Journal List > Korean J Physiol Pharmacol > v.14(6) > 1025702

Li, Kothandan, Cho, Huong, Nan, Lee, Shin, Yea, and Jeon: Magnolol Inhibits LPS-induced NF-κB/Rel Activation by Blocking p38 Kinase in Murine Macrophages

Abstract

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-κB/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-κB/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-κB/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-κB/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-κB/Rel and p38 kinase signaling.

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Fig. 1.
Inhibition of macrophage activation by magnolol. (A) RAW 264.7 cells (5×105 cells/ml) incubated with magnolol (50μM) in the presence of LPS (200 ng/ml) for 24 hr on cover slide in 12 well plates. Cells were subjected to immunohistochemical staining using an antibody specific for murine iNOS. Immunoreactivity of iNOS was localized along the margins of the cytoplasm in control group. (B) Cells were treated with magnolol in the presence of LPS (200 ng/ml) for 24 hr. Cell lysates were then prepared and subjected to Western immunoblotting. (C) RAW 264.7 cells were transfected with p(NF-κB/Rel)3-CAT by DEAE dextran method. Twenty-four hours after transfection, cells were treated with the magnolol in the presence or absence of LPS (200 ng/ml) for 18 hr. Cell extracts were then prepared and analyzed for the expression of CAT using CAT ELISA kit. (D) Cells (5×105 cells/ml) were incubated with magnolol (50μM) in the presence or absence of LPS (200 ng/ml) for 2 hr. Nuclear extracts (5μg/ml) were then isolated and analyzed for the activity of NF-κB/Rel and Oct.
kjpp-14-353f1.tif
Fig. 2.
Molecular docking and pose generation. (A) A docking study was performed using SYBYL8.1 molecular modeling package as described in Materials and methods. Magnolol was docked with p38 kinase structure (PDB code: 3GC7). The ligand is represented in space fill model and the macromolecule is in ribbon and tube. (B) The proposed binding pose of Magnolol showed the interaction with the backbone of hinge residues M109 and G110. (C) Chemical structure of magnolol is shown. (D) Flag-p38 expression vectror was transiently transfected to 293 cells. Cell lysates were then prepared, used for pull-down assay using magnolol-Sepharose 4B beads and subjected to Western blot analysis.
kjpp-14-353f2.tif
Fig. 3.
Effects of SB203580 and PD98059 on NF-κB/Rel activation in LPS-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were transfected with p(NF-κB/Rel)3-CAT by DEAE dextran method. Twenty-four hours after transfection, cells were treated with SB203580 (30μM), PD98059 (50μM), or magnolol (50μM) in the presence of LPS (200 ng/ml) for 18 hr. Cell extracts were then prepared and analyzed for the expression of CAT using CAT ELISA kit. (B) RAW 264.7 cells were pretreated with SB203580 (30μM), PD98059 (50μM), or magnolol (50μM) for 30 min before incubation with LPS (200 ng/ml) for 2 hr. Nuclear extracts were then isolated and analyzed for the activity of NF-κB/Rel and Oct.
kjpp-14-353f3.tif
Table 1.
Ligands with Biphenyl Moiety bound to p38 and their resolution
kjpp-14-353f4.tif
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