Graphical scheme of the anti-inflammatory mechanism of eupafolin in LPS-treated RAW264.7 macrophages.

<p>Graphical scheme of the anti-inflammatory mechanism of eupafolin in LPS-treated RAW264.7 macrophages.</p

Effects of eupafolin on the LPS-induced NF-κB p65 and AP-1/c-fos nuclear translocation in RAW264.7 macrophages.

<p>The RAW264.7 macrophages were pretreated for 1 h with 60 μM eupafolin or with 30 μM of the MAPK inhibitors and were then treated with 1 μg/ml of LPS for 30 min. Western blot analysis (n = 3) was performed to evaluate the subcellular and nuclear localization of (A) phosphorylated (P-p65) and total p65 (t-p65) (total cell lysate) and (B) p65 and c-fos (nuclear fraction). GAPDH and PCNA (proliferating cell nuclear antigen) were processed in parallel as internal controls for protein loading. *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the LPS-treated cells. Immunofluorescence staining (n = 3) was performed to show the subcellular localization of (C) t-p65 and (D) c-fos. Bar = 50 μm.</p

Eupafolin inhibited the LPS-induced activation of MAPKs in RAW264.7 macrophages.

<p>The RAW264.7 macrophages were pretreated with 1 μg/ml of LPS for various times as indicated. The phosphorylated and total (A) ERK, (B) JNK, (C) p38, or (D) AKT levels were determined by Western blot analysis. Total ERK (t-ERK), total JNK (t-JNK), total p38 (t-p38), or total AKT (t-AKT) protein was used as the loading control. The data are shown as the means ± SEM (n = 6). *<i>P</i><0.05 vs. the untreated control. The cells were treated for 1 h with 60 μM eupafolin and were then incubated with 1 μg/ml of LPS for 30 min. The phosphorylated (E) ERK, (F) JNK, (G) p38, or (H) AKT levels were determined by Western blot analysis. Total ERK (t-ERK), total JNK (t-JNK), total p38 (t-p38), or total AKT (t-AKT) protein was used as the loading control. The data are shown as the means ± SEM (n = 6). *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the LPS-treated cells.</p

Eupafolin inhibited LPS-induced paw edema and inflammation in mice.

<p>(A) Representative cross-sections of the mouse paws (n = 4) were stained with hematoxylin and eosin. The degree of paw edema is shown between the two arrows. Bar = 100 μm. (B) Quantification of the degree of paw edema. The values are shown as the means ± SEM. *<i>P</i><0.05 vs. the saline-treated mice. ^†<i>P</i><0.05 vs. the LPS-treated mice. (C-D) Immunohistochemical staining with the antibodies to COX-2, iNOS and Iba1 in paw sections. The positive reactions are indicated by arrowheads. Bar = 50 μm. Eupafolin inhibited the LPS-induced paw inflammation in mice. (E) Representative cross-sections of lungs were stained with hematoxylin and eosin. Bar = 50 μm. (F) Quantification of the degree of inflammatory cell infiltration into the lung. The values are shown as the means ± SEM. *<i>P</i><0.05 vs. the saline-treated mice. ^†<i>P</i><0.05 vs. the LPS-treated mice. (G-H) Immunohistochemical staining with the antibodies to COX-2, iNOS and Iba1 in lung sections. The positive reactions are indicated by arrowheads. The sections were stained for COX-2 or iNOS (arrows) and Iba1 (macrophage marker, arrows) antibodies by double immunofluorescent staining. Nuclei were stained with DAPI (arrows). The stronger COX-2 or iNOS expression was present in the macrophage (arrows, the merged image). Bar = 50 μm.</p

Eupafolin inhibited the LPS-induced NO, iNOS and COX-2 expression in RAW264.7 macrophages.

<p>(A) Chemical structure of eupafolin. RAW264.7 macrophages were pretreated with 1, 1.5, or 2 μg/ml of LPS for 24 h. The control group included cells grown in medium without LPS. (B) COX-2 and iNOS protein expression was determined by Western blot analysis. GAPDH or β-actin was processed in parallel as an internal control for protein loading. (C) The RAW264.7 macrophages were pretreated with 0, 20, 40, or 60 μM eupafolin for 1 h and then with 1 μg/ml of LPS for another 24 h. The COX-2 expression was analyzed by Western blotting (n = 4). (D) Immunofluorescence staining was performed to show the COX-2 expression. (n = 4) (E) The RAW264.7 macrophages were pretreated with 0, 20, 40, or 60 μM eupafolin for 1 h and then with 1 μg/ml of LPS for another 24 h. The iNOS expression was measured using Western blot analysis (n = 4). (F) The Griess assay was performed to evaluate the NO production. In B-C and E-F, the data are shown as the means ± SEM (n = 4). *<i>P</i><0.05 vs. the untreated group; ^†<i>P</i><0.05 vs. the LPS-treated group. The scale bars in D = 25 μm.</p

Eupafolin inhibited the LPS-induced cytokine expression in RAW264.7 macrophages.

