Double immunofluorescent evidence that oxidative stress-associated activation of JNK/AP-1 signaling participates in neuropeptide-mediated appetite control.

Amphetamine (AMPH), an appetite suppressant, alters expression levels of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. This study explored the potential role of cJun-N-terminal kinases (JNK) in appetite control, mediated by reactive oxygen species (ROS) and activator protein-1 (AP-1) in AMPH-treated rats. Rats were given AMPH daily for 4 days. Changes in feeding behavior and expression levels of hypothalamic NPY, CART, cFos, cJun, phosphorylated JNK (pJNK), as well as those of anti-oxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione S-transferase (GST), were examined and compared. Following AMPH treatment, food intake and NPY expression decreased, whereas the other proteins expression and AP-1/DNA binding activity increased. Both cerebral cJun inhibition and ROS inhibition attenuated AMPH anorexia and modified detected protein, revealing a crucial role for AP-1 and ROS in regulating AMPH-induced appetite control. Moreover, both pJNK/CART and SOD/CART activities detected by double immunofluorescent staining increased in hypothalamic arcuate nucleus in AMPH-treated rats. The results suggested that pJNK/AP-1 signaling and endogenous anti-oxidants participated in regulating NPY/CART-mediated appetite control in rats treated with AMPH. These findings advance understanding of the molecular mechanism underlying the role of pJNK/AP-1 and oxidative stress in NPY/CART-mediated appetite suppression in AMPH-treated rats.

Downregulation of Type 3 Deiodinase in the Hypothalamus During Inflammation.

Background: Inflammation is associated with marked changes in cellular thyroid hormone (TH) metabolism in triiodothyronine (T3) target organs. In the hypothalamus, type 2 deiodinase (D2), the main T3 producing enzyme, increases upon inflammation, leading to an increase in local T3 availability, which in turn decreases thyrotropin releasing hormone expression in the paraventricular nucleus. Type 3 deiodinase (D3), the T3 inactivating enzyme, decreases during inflammation, which might also contribute to the increased T3 availability in the hypothalamus. While it is known that D2 is regulated by nuclear factor κB (NF-κB) during inflammation, the underlying mechanisms of D3 regulation are unknown. Therefore, the aim of the present study was to investigate inflammation-induced D3 regulation using in vivo and in vitro models. Methods: Mice were injected with a sublethal dose of bacterial endotoxin (lipopolysaccharide [LPS]) to induce a systemic acute-phase response. A human neuroblastoma (SK-N-AS) cell line was used to test the involvement of the thyroid hormone receptor alpha 1 (TRα1) as well as the activator protein-1 (AP-1) and NF-κB inflammatory pathways in the inflammation-induced decrease of D3. Results: D3 expression in the hypothalamus was decreased 24 hours after LPS injection in mice. This decrease was similar in mice lacking the TRα. Incubation of SK-N-AS cells with LPS robustly decreased both D3 mRNA expression and activity. This led to increased intracellular T3 concentrations. The D3 decrease was prevented when NF-κB or AP-1 was inhibited. TRα1 mRNA expression decreased in SK-N-AS cells incubated with LPS, but knockdown of the TRα in SK-N-AS cells did not prevent the LPS-induced D3 decrease. Conclusions: We conclude that the inflammation-induced D3 decrease in the hypothalamus is mediated by the inflammatory pathways NF-κB and AP-1, but not TRα1. Furthermore, the observed decrease modulates intracellular T3 concentrations. Our results suggest a concerted action of inflammatory modulators to regulate both hypothalamic D2 and D3 activities to increase the local TH concentrations.

Quality marker identification based on standard decoction of differently processed materials of Ephedrae Herba.

ETHNOPHARMACOLOGICAL RELEVANCE: The quality control of Traditional Chinese medicine (TCM) is a scientific problem and an industrial issue, which hampers the development of evidence based TCM. The concept of quality markers (Q-markers) is proposed and has been applied to the quality evaluation of TCM based on its clinical efficacy. However, more specific methods are needed to put this idea into practice. The standard decoction is a representative of decoction used in clinical practice and it can be used for the discovery of Q-markers related to the clinical efficacy of TCM. AIM OF THE STUDY: In this study, a systemic strategy was established to discover Q-markers related to the clinical efficacy of TCM Ephedrae Herba (EH), dried stem of Ephedra sinica Stapf. The different processed materials of EH have different clinical applications, though originating from the same medicinal herb. MATERIALS AND METHODS: The standard decoction of each of the processed materials was prepared and a 1HNMR metabolomics approach and total polysaccharide analysis were used to identify potential Q-markers related to the different clinical applications of EH. Correlation analysis was made of the measured biological activity and the holistic chemical profile. RESULTS: The results showed that total polysaccharides and alkaloids were Q-markers for EH preparations. CONCLUSION: This study demonstrates that the standard decoction is a reasonable research objective to explore chemical markers that correlate with the clinical efficacy of TCM.

MAPK/AP-1-Targeted Anti-Inflammatory Activities of Xanthium strumarium.

