Gallic Acid-L-Leucine Conjugate Protects Mice against LPS-Induced Inflammation and Sepsis via Correcting Proinflammatory Lipid Mediator Profiles and Oxidative Stress

Figure 2

Effects of GAL on the production of proinflammatory mediators (i.e., NO and lipid mediators). (a) Effect of GAL on LPS-induced NO production in RAW264.7 cell and primary macrophages. Following 24 h treatment with LPS and GAL, the intracellular NO was detected by fluorescent probe DAF-FM diacetate and subsequently imaged on a Zeiss fluorescence microscopy (Zeiss, Jena, Germany). Representative images were shown and quantified by Image J software (http://imagej.nih.gov/ij/). The results represent the mean ± SD of three independent experiments. ; (sample versus LPS alone); scale bar, 20 μm. (b) Effect of GAL on LPS-induced NO production in RAW264.7 cell and culture medium. Following 24 h treatment with LPS and GAL, the intracellular NO was detected by fluorescent probe DAF-FM diacetate on Fusion™α-FP microplate reader (Packard BioScience Company, USA). The NO levels in cell culture medium were assayed by using Griess reagent kit (Thermo Fisher Scientific, MA USA). (c) Scheme illustrating the metabolic pathways for the generation of lipid mediators. (d) LC-MS/MS determination of proinflammatory lipid mediators. After the treatments with GAL and LPS, the lipids were isolated from RAW264.7 cells and culture medium and subsequently quantified by a MRM method on a LC-MS/MS system.

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