Gil-Saeng Jeong

Anti-inflammatory effects of apigenin on nicotine- and lipopolysaccharide-stimulated human periodontal ligament cells via heme oxygenase-1.

BACKGROUND AND OBJECTIVESnAlthough apigenin exhibits various biological effects, its anti-inflammatory role in the periodontal field remains unknown. We examined the anti-inflammatory effects of apigenin and the underlying mechanism in nicotine- and lipopolysaccharide (LPS)-stimulated human periodontal ligament (hPDL) cells.nnnMATERIALS AND METHODSnWestern blotting was used to examine the effect of apigenin (10-40 microM) on the LPS- and nicotine-induced expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1), as well as the phosphorylation of mitogen-activated protein kinases (MAPKs), in hPDL cells. Pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-12 were monitored using Griess reagents and ELISA.nnnRESULTSnIncubation of hPDL cells with apigenin decreased LPS- and nicotine-induced HO-1 protein expression and activity. Apigenin significantly inhibited the nicotine- and LPS-induced production of NO, PGE2, IL-1beta, TNF-alpha, IL-6, and IL-12, and the upregulation of iNOS and COX-2 in hPDL cells. Hemin, a selective HO-1 inducer, reversed the apigenin-mediated suppression of nicotine- and LPS-induced NO, PGE2 and cytokine production. Treatment with inhibitors of the phosphoinositide 3-kinase, MAPKs, p38, and JNK, as well as a protein kinase C inhibitor, blocked the anti-inflammatory effects of apigenin in nicotine- and LPS-treated cells.nnnCONCLUSIONSnApigenin possesses anti-inflammatory activity in hPDL cells and works through a novel mechanism involving the action of HO-1. Thus, apigenin may have potential benefits as a host modulatory agent in the prevention and treatment of periodontal disease associated with smoking and dental plaque.

Roles of heme oxygenase-1 in curcumin-induced growth inhibition in rat smooth muscle cells.

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-α and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.

Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection

Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non-stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages.

Neuroprotective and anti-inflammatory effects of mollugin via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells.

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L. (Rubiaceae), exhibits antimutagenic activity, antitumor activity, antiviral activity, and inhibitory activity in arachidonic acid- and collagen-induced platelet aggregation. In this study, we investigated the effects of mollugin as a neuroprotective agent in glutamate-induced neurotoxicity in the mouse hippocampal HT22 cell line and as an anti-inflammatory agent in lipopolysaccharide-induced microglial activation in BV2 cells. Mollugin showed potent neuroprotective effects against glutamate-induced neurotoxicity and reactive oxygen species generation in mouse hippocampal HT22 cells. In addition, the anti-inflammatory effects of mollugin were demonstrated by the suppression of pro-inflammatory mediators, including pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) and cytokines (tumor necrosis factor-α and interleukin-6). Furthermore, we found that the neuroprotective and anti-inflammatory effects of mollugin were linked to the up-regulation of the expression of heme oxygenase (HO)-1 and the activity of HO in HT22 and BV2 cells. In addition, the effects of mollugin resulted in the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) in HT22 and BV2 cells. Furthermore, mollugin also activated the p38 mitogen-activated protein kinase (MAPK) pathway both in HT22 and BV2 cells. These results suggest that mollugin may be a promising candidate for the treatment of neurodegenerative diseases related to neuroinflammation.

Anti-inflammatory effects of sulfuretin from Rhus verniciflua Stokes via the induction of heme oxygenase-1 expression in murine macrophages

Rhus verniciflua Stokes (Anacardiaceae) has traditionally been used as an ingredient in East Asian medicines used to treat oxidative damage and cancer. Sulfuretin is one of the major flavonoid components isolated from R. verniciflua. In the present study, we isolated sulfuretin from R. verniciflua and demonstrated that sulfuretin inhibited inducible nitric oxide synthase (iNOS) protein and mRNA expression, reduced iNOS-derived NO, suppressed COX-2 protein and mRNA expression, and reduced COX-derived PGE(2) production in lipopolysaccharide (LPS)-stimulated RAW264.7 and murine peritoneal macrophages. Similarly, sulfuretin reduced tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) production. In addition, sulfuretin suppressed the phosphorylation and degradation of I kappaB-alpha as well as the nuclear translocation of p65 by the stimulation of LPS in RAW264.7 macrophages. Furthermore sulfuretin induced heme oxygenase (HO)-1 expression through nuclear translocation of nuclear factor E2-related factor 2 (Nrf)2 and increased heme oxygenase (HO) activity in RAW264.7 macrophages. The effects of sulfuretin on LPS-induced NO, PGE(2), TNF-alpha, and IL-1 beta production were partially reversed by the HO-1 inhibitor, tin protoporphyrin (SnPP). Therefore, it is suggested that sulfuretin-induced HO-1 expression plays a role of the resulting anti-inflammatory effects in macrophages. This indicated that the anti-inflammatory effects of sulfuretin in macrophages might be exerted through a novel mechanism that involves HO-1 expression.

Sulfuretin protects against cytokine-induced

NF-κB activation has been implicated as a key signaling mechanism for pancreatic β-cell damage. Sulfuretin is one of the main flavonoids produced by Rhus verniciflua, which is reported to inhibit the inflammatory response by suppressing the NF-κB pathway. Therefore, we isolated sulfuretin from Rhus verniciflua and evaluated if sulfuretin could inhibit cytokine- or streptozotocin-induced β-cell damage. Rat insulinoma RINm5F cells and isolated rat islets were treated with IL-1β and IFN-γ to induce cytotoxicity. Incubation of cells and islets with sulfuretin resulted in a significant reduction of cytokine-induced NF-κB activation and its downstream events, iNOS expression, and nitric oxide production. The cytotoxic effects of cytokines were completely abolished when cells or islets were pretreated with sulfuretin. The protective effect of sulfuretin was further demonstrated by normal insulin secretion of cytokine-treated islets in response to glucose. Treatment of mice with streptozotocin resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining of islets. However, the diabetogenic effects of streptozotocin were completely prevented when mice were pretreated with sulfuretin. The anti-diabetogenic effects of sulfuretin were also mediated by suppression of NF-κB activation. Collectively, these results indicate that sulfuretin may have therapeutic value in preventing β-cell damage.