Xanthium strumarium L. (Asteraceae), a traditional Chinese medicine, is prescribed to treat arthritis, bronchitis, and rhinitis. Although the plant has been used for many years, the mechanism by which it ameliorates various inflammatory diseases is not yet fully understood. To explore the anti-inflammatory mechanism of methanol extracts of X. strumarium (Xs-ME) and its therapeutic potential, we used lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells and human monocyte-like U937 cells as well as a LPS/D-galactosamine (GalN)-induced acute hepatitis mouse model. To find the target inflammatory pathway, we used holistic immunoblotting analysis, reporter gene assays, and mRNA analysis. Xs-ME significantly suppressed the up-regulation of both the activator protein (AP)-1-mediated luciferase activity and the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1[Formula: see text], IL-6, and tumor necrosis factor (TNF)-[Formula: see text]. Moreover, Xs-ME strongly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW264.7 and U937 cells. Additionally, these results highlighted the hepatoprotective and curative effects of Xs-ME in a mouse model of LPS/D-GalN-induced acute liver injury, as assessed by elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and histological damage. Therefore, our results strongly suggest that the ethnopharmacological roles of Xs-ME in hepatitis and other inflammatory diseases might result from its inhibitory activities on the inflammatory signaling of MAPK and AP-1.

Effects of Salidroside on Myocardial Injury In Vivo In Vitro via Regulation of Nox/NF-κB/AP1 Pathway.

Salidroside (Sal), a phenylpropanoid glycoside isolated from a popular traditional Chinese medicinal plant Rhodiola rosea L., possesses multiple pharmacological actions. This aim of this study is to investigate the effects of Sal against isoproterenol (ISO)-induced myocardial ischemia. Fifty male Sprague-Dawley rats were randomized equally to five groups: control group, ISO group, Sal (20 mg/kg; 40 mg/kg) treatments groups, and propranolol (Pro, 15 mg/kg) group. Rats were treated for 14 days and then given ISO (80 mg/kg) for 2 consecutive days by subcutaneous injection. In vitro, we used H9C2 cells to investigate the effects of Sal against hypoxia-reoxygenation. ST-segment elevation was measured after the last administration. Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), superoxide dismutase (SOD), and malondialdehyde (MDA); levels of NADPH oxidases 2 and 4 (Nox2 and Nox4), NF-κBP65, and AP1 in heart, and H9C2 cells were measured by Western blot. The hearts were excised for determining microscopic examination, SOD, and MDA measurements. Sal decreased the ST elevation induced by ISO, decreased serum levels of CK-MB, LDH, TNF-α, IL-6, SOD, and MDA. In addition, Sal increased SOD activity and decreased MDA content in myocardial tissue. Sal also decreased Nox2 and 4, NF-κBP65, P-NF-κBP65, and AP1 protein levels in the heart. The results support a further study of Sal as potential treatments for ischemic heart disease.

Azadirachta indica exerts chemopreventive action against murine skin cancer: studies on histopathological, ultrastructural changes and modulation of NF-kappaB, AP-1, and STAT1.

The present study reports the histopathological, ultrastructural changes and modulation of NF-kappaB, AP-1, and STAT 1 during skin carcinogenesis in LACA mice and its intervention with Azadirachta indica. Skin tumors were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) (500 nmol/100 microl for 2 weeks) followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 microl of acetone, twice weekly) as a promoter. Male LACA mice were divided into four groups: Control, DMBA/TPA, aqueous Azadirachta indica leaf extract (AAILE), and AAILE + DMBA/TPA. AAILE was administered orally at a dose level of 300 mg/kg body weight three times a week for 20 weeks. Topical application of DMBA/ TPA to the skin resulted in well-developed squamous cell carcinomas characterized by hyperproliferation, hyperkeratosis, and corrugation of the epidermis. Degenerative changes were observed in the tumors of AAILE + DMBA/TPA-treated animals. Scanning electron microscopy revealed surface disruptions and certain rounded structures on the skin tumors of DMBA/TPA-treated animals. Topographical changes were also observed in the tumors of AAILE + DMBA/TPA-treated animals, which resembled regions of degeneration. Tumors obtained in DMBA/TPA group were associated with enhanced expression of NF-kappaB and AP-1 when compared to the control counterparts. Inhibition in tumorigenesis in response to A. indica treatment was accompanied by an overexpression of STAT 1 and AP-1 and decrease in NF-kappaB expression. The results of the present study provide a basis for the chemopreventive potential of A. indica against murine skin carcinogenesis.

Signal transduction in the hypothalamic corticotropin-releasing factor system and its clinical implications.

