Abstract:

The objective of this research is to investigate the suppressive effect of silymarin on vitro cell culture model of inflammatory macrophage RAW264.7 induced by Kdo2-Lipid A, and explore its mechanism based on cell metabonomics. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was used in the cell metabonomic assay to quantitative analysis of metabolites related to eicosanoids pathway. Then chemometric approaches such as principal component analysis were used to process the metabolic data. Within the established method, a total of 59 eicosanoids standards (containing 15 deuterated internal standards) were simultaneously separated in a single 5 min run, and the analytical method is proved to be rapid, sensitive and accurate. Whereafter, the metabolites with VIP> 1 and P value< 0.05 were considered as biomarkers. 12-OxoLeukotriene B₄ (12-OxoLTB₄) was eventually identified as metabolic biomarkers of silymarin treatment group in this research, and according to the related inflammatory pathways, we speculated silymarin has anti-inflammatory activities by inhibiting the 5-lipoxygenase (5-LOX) activity and blocking lipid peroxidation in 5-LOX metabolic pathways to reduce the formation of peroxides and oxygen free radicals. This study provide a novel approach to the mechanism research on the silymarin treatment on RAW264.7 cells based on cell metabonomics.

Key words: deuterated internal standards, eicosanoids, inflammatory, metabonomics, silymarin, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)