapping metabolites between CF and four fractions illustrated by Venn diagram. Model group vs. Normal group (Model/Control), CF group vs. Model group (CF/Model), SX group vs. Model group (SX/Model), CF30 group vs. Model group (CF30/Model), CF60 group vs. Model group (CF60/Model), CF90 group vs. Model group (CF90/Model)

Fig.7

Pearson correlation analysis of differential metabolites and anti-hyperuricemia efficacy. *p < 0.05, **p < 0.01

Fig.8

Metabolic pathway analysis of significantly altered metabolites in plasma and kidney

Fig.9

Network of the key biomarkers and pathways according to the KEGG pathway databases