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

Figure 5

Effect of GAL on LPS-induced ROS production and MAP kinase activation. (a) Detection of intracellular superoxide ion. Following GAL pretreatment for 2 h and LPS stimulation for another 2 h, RAW264.7 cells were incubated with DHE and imaged under a fluorescence microscope (Carl-Zeiss, Jena, Germany). The fluorescence was quantified by using NIH Image J software (http://imagej.nih.gov/ij/). Results represent means ± SD of three independent experiments. ; (GAL + LPS versus LPS). Scale bar, 20 μm. (b) Detection of intracellular ROS. Following GAL pretreatment for 2 h and LPS stimulation for another 2 h, RAW264.7 cells were incubated with DCFH2-DA and imaged under a fluorescence microscope (Carl-Zeiss, Jena, Germany). The fluorescence was quantified by using NIH Image J software (http://imagej.nih.gov/ij/). Results represent means ± SD of three independent experiments. ; (GAL + LPS versus LPS). (c) Western blot analysis of MAP kinase activation. Following GAL pretreatment for 2 h and LPS stimulation for another 20 min, the cellular proteins were isolated from RAW264.7 cells and primary macrophages and subsequently analyzed by Western blotting using specific antibodies. Western blots were quantified by a densitometric method. ; (GAL + LPS versus LPS).

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