Histological appearances of mouse hind footpads after subcutaneously injecting 0.9% saline (Control group) or Carr, and then stained with H&E stain, while others were processed for iNOS and COX-2 immunohistochmistry staining.

<p>(A). Control mice: show the normal appearance of dermis and subdermis without any significant lesions, (F) iNOS and (J) COX-2 immunoreactive cells existed in the paws of normal mice; (B). Carr Only: Hemorrhage with moderately extravascular red blood cell and large amounts of inflammatory leucocytes, mainly neutrophils infiltrating the subdermis interstitial tissue. Moreover, the detail of the subdermis layer show enlargement of the interstitial space caused by the exudate fluid in the edema, (G) numerous iNOS and (K) COX-2 immunoreactive cells were observed in the brown site of paw tissue; (C). Carr + Indo 10 mg/kg (<i>i.p.</i>) (100×): there were obvious morphological alterations and improvements, (H) iNOS and (L) COX-2 immunoreactive cells; (D). Carr + inotilone: there were significant morphological alterations compared to the tissue with Carr treatment only. The lesions showed no hemorrhage and the number of neutrophils infiltrating the subdermis interstitial tissue was markedly reduced and also in (I) iNOS and (M) COX-2 immunoreactive cells in paws. Scale bar = 100 µm. There were markedly fewer inflammatory cells, and iNOS and COX-2 immunoreactive cells in the paws of Carr treated mice. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicated the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.</p

Proposed mechanism of inotilone inhibition of LPS-induced inflammation in RAW 264.7 cells.

<p>Inotilone abrogates the phosphorylation of MAPKs/IKK and subsequently inactivates NF-κB, which may result from inotilone down-regulation of iNOS and COX-2. Arrows indicate the main inflammatory pathway activated by LPS stimulation. The prohibition signs indicate the inhibitory effects of inotilone. TLR4; Toll-like receptor 4.</p

Inhibition of iNOS, COX-2 (A), MMP-9 (B), NF-κB (C), and MAPK (JNK, p38, and ERK) (D) protein expressions by inotilone induced by Carr of foot at the 5^th

<p> <b>h in mice.</b> Suspended tissue were then prepared and subjected to Western blotting using an antibody specific for iNOS and COX-2. β-actin was used as an internal control. A representative Western blot from two separate experiments is shown. Relative iNOS, COX-2, MMP-9, NF-κB, and MAPK (JNK, p38, and ERK) protein levels were calculated with reference to a Carr-injected mouse. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ^###<i>p</i><0.001 as compared with the control group. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 as compared with the Carr group (one-way ANOVA followed by Scheffe’s multiple range test).</p

Effects of inotilone and Indo on hind paw edema induced by Carr in mice (A), the tissue MDA concentration of foot in mice (B), Carr-induced NO (C), and TNF-α (D) concentrations of serum at the 5^th

<p> <b>h in mice.</b> Each value represents as mean ± S.E.M. ^###<i>p</i><0.001 as compared with the control group. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 as compared with the Carr group (one-way ANOVA followed by Scheffe’s multiple range test).</p

Effects of inotilone and indomethacin (Indo) on changes in CAT, SOD and GPx activities was studied on Carr-induced mice paw edema (5^th h).

<p>Each value represents as mean ± S.E.M.^###<i>p</i><0.001 as compared with the control group. *<i>p</i><0.05 and **<i>p</i><0.01 as compared with the Carr group (one-way ANOVA followed by Scheffe’s multiple range test).</p

The chemical structure of inotilone (A) and the effects of inotilone on lipopolysaccharide (LPS)-induced cell viability (B), NO production (C), inhibition of iNOS and COX-2 protein expression (D), and MAPK (JNK, p38, and ERK) protein expression (E) were evaluated in RAW264.7 cells.

<p>Cells were incubated for 24 h or 5, 10, 15, 30, and 60 mins with 100 ng/mL of LPS in the absence or presence of inotilone (0, 1.56, 3.12, 6.25, 12.5, and 25 µM). Inotilone was added 1 h before the incubation with LPS. Cell viability was performed by using MTT assay. Nitrite concentration in the medium was determined by using Griess reagent. Lysed cells were then prepared and subjected to Western blotting by using an antibody specific for iNOS, COX-2, and MAPK. β-actin was used as an internal control. The data were presented as mean ± S.D. for three different experiments performed in triplicate. **<i>p</i><0.01 and ***<i>p</i><0.001 were compared with LPS-alone group.</p

Inotilone suppresses LPS-induced MMP-9 activities (A), MMP-9 protein (B), and NF-κB expressions (C) in RAW264.7 cells.

