UPLC MS/MS Based Profiling of Eicosanoids in RAW264.7 Cells Treated with Lipopolysaccharide
While both the pro and anti inflammatory effects of several eicosanoids have been widely studied, the degree of inflammation in cells that results from various eicosanoids has yet to be comprehensively studied. The objective of this study was to assess the effect of lipopolysaccharide (LPS) treatment on eicosanoid content in RAW264.7 cells. An Ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC MS/MS) based profiling method was used to analyze the eicosanoid contents of RAW264.7 cells treated with different LPS concentrations. The profiling data were subjected to statistical analyses, such as principal component analysis (PCA) and hierarchical clustering analysis. LPS treatment increased nitric oxide production and secretion of pro inflammatory cytokines, such as tumor necrosis factor a and interleukin 6, in a concentration dependent manner. In total, 79 eicosanoids were identified in the cells. RAW264.7 cells treated with different LPS concentrations were well differentiated in the PCA score plot. A heatmap was used to identify the eicosanoids that were up or down regulated according to the degree of inflammation and LPS concentration. Thirty nine eicosanoids were upregulated and seven were down regulated by LPS treatment in a concentration dependent manner. Our novel UPLC MS/MS technique can profile eicosanoids, and can evaluate the correlations between inflammation and eicosanoid metabolism.
International journal of molecular sciences