Anti-Inflammatory Activities of Cinnamomum cassia Constituents In Vitro and In Vivo

We have investigated the anti-inflammatory effects of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) and carrageenan (Carr)-induced mouse paw edema model. When RAW264.7 macrophages were treated with cinnamic aldehyde together with LPS, a significant concentration-dependent inhibition of nitric oxide (NO), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) levels productions were detected. Western blotting revealed that cinnamic aldehyde blocked protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-κB), and IκBα, significantly. In the anti-inflammatory test, cinnamic aldehyde decreased the paw edema after Carr administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw tissue. We also demonstrated cinnamic aldehyde attenuated the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity in the edema paw after Carr injection. Cinnamic aldehyde decreased the NO, TNF-α, and PGE2 levels on the serum level after Carr injection. Western blotting revealed that cinnamic aldehyde decreased Carr-induced iNOS, COX-2, and NF-κB expressions in the edema paw. These findings demonstrated that cinnamic aldehyde has excellent anti-inflammatory activities and thus has great potential to be used as a source for natural health products

Analgesic Effects and the Mechanisms of Anti-Inflammation of Hispolon in Mice

Hispolon, an active ingredient in the fungi Phellinus linteus was evaluated with analgesic and anti-inflammatory effects. Treatment of male ICR mice with hispolon (10 and 20 mg/kg) significantly inhibited the numbers of acetic acid-induced writhing response. Also, our result showed that hispolon (20 mg/kg) significantly inhibited the formalin-induced pain in the later phase (P<.01). In the anti-inflammatory test, hispolon (20 mg/kg) decreased the paw edema at the fourth and fifth hour after λ-carrageenin (Carr) administration, and increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRx) in the liver tissue. We also demonstrated that hispolon significantly attenuated the malondialdehyde (MDA) level in the edema paw at the fifth hour after Carr injection. Hispolon (10 and 20 mg/kg) decreased the nitric oxide (NO) levels on both the edema paw and serum level at the fifth hour after Carr injection. Also, hispolon (10 and 20 mg/kg) diminished the serum TNF-α at the fifth hour after Carr injection. The anti-inflammatory mechanisms of hispolon might be related to the decrease in the level of MDA in the edema paw by increasing the activities of SOD, GPx and GRx in the liver. It probably exerts anti-inflammatory effects through the suppression of TNF-α and NO

Antinociceptive Activities and the Mechanisms of Anti-Inflammation of Asiatic Acid in Mice

Asiatic acid (AA), a pentacyclic triterpene compound in the medicinal plant Centella asiatica, was evaluated for antinociceptive and anti-inflammatory effects. Treatment of male ICR mice with AA significantly inhibited the numbers of acetic acid-induced writhing responses and the formalin-induced pain in the late phase. In the anti-inflammatory test, AA decreased the paw edema at the 4th and 5th h after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. AA decreased the nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels on serum level at the 5th h after Carr injection. Western blotting revealed that AA decreased Carr-induced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and nuclear factor-κB (NF-κB) expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with AA also diminished neutrophil infiltration into sites of inflammation as did indomethacin (Indo). The anti-inflammatory mechanisms of AA might be related to the decrease in the level of MDA, iNOS, COX-2, and NF-κB in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver

Anti-Inflammatory Activities of Inotilone from Phellinus linteus through the Inhibition of MMP-9, NF-κB, and MAPK Activation In Vitro and In Vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK) [extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4th and the 5th h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5th h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5th h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs

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 MARK pathway and hispolon-induced cell cycle arrest and apoptosis in Hep3B cells

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

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

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