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We previously reported that a chloroform extract of Caesalpinia sappan L. induces apoptosis in oral cancer cells but not in normal epithelial cell lines. In the present study, we explored the effects of a single compound isolated from C. sappan heartwood, isoliquiritigenin 2-methyl ether (ILME), on cultured primary and metastatic oral cancer cell lines using MTT assays, fluorescence microscopy, flow cytometry, and Western blotting. ILME inhibited the growth of the oral cancer cells in a time- and dose-dependent manner. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of sub-G(1)-phase arrest and by annexin V-FITC and propidium iodide staining. ILME time-dependently activated NF-kappaB transcription factors, phospholated the MAP kinases JNK (c-Jun N-terminal kinase) and ERK (extracellular signal-regulated kinase). Furthermore, ILME treatment upregulated HO-1 expression though activation of Nrf2 (NF-E2-related factor 2) pathway, and induced the expression of heme oxygenase-1 (HO-1). Tin protoporphyrin, an HO-1 inhibitor, dose-dependently attenuated the growth-inhibitory effect of ILME and blocked ILME-induced expression of the p21 and p53 cell cycle-regulatory proteins. These results provide the first evidence that the anti-oral cancer effects of ILME may involve a mechanism in which HO-1 is upregulated via a pathway involving MAP kinases, NF-kappaB, and Nrf2. Thus, ILME could be considered to be a potential chemotherapeutic target for anti-oral cancer treatment strategies.

-cell damage and prevents streptozotocin-induced diabetes

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata Bureau (Moraceae) has an isoprenylated xanthone skeleton that is known to exert a variety of biological activities. In the present study, we demonstrated that CTXA inhibited cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) expression, and thereby reduced COX-2-derived prostaglandin E2 (PGE(2)) and iNOS-derived NO production in lipopolysaccharide (LPS)-stimulated macrophages. Similarly, CTXA suppressed tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) production. Moreover, CTXA inhibited the induced phosphorylation and degradation of IkappaB-alpha as well as the LPS-induced increase in p65 in the nuclear fraction of macrophages. CTXA also induced heme oxygenase-1 (HO-1) expression and increased heme oxygenase (HO) activity in RAW264.7 macrophages. We also demonstrated that the effects of CTXA on LPS-induced PGE(2), NO, TNF-alpha, and IL-1beta production were partially reversed by the HO-1 inhibitor tin protoporphyrin, suggesting that CTXA-induced HO-1 expression was partly responsible for the resulting anti-inflammatory effects of the drug. Thus CTXA was shown to be an effective HO-1 inducer, capable of inhibiting macrophage-derived pro-inflammatory mediators.

Isoliquiritigenin 2'-methyl ether induces growth inhibition and apoptosis in oral cancer cells via heme oxygenase-1.

Abstract.Objectives:Heme oxygenase (HO)-1 expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation has an ability to inhibit tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. Costunolide has been reported to inhibit IL-1 production, but whether other cytokines could be inhibited remains to be confirmed. We investigated the effects of costunolide and its components (α-methylene-γ-butyrolactone; CH2-BL, α-methyl-γ-butyrolactone; CH3-BL, and γ-butyrolactone; BL) on HO-1 expression as well as TNF-α and IL-6 production in RAW264.7 macrophages.Methods:HO-1 expression and Nrf2 nuclear accumulation were analyzed by Western blot analysis. The production of TNF-α and IL-6 in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) was assayed by ELISA.Results:Costunolide and CH2-BL induced HO-1 expression and Nrf2 nuclear accumulation, whereas CH3-BL and BL did not. Pre-incubation with costunolide inhibited LPS-induced production of TNF-α and IL-6. The inhibitory effects of costunolide on TNF-α and IL-6 production were abrogated by tin protoporphyrin, an HO inhibitor.Conclusions:Costunolide is an effective HO-1 inducer capable of inhibiting macrophage-derived pro-inflammatory cytokines. CH2-BL moiety of costunolide is essential for Nrf2 activation leading to HO-1 expression.

Cudratricusxanthone A from Cudrania tricuspidata suppresses pro-inflammatory mediators through expression of anti-inflammatory heme oxygenase-1 in RAW264.7 macrophages

This study was conducted to investigate the effects of mechanical stress, particularly cyclic strain, on proinflammatory cytokines as well as antioxidant properties and their interactions with cellular defense systems in human dental pulp (HDP) cells. Exposure of HDP cells to mechanical strain induced inflammatory cytokines such as interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6, as well as antioxidant genes such as heme oxygenase-1, superoxide dismutases, reduced nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1, and glutathione peroxidases. In addition, treatment with N-acetylcysteine, indomethacin, and heme oxygenase-1 inhibitors blocked reactive oxygen species production, antioxidant response element (ARE) gene expression, and Nrf2 accumulation that occurred in response to mechanical stress. These data demonstrate that mechanical strain activates inflammatory cytokines and oxidative stress, which then act in concert to induce the Nrf2-/ARE-mediated antioxidant enzymes. Therefore, we suggest that the activation of a compensatory adaptation or defense antioxidant system might represent a novel mechanism for protecting HDP cells against mechanical stress.