Jae Gwang Park

Anti-inflammatory activities and mechanisms of Artemisia asiatica ethanol extract

ETHNOPHARMACOLOGICAL RELEVANCE Artemisia asiatica Nakai (Compositae) is a representative herbal plant used to treat infection and inflammatory diseases. Although Artemisia asiatica is reported to have immunopharmacological activities, the mechanisms of these activities and the effectiveness of Artemisia asiatica preparations in use are not known. MATERIALS AND METHODS To evaluate the anti-inflammatory activities of Artemisia asiatica ethanol extract (Aa-EE), we assayed nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2) in macrophages and measured the extent of tissue injury in a model of gastric ulcer induced in mice by treatment with HCl in EtOH. Putative enzymatic mediators of Aa-EE activities were identified by nuclear fractionation, reporter gene assay, immunoprecipitation, immunoblotting, and kinase assay. Active compound in Aa-EE was identified using HPLC. RESULTS Treatment of RAW264.7 cells and peritoneal macrophages with Aa-EE suppressed the production of NO, PGE2, and TNF-α in response to lipopolysaccharide (LPS) and induced heme oxygenase-1 expression. The Aa-EE also ameliorated symptoms of gastric ulcer in HCl/EtOH-treated mice. These effects were associated with the inhibition of nuclear translocation of nuclear factor (NF)-κB and activator protein (AP)-1, implying that the anti-inflammatory action of the Aa-EE occurred through transcriptional inhibition. The upstream regulatory signals Syk and Src for translocation of NF-κB and TRAF6 for AP-1 were identified as targets of this effect. Analysis of Aa-EE by HPLC revealed the presence of luteolin, known to inhibit NO and PGE2 activity. CONCLUSION The anti-inflammatory activities attributed to Artemisia asiatica Nakai in traditional medicine may be mediated by luteolin through inhibition of Src/Syk/NF-κB and TRAF6/JNK/AP-1 signaling pathways.

The Dietary Flavonoid Kaempferol Mediates Anti-Inflammatory Responses via the Src, Syk, IRAK1, and IRAK4 Molecular Targets

Even though a lot of reports have suggested the anti-inflammatory activity of kaempferol (KF) in macrophages, little is known about its exact anti-inflammatory mode of action and its immunopharmacological target molecules. In this study, we explored anti-inflammatory activity of KF in LPS-treated macrophages. In particular, molecular targets for KF action were identified by using biochemical and molecular biological analyses. KF suppressed the release of nitric oxide (NO) and prostaglandin E2 (PGE2), downregulated the cellular adhesion of U937 cells to fibronectin (FN), neutralized the generation of radicals, and diminished mRNA expression levels of inflammatory genes encoding inducible NO synthase (iNOS), TNF-α, and cyclooxygenase- (COX-) 2 in lipopolysaccharide- (LPS-) and sodium nitroprusside- (SNP-) treated RAW264.7 cells and peritoneal macrophages. KF reduced NF-κB (p65 and p50) and AP-1 (c-Jun and c-Fos) levels in the nucleus and their transcriptional activity. Interestingly, it was found that Src, Syk, IRAK1, and IRAK4 responsible for NF-κB and AP-1 activation were identified as the direct molecular targets of KF by kinase enzyme assays and by measuring their phosphorylation patterns. KF was revealed to have in vitro and in vivo anti-inflammatory activity by the direct suppression of Src, Syk, IRAK1, and IRAK4, involved in the activation of NF-κB and AP-1.

Novel anti-inflammatory function of NSC95397 by the suppression of multiple kinases.

NSC95397 (2,3-bis-[(2-hydroxyethyl)thio]-1,4-naphthoquinone) is a CDC25 inhibitor with anti-cancer properties. Since the anti-inflammatory activity of this compound has not yet been explored, the aim of this study was to examine whether this compound is able to modulate the inflammatory process. Toll like receptor (TLR)-mediated inflammatory responses were induced by lipopolysaccharide (LPS), a TLR4 ligand, and pam3CSK, a TLR2 ligand, in peritoneal macrophages and RAW264.7. The molecular mechanism of NSC95397s anti-inflammatory activity was studied using immunoblotting analysis, nuclear fractionation, immunoprecipitation, overexpression strategies, luciferase reporter gene assays, and kinase assays. NSC95397 dose-dependently suppressed the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin (PG)E2, and diminished the mRNA expression of inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, interferon (IFN)-β, and TNF-α in peritoneal macrophages and RAW264.7 cells that were stimulated by LPS and pam3CSK. This compound also clearly blocked the activation of NF-κB (p65), AP-1 (c-Fos/c-Jun), and IRF-3 in LPS-treated RAW264.7 cells and TRIF- and MyD88-overexpressing HEK293 cells. In addition, biochemical and molecular approaches revealed that this compound targeted AKT, IKKα/β, MKK7, and TBK1. Therefore, these results suggest that the anti-inflammatory function of NSC95397 can be attributed to its inhibition of multiple targets such as AKT, IKKα/β, MKK7, and TBK1.