Corticotropin-releasing factor (CRF) is a major regulatory peptide in the hypothalamic-pituitary-adrenal (HPA) axis under stress conditions. In response to stress, CRF is produced in the hypothalamic paraventricular nucleus. Forskolin- or pituitary adenylate cyclase-activating polypeptide-stimulated CRF gene transcription is mediated by the cyclic AMP (cAMP) response element on the CRF 5'-promoter region. Estrogens enhance activation of the CRF gene in stress, while inducible cAMP-early repressor suppresses the stress response via inhibition of the cAMP-dependent CRF gene. Glucocorticoid-dependent repression of cAMP-stimulated CRF promoter activity is mediated by both the negative glucocorticoid-response element and the serum-response element, while interleukin-6 (IL-6) stimulates the CRF gene. Suppressor of cytokine signaling-3, stimulated by IL-6 and cAMP, is involved in the negative regulation of CRF gene expression. Such complex mechanisms contribute to stress responses and homeostasis in the hypothalamus. Moreover, disruption of the HPA axis may cause a number of diseases related to stress. For example, CRF-induced p21-activated kinase 3 mRNA expression may be related to the proliferation of corticotrophs in Nelson's syndrome. A higher molecular weight form of immunoreactive ß-endorphin, putative proopiomelanocortin (POMC), is increased in CRF-knockout mice, suggesting the important role of CRF in the processing of POMC through changes in prohormone convertase type-1 expression levels.

p38-targeted inhibition of interleukin-12 expression by ethanol extract from Cordyceps bassiana in lipopolysaccharide-activated macrophages.

Cordyceps species have been known as ethnopharmacologically valuable mushroom in Korea, China, and Japan. This plant has been reported to exhibit a variety of pharmacological activities such as antioxidative, anticancer, anti-inflammatory, antidiabetic, and antiobesity effects. Although numerous pharmacological potentials of Cordyceps spp. have been demonstrated, immunomodulatory effect of Cordyceps bassiana has not been published yet. To evaluate its immunomodulatory activity, macrophages activated by lipopolysaccharide (LPS) were employed and the production of interleukin-12 (IL-12) was explored in terms of understanding its molecular inhibitory mechanism. Seventy percent of ethanol extract from Cordyceps bassiana (Cb-EE) was able to suppress the expression of IL-12, a cytokine regulating interferon-γ (IFN-γ)-producing T helper type 1 (Th1) polarization response, at the transcriptional levels. The inhibitory effect of Cb-EE seemed to be due to activator protein-1 (AP-1) translocation inhibition, according to immunoblotting analysis with nuclear fraction and luciferase assay. In agreement with this, Cb-EE strongly suppressed the phosphorylation of p38, a prime signal to stimulate AP-1 translocation and IL-12 production, strongly suppressed by SB203580, a p38 inhibitor. Furthermore, this extract also suppressed IFN-γ production in both phytohemaglutinin A and LPS-activated splenocytes. Our results suggest that Cb-EE can be applied as a Th1 response regulatory herbal medicine.

Ethanol extract of Lycoris radiata induces cell death in B16F10 melanoma via p38-mediated AP-1 activation.

Some active alkaloids isolated from Lycoris, a bulbous perennial herb, was shown to possess various anti-tumor and anti-inflammatory activities. In this study, we evaluated the in vitro apoptotic effect of ethanol extract from Lycoris radiata (LRE) and further probed the underlying molecular mechanisms of LRE effects. The survival rate of B16F10 melanoma cells exposed to LRE was decreased in a dose-dependent manner, cell growth was retarded by arresting cell cycle at G1 phase and apoptotic appearance such as caspase-3 activation as well as DNA fragmentation was observed by LRE treatment. In addition, LRE induced p38 and c-Jun phosphorylation, followed by activation of transcription factor AP-1. Pretreatment with the p38 inhibitor (SB203580) blocked LRE-induced AP-1 transcriptional activity, and curcumin, AP-1 inhibitor, dramatically inhibited LRE-induced apoptosis in B16F10 melanoma cells. Our results collectively indicate that LRE-mediated apoptosis occurs through the activation of p38 and AP-1 pathway and potentially LRE exhibits anti-cancer activity against B16F10 melanoma cells.

Rhizoma Coptidis inhibits LPS-induced MCP-1/CCL2 production in murine macrophages via an AP-1 and NFkappaB-dependent pathway.

INTRODUCTION: The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. METHODS: We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFkappaB was analyzed in nuclear extracts, secretion of MCP-1/CCL2 was measured in supernatants. RESULTS: Incubation with Rhizoma coptidis and berberine strongly inhibited LPS-induced monocyte chemoattractant protein (MCP)-1 production in RAW cells. Activation of the transcription factors AP-1 and NFkappaB was inhibited by Rhizoma coptidis in a dose- and time-dependent fashion. CONCLUSIONS: Rhizoma coptidis extract inhibits LPS-induced MCP-1/CCL2 production in vitro via an AP-1 and NFkappaB-dependent pathway. Anti-inflammatory action of the extract is mediated mainly by its alkaloid compound berberine.

Hop bitter acids efficiently block inflammation independent of GRalpha, PPARalpha, or PPARgamma.

Hop (Humulus lupulus L.) is an essential ingredient of beer, where it provides the typical bitter taste, but is also applied in traditional folk medicine for sedative and antibacterial purposes. In this study, we demonstrate and compare the anti-inflammatory effect of various classes of hop bitter acids (HBA), including alpha-acids (AA), beta-acids (BA), and iso-alpha-acids (IAA), in fibroblasts, which are important players in the inflammatory response. All three studied classes of HBA blocked the tumor necrosis factor alpha (TNF)-induced production of the cytokine IL6, and inhibited the transactivation of the pro