Targeting MAPK/NF-κB Pathways in Anti-Inflammatory Potential of Rutaecarpine: Impact on Src/FAK-Mediated Macrophage Migration

Abstract

Studies have discovered that different extracts of Evodia rutaecarpa and its phytochemicals show a variety of biological activities associated with inflammation. Although rutaecarpine, an alkaloid isolated from the unripe fruit of E. rutaecarpa, has been exposed to have anti-inflammatory properties, the mechanism of action has not been well studied. Thus, this study investigated the molecular mechanisms of rutaecarpine (RUT) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. RUT reserved the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL)-1β in the LPS-induced macrophages. RUT showed an inhibitory effect on the mitogen-activated protein kinases (MAPKs), and it also inhibited nuclear transcription factor kappa-B (NF-κB) by hindering IκBα and NF-κB p65 phosphorylation and p65 nuclear translocation. The phospho-PI3K and Akt was concentration-dependently suppressed by RUT. However, RUT not only suggestively reduced the migratory ability of macrophages and their numbers induced by LPS but also inhibited the phospho-Src, and FAK. Taken together, these results indicate that RUT participates a vital role in the inhibition of LPS-induced inflammatory processes in RAW 264.7 macrophages and that the mechanisms involve PI3K/Akt and MAPK-mediated downregulation of NF-κB signaling pathways. Notably, reducing the migration and number of cells induced by LPS via inhibiting of Src/FAK pathway was also included to the anti-inflammatory mechanism of RUT. Therefore, RUT may have potential benefits as a therapeutic agent against chronic inflammatory diseases.

Keywords: MAPK; NF-κB; PI3K/Akt; Src/FAK; anti-inflammation; cell migration; molecular mechanism; rutaecarpine.

Figure 1

Effects of rutaecarpine (RUT) on cell viability in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. (A) Chemical structure of RUT. (B) Cells were treated with 0.1% DMSO or pretreated with RUT (10–100 μM) for 24 h. (C) Cells were treated with 0.1% DMSO or pretreated with RUT (10–100 μM) for 20 min and then treated with LPS (1 μg/mL) for 24 h. Cell viability was evaluated as described in the Methods section. Data are presented as the means ± SEM (n = 4). *** p < 0.001, compared with the control group; ^### p < 0.001, compared with the LPS group.

Figure 2

Effects of RUT on nitric oxide (NO) production, and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) in LPS-stimulated RAW cells. Cells were pretreated with RUT (10 and 20 μM) for 20 min and then stimulated by LPS (1 μg/mL) for 24 h. (A) NO was measured using Griess reagent. (B–F) The levels of (C) iNOS, (D) COX-2, (E) TNF-α, and (F) IL-1β protein expression were evaluated as described in the Methods section. Data are presented as the means ± SEM (n = 4); * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with the control group; ^# p <0.05 and ^## p < 0.01, compared with the LPS group.

Figure 3

Influence of RUT on LPS-induced phosphorylation of c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinase (ERK) in RAW cells (A–D). Cells were treated with 0.1% DMSO or RUT (10 and 20 μM) for 20 min, followed by LPS (1 μg/mL) for 30 min, and the phosphorylation of (B) JNK, (C) p38 MAPK, and (D) ERK were evaluated by immunoblotting assay as described in the Methods. Data are presented as the means ± SEM (n = 4). ** p < 0.01 and *** p < 0.001, compared with the control group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with the LPS group.

Figure 4

Effect of RUT on LPS-induced phosphoinositide 3-kinase (PI3K)/Akt signaling pathways in RAW cells. RAW cells were treated with 0.1% DMSO or RUT (10 and 20 μM) for 20 min, followed by LPS (1 μg/mL) for 30 min. The expression of phosphorylated (A) PI3K and (B) Akt were detected by immunoblotting. Data are expressed as the mean ± SEM (n = 4). ** p < 0.01, compared with the control group; ^# p < 0.05 and ^## p < 0.01, compared with the LPS group.

Figure 5

Effects of RUT on LPS-induced IκBα and p65 phosphorylation, and nuclear translocation of NF-κB p65 in RAW cells. (A,B) Cells were treated with 0.1% DMSO or RUT (10 and 20 μM) for 20 min, followed by LPS (1 μg/mL) for 30 min. The phosphorylation of (A) IκBα and (B) p65 were determined by immunoblotting. (C,D) Cells were treated with 0.1% DMSO or RUT (20 μM) for 20 min, followed by LPS (1 μg/mL) for 30 min. The cytosolic and nuclear fractions were isolated using the NE-PER kit and then subjected to Western blotting to detect p65 expression. α-tubulin and Lamin B1 were used as internal controls for the nucleus and cytosol, respectively. Data are presented as the means ± SEM (n = 4). * p < 0.05 and ** p < 0.01, compared with the control group; ^# p < 0.05 and ^## p < 0.01, compared with the LPS group.

Figure 6

Effects of RUT on LPS induced NF-κB p65 nuclear translocation in RAW cells. Cells were treated with 0.1% DMSO, RUT (20 μM) for 20 min, followed by LPS (1 μg/mL) for 30 min. (A) The immunofluorescence staining analysis was performed with an anti-p65 antibody and FITC-conjugated anti-rabbit IgG antibody (green). 4′,6-diamidino-2-phenylindole (DAPI) was used to label the nuclei (blue). The images were captured by confocal microscopy (scale bar = 5 μm). (B) Data were graphed by pooling multiple images, with each individual data point corresponding to the mean fluorescence intensity of each individual cell nucleus. Data are presented as the means ± SEM (n = 4). ** p < 0.01, compared with the control group; ^# p < 0.05, compared with the LPS group.

Figure 7

Effects of RUT on migration and Src/FAK expression in LPS-treated RAW cells. (A–C) Cells were treated with 0.1% DMSO or RUT (20 μM) for 20 min, followed by LPS (1 μg/mL) for 24 h. Cell migration was measured by scratch wound healing assay. (A) Images of cell migration at 0 and 24 h. (B) Src and (C) FAK phosphorylation were determined by immunoblotting. Data are presented as the means ± SEM (n = 4). ** p < 0.01 and *** p < 0.001, compared with the control group; ^# p < 0.05 and ^## p < 0.01, compared with the LPS group.