IRAK1/4-Targeted Anti-Inflammatory Action of Caffeic Acid

Caffeic acid (CA) is a phenolic compound that is frequently present in fruits, grains, and dietary supplements. Although CA has been reported to display various biological activities such as anti-inflammatory, anti-cancer, anti-viral, and anti-oxidative effects, the action mechanism of CA is not yet fully elucidated. In this study, the anti-inflammatory action mechanism of CA was examined in lipopolysaccharide (LPS) treated macrophages (RAW264.7 cells) and HCl/EtOH-induced gastritis. CA was found to diminish nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-stimulated RAW264.7 cells. Additionally, mRNA levels of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and inducible NO synthase (iNOS) were downregulated by CA. CA also strongly suppressed the nuclear translocation of AP-1 family proteins and the related upstream signaling cascade composed of interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, TGF-β-activated kinase 1 (TAK1), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase (JNK). In a direct kinase assay, CA was revealed to directly inhibit IRAK1 and IRAK4. CA also ameliorated HCl/EtOH-induced gastric symptoms via the suppression of JNK, IRAK1, and IRAK4. Therefore, our data strongly suggest that CA acts as an anti-inflammatory drug by directly suppressing IRAK1 and IRAK4.

Anticancer effect of joboksansam, Korean wild ginseng germinated from bird feces.

Background Joboksansam, Korean bird wild ginseng, is an artificially cultivated wild ginseng germinated from bird feces. Although numerous pharmacologic activities of wild ginsengs have been reported, the beneficial effect of joboksansam in cancer has not been elucidated. In this study, we investigated the in vivo and in vitro anticancer activities of joboksansam powder. Methods To evaluate the in vivo anticancer activity of joboksansam, we established a xenograft mouse model bearing RMA cell-derived cancer. Direct cytotoxicity induced by joboksansam powder was also investigated in vitro using (3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. The inhibitory activity of this powder on the activation of cell survival signaling involving Akt and Src was examined with immunoblot analysis. Results Joboksansam powder displayed strong inhibitory activity against the increased tumor size, increased weight of total body and cancer tissues, and mortality of tumor-bearing mice. Joboksansam powder also suppressed the activation of survival regulatory enzymes Akt and Src, as assessed by phosphorylation levels in the immunoblot analysis of tumor tissues. Interestingly, the viability of RMA cells in vitro was directly decreased by joboksansam treatment. Conclusion Overall, our results strongly suggest that joboksansam powder has the potential to protect against cancer generation by direct cytotoxic effects on cancer cells resulting from suppression of cell survival signaling.

Korean Red Ginseng water extract arrests growth of xenografted lymphoma cells

Background Although numerous studies of the anticancer activities of Korean Red Ginseng (KRG) have been performed, the therapeutic effect of KRG on leukemia has not been fully elucidated. In this study, we investigated the antileukemia activities of KRG and its cellular and molecular mechanisms. Methods An established leukemia tumor model induced by xenografted T cell lymphoma (RMA cells) was used to test the therapeutic activity of KRG water extract (KRG-WE). Direct cytotoxic activity of KRG-WE was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The immunomodulatory activities of KRG-WE were verified by immunohistochemistry, nitric oxide production assay. The inhibitory effect of KRG-WE on cell survival signaling was also examined. Results Orally administered KRG-WE reduced the sizes of tumor masses. Levels of apoptosis regulatory enzymes and cleaved forms of caspases-3 and -8 were increased by this extract. In addition, expression of matrix metalloproteinase-9, a metastasis regulatory enzyme, was decreased by KRG-WE treatment. The proportion of CD11c+ cells was remarkably increased in the KRG-treated group compared to the control group. However, KRG-WE did not show significant direct cytotoxicity against RMA cells. Conclusion Our results strongly suggest that the KRG might have antileukemia activity through CD11c+ cell-mediated antitumor immunity.

