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

Figure 6

Effects of GAL on LPS-induced activation of NF-κB and AP-1 pathways. (a) Immunofluorescence staining of intracellular NF-κB subunit p65 in RAW264.7 cells. Following the treatments of RAW264.7 cells with GAL for 2 h and LPS for 2 h, NF-κB p65 subunit was stained by specific antibodies, whereas the nuclei were stained by DAPI. Images were captured under a fluorescence microscopy (Carl-Zeiss, Jena, Germany). (b) Immunofluorescence staining of intracellular NF-κB subunit p65 in primary macrophages. Primary peritoneal macrophages were similarly treated with GAL and LPS. NF-κB p65 subunit was detected and imaged as described in (a). (c) Western blot analysis of intracellular NF-κB subunit p65. After the treatments of RAW264.7 cells with GAL and LPS, cytosolic and nuclear proteins were isolated, analyzed by Western blotting, and determined by a densitometric method. The values represent means ± SD of three independent experiments. ; (GAL + LPS versus LPS). (d) Western blot analysis nuclear of NF-κB and c-fos in primary macrophages. Primary peritoneal macrophages were similarly treated with GAL and LPS. NF-κB and c-fos were determined as described in (c). (e) Western blot analysis of c-fos and c-jun phosphorylation. After the treatments of RAW264.7 cells with GAL and LPS, the cellular proteins were analyzed by Western blotting with specific antibodies. The protein levels were determined by a densitometric method. The values represent means ± SD of three independent experiments. ; (GAL + LPS versus LPS). (f) Western blot analysis of c-fos and c-jun in the cell nucleus. After the treatments of RAW264.7 cells with GAL and LPS, the nuclear proteins were isolated and analyzed by Western blotting with specific antibodies. The protein levels were determined by a densitometric method. The values represent means ± SD of three independent experiments. ; (GAL + LPS versus LPS). LB: lamin B.

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