<p>Cells were incubated for 24 h or 1 h with 100 ng/mL of LPS in the absence or the presence of inotilone (0, 6.25, 12.5, and 25 µM). Inotilone was added 1 h before the incubation with LPS. The conditioned media were collected MMP-9 activities determined by gelatin zymography. MMP-9 activities were quantified by densitometric analysis. Representative Western blot from two separate experiments was shown. MMP-9 and NF-κB levels were calculated with reference to a LPS-stimulated culture. The data were presented as mean ± S.D. for three different experiments performed in triplicate.^###compared with sample of control group. **<i>p</i><0.01 and ***<i>p</i><0.001 were compared with LPS-alone group.</p

Sclareol Exhibits Anti-inflammatory Activity in Both Lipopolysaccharide-Stimulated Macrophages and the λ-Carrageenan-Induced Paw Edema Model

Sclareol (<b>1</b>) is a natural fragrance compound used widely in the cosmetic and food industries. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and the λ-carrageenan-induced edema mouse paw model were applied to examine the anti-inflammatory potential of <b>1</b> and its possible molecular mechanisms. The experimental results obtained demonstrated that this compound inhibited cell growth, nitric oxide (NO) production, and the expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in LPS-stimulated macrophages. Compound <b>1</b> also reduced paw edema, the tissue content of NO, tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), iNOS and COX-2 protein expression, and neutrophil infiltration within the tissues after λ-carrageenan stimulation. The present study suggests that the anti-inflammatory mechanisms of <b>1</b> might be related to a decrease of inflammatory cytokines and an increase of antioxidant enzyme activity

Hispolon Induces Apoptosis and Cell Cycle Arrest of Human Hepatocellular Carcinoma Hep3B Cells by Modulating ERK Phosphorylation

Hispolon is an active phenolic compound of Phellinus igniarius, a mushroom that has recently been shown to have antioxidant, anti-inflammatory, and anticancer activities. This study investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma Hep3B cells by using the MTT assay, DNA fragmentation, DAPI (4,6-diamidino-2-phenylindole dihydrochloride) staining, and flow cytometric analyses. Hispolon inhibited cellular growth of Hep3B cells in a time-dependent and dose-dependent manner, through the induction of cell cycle arrest at S phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA laddering. Hispolon-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclins A and E and cyclin-dependent kinase (CDK) 2, with concomitant induction of p21waf1/Cip1 and p27Kip1. Exposure of Hep3B cells to hispolon resulted in apoptosis as evidenced by caspase activation, PARP cleavage, and DNA fragmentation. Hispolon treatment also activated JNK, p38 MAPK, and ERK expression. Inhibitors of ERK (PB98095), but not those of JNK (SP600125) and p38 MAPK (SB203580), suppressed hispolon-induced S-phase arrest and apoptosis in Hep3B cells. These findings establish a mechanistic link between the MAPK pathway and hispolon-induced cell cycle arrest and apoptosis in Hep3B cells

Bioassay Guided Isolation and Identification of Anti-inflammatory Active Compounds from the Root of Ficus formosana

Activity-directed fractionation and purification processes were employed to identify the anti-inflammatory active compounds using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) in vitro. Air-dried roots of Ficus formosana were extracted with methanol and separated into <i>n</i>-hexane, chloroform, ethyl acetate, <i>n</i>-butanol, and water layers. Among them, the chloroform layer showed strong activity and was subjected to separation and purification by using various chromatographic techniques. Five compounds showing potent activity were identified by comparing spectral data to be β-sitosterol, stigmasterol, psoralen, kaempferol, carpachromene, and syringic aldehyde. When macrophages were treated with psoralen and kaempferol together with LPS, a concentration-dependent inhibition of nitric oxide (NO) and tumor necrosis factor (TNF-α) productions were detected. Western blotting revealed that kaempferol, psoralen, and carpachromene blocked protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated macrophages. The results confirmed that the traditional use of F. formosana could be a potential anti-inflammatory agent