Radical Scavenging Activity-Based and AP-1-Targeted Anti-Inflammatory Effects of Lutein in Macrophage-Like and Skin Keratinocytic Cells

Lutein is a naturally occurring carotenoid with antioxidative, antitumorigenic, antiangiogenic, photoprotective, hepatoprotective, and neuroprotective properties. Although the anti-inflammatory effects of lutein have previously been described, the mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, in the present study, we aimed to investigate the regulatory activity of lutein in the inflammatory responses of skin-derived keratinocytes or macrophages and to elucidate the mechanism of its inhibitory action. Lutein significantly reduced several skin inflammatory responses, including increased expression of interleukin-(IL-) 6 from LPS-treated macrophages, upregulation of cyclooxygenase-(COX-) 2 from interferon-γ/tumor necrosis-factor-(TNF-) α-treated HaCaT cells, and the enhancement of matrix-metallopeptidase-(MMP-) 9 level in UV-irradiated keratinocytes. By evaluating the intracellular signaling pathway and the nuclear transcription factor levels, we determined that lutein inhibited the activation of redox-sensitive AP-1 pathway by suppressing the activation of p38 and c-Jun-N-terminal kinase (JNK). Evaluation of the radical and ROS scavenging activities further revealed that lutein was able to act as a strong anti-oxidant. Taken together, our findings strongly suggest that lutein-mediated AP-1 suppression and anti-inflammatory activity are the result of its strong antioxidative and p38/JNK inhibitory activities. These findings can be applied for the preparation of anti-inflammatory and cosmetic remedies for inflammatory diseases of the skin.

Kaempferol, a dietary flavonoid, ameliorates acute inflammatory and nociceptive symptoms in gastritis, pancreatitis, and abdominal pain

Kaempferol (KF) is the most abundant polyphenol in tea, fruits, vegetables, and beans. However, little is known about its in vivo anti-inflammatory efficacy and mechanisms of action. To study these, several acute mouse inflammatory and nociceptive models, including gastritis, pancreatitis, and abdominal pain were employed. Kaempferol was shown to attenuate the expansion of inflammatory lesions seen in ethanol (EtOH)/HCl- and aspirin-induced gastritis, LPS/caerulein (CA) triggered pancreatitis, and acetic acid-induced writhing.

Syk and Src are major pharmacological targets of a Cerbera manghas methanol extract with kaempferol-based anti-inflammatory activity

ETHNOPHARMACOLOGICAL RELEVANCE Cerbera manghas L. (Apocynaceae), a semi-mangrove medicinal plant distributed throughout tropical and subtropical countries, is traditionally known to possess analgesic, anti-inflammatory, anti-convulsant, cardiotonic, and hypotensive activity. In vitro and in vivo anti-inflammatory activities of a methanol extract of the leaves of Cerbera manghas and the underlying molecular mechanisms were investigated to validate the ethnopharmacological use of this plant. MATERIALS AND METHODS The effect of Cerbera manghas methanol extract (Cm-ME) on the production of inflammatory mediators and the induction of HCl/EtOH-treated gastritis was explored using macrophages, HEK293 cells, and ICR mice. The molecular targets of this extract and potential active components in Cm-ME were also investigated. RESULTS Cm-ME inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-treated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. This extract also suppressed the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2. NF-κB-mediated enhancement of luciferase activity, nuclear translocation of p50 and p65, and phosphorylation of IκBα were markedly reduced by Cm-ME treatment. Direct enzyme assays, reporter gene assays, and immunoprecipitation analysis of kinases revealed Syk and Src as immunopharmacological targets of Cm-ME. Moreover, this extract strongly ameliorated the gastric symptoms induced by HCl/EtOH treatment of mice. Finally, HPLC analysis and pharmacological tests identified kaempferol as an active component of the extract with Src/Syk inhibitory activities. CONCLUSION Inhibition of Syk/Src and the NF-κB pathway by kaempferol could play a key role in the anti-inflammatory pharmacological action of Cerbera manghas.

Lancemaside A from Codonopsis lanceolata Modulates the Inflammatory Responses Mediated by Monocytes and Macrophages

In this study, we aimed to examine the cellular and molecular mechanisms of lancemaside A from Codonopsis lanceolata (Campanulaceae) in the inflammatory responses of monocytes (U937 cells) and macrophages (RAW264.7 cells). Lancemaside A significantly suppressed the inflammatory functions of lipopolysaccharide- (LPS-) treated RAW264.7 cells by suppressing the production of nitric oxide (NO), the expression of the NO-producing enzyme inducible NO synthase (iNOS), the upregulation of the costimulatory molecule CD80, and the morphological changes induced by LPS exposure. In addition, lancemaside A diminished the phagocytic activity of RAW264.7 cells and boosted the neutralizing capacity of these cells when treated with the radical generator sodium nitroprusside (SNP). Interestingly, lancemaside A strongly blocked the adhesion activity of RAW264.7 cells to plastic culture plates, inhibited the cell-cell and cell-fibronectin (FN) adhesion of U937 cells that was triggered by treatment with an anti-β1-integrin (CD29) antibody and immobilized FN, respectively. By evaluating the activation of various intracellular signaling pathways and the levels of related nuclear transcription factors, lancemaside A was found to block the activation of inhibitor of κB kinase (IKK) and p65/nuclear factor- (NF-) κB. Taken together, our findings strongly suggest that the anti-inflammatory function of lancemaside A is the result of its strong antioxidative and IKK/NF-κB inhibitory activities.