<p>The RAW264.7 macrophages were pretreated with 60 μM eupafolin for 1 h and then with 1 μg/ml of LPS for another 24 h. The cytokine expression was regulated by LPS and eupafolin treatment. Protein extracts (A) and cultured medium (C) were collected to perform the cytokine membrane array assay. (B, D) Quantification of the cytokine expression.</p

Effects of p38-, JNK- and AKT-specific siRNA on the COX-2 and iNOS expression in LPS-treated RAW264.7 macrophages.

<p>The expression of (E, G) COX-2 and (F, H) iNOS was determined by Western blot analysis after p38 or JNK silencing followed by LPS treatment. p38-, JNK- and AKT-specific siRNA reduced the total p38 (t-p38), JNK (t-JNK) and AKT (t-AKT) protein expression, respectively, compared with that in the non-treated cells. The values are shown as the means ± SEM (n = 3–6). *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the LPS-treated cells. ^‡<i>P</i><0.05 vs. the LPS+EUP treated cells.</p

The eupafolin-mediated inhibition of the LPS-induced NO, iNOS, COX-2 and PGE₂ expression in RAW264.7 macrophages involved MAPK activation.

<p>The RAW264.7 macrophages were treated for 1 h with 30 μM of the MAPK inhibitors or 10 μM of the PI3K/AKT inhibitor and were then incubated with 1 μg/ml of LPS for 24 h. (A) COX-2 and (B) iNOS protein expression was determined by Western blot analysis. β-actin was processed in parallel as an internal control for protein loading. (C) NO was measured with the Griess assay, and (D) PGE₂ was measured with an ELISA assay. The data are shown as the means ± SEM (n = 5–8). *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the LPS-treated cells. ^‡<i>P</i><0.05 vs. the LPS+EUP treated cells.</p

Viscolin Inhibits <i>In Vitro</i> Smooth Muscle Cell Proliferation and Migration and Neointimal Hyperplasia <i>In Vivo</i>

<div><p>Viscolin, an extract of <i>Viscum coloratum</i>, has anti-inflammatory and anti-proliferative properties against harmful stimuli. The aim of the study was to examine the anti-proliferative effects of viscolin on platelet derived growth factor-BB (PDGF)-treated human aortic smooth muscle cells (HASMCs) and identify the underlying mechanism responsible for these effects. Viscolin reduced the PDGF-BB-induced HASMC proliferation and migration <i>in vitro</i>; it also arrested HASMCs in the G0/G1 phase by decreasing the protein expression of Cyclin D1, CDK2, Cyclin E, CDK4, and p21^Cip1 as detected by Western blot analysis. These effects may be mediated by reduced PDGF-induced phosphorylation of ERK1/2, JNK, and P38, but not AKT as well as inhibition of PDGF-mediated nuclear factor (NF)-κB p65 and activator protein 1 (AP-1)/c-fos activation. Furthermore, viscolin pre-treatment significantly reduced neointimal hyperplasia of an endothelial-denuded femoral artery <i>in vivo</i>. Taken together, viscolin attenuated PDGF–BB-induced HASMC proliferation <i>in vitro</i> and reduced neointimal hyperplasia <i>in vivo</i>. Thus, viscolin may represent a therapeutic candidate for the prevention and treatment of vascular proliferative diseases.</p></div

Effects of MAPK inhibitors on cell cycle regulator protein expression in PDGF-BB-treated HASMCs.

<p>Cells were treated with 30 μM MAPK inhibitors for 1 h, and then incubated with 30 ng/mL PDGF-BB for 24 h. The expression of (A) Cyclin D1, (B) Cyclin E, (C) p27^Kip1, (D) CDK4, (E) CDK2, (F) or p21^Cip1 proteins was determined by Western blot analysis. β-actin was processed in parallel as an internal control for protein loading. The histograms show the phosphorylated band/β-actin ratio (A-F). *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the PDGF-BB-treated cells.</p