Serum metabonomics coupled with HPLC-LTQ/orbitrap MS and multivariate data analysis on the ameliorative effects of Bidens bipinnata L. in hyperlipidemic rats

Abstract

Ethnopharmacological relevance: Hyperlipidemia (HLP) is a prevalence chronic cardiovascular disease, which is treated by traditional Chinese medicine (TCM) in China. More and more attention has been paid to the application of metabolomics in the study of TCM. Bidens bipinnata L. (BBL), a folk medicine in contemporary China, has the efficacy in the treatment of hyperlipidemia (HLP) in China. However, little is known of the underlying mechanism of BBL. This research aimed to investigate ameliorative effects of BBL on hyperlipidemic rats and explore the mechanism by metabolomics method.

Materials and methods: Hyperlipidemic rats were established by high fat diet (HFD). Biochemical assay was used to evaluate the efficacy of BBL. A metabolomics approach based on high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry (HPLC-LTQ/orbitrap MS) was performed to analyze the serum biomarkers from model group, control group and BBL group. Principle component analysis (PCA) and partial least-squares discriminate analysis (PLS-DA) were utilized to identify differences of metabolic profiles in rats among the three groups. In order to identify possible pathways that were affected by HLP, the identified endogenous metabolites were analyzed by using MetaboAnalyst. In the network pharmacology study, our research group found that PPAR signaling pathway was the most important pathway of BBL in the treatment of HLP. Then, it was found that changes in the major metabolic pathways would affect the PPAR signaling pathway through comprehensive analysis based on KEGG database. Therefore, the expression of key genes in the PPAR signaling pathway was detected by real-time quantitative fluorescence PCR (RT-qPCR).

Results: Six metabolites, which showed a significantly restoring trend from HLP to normal condition, were regarded as potential biomarkers of BBL treatment. The levels of phosphorylcholine, mevalonic acid and leukotriene B₄ (LTB₄) increased significantly (P < 0.01) in hyperlipidemic rats, while the levels of linoleic acid, arachidonic acid (AA) and lysophosphatidylcholine (18:0) (Lyso PC (18:0)) decreased significantly (P < 0.01) in comparison with control rats. Those endogenous metabolites were chiefly involved in linoleic acid metabolism, AA metabolism and terpenoid backbone biosynthesis. According to the results of RT-qPCR analysis, the mRNA expressions of PPAR α, PPAR β and PPARγ in model group were difference compared with control group. And the expression difference could be regulated closer to normal level after BBL intervention.

Conclusions: The results of biochemical assay, serum metabolic pattern and RT-qPCR analysis showed that BBL could exert a significant improvement on lipid levels, liver function, renal function, as well as the mRNA expression level of PPAR signaling pathway.

Keywords: Bidens bipinnata L.; HPLC-LTQ/Orbitrap MS; Hyperlipidemia; Metabonomics; PPAR signaling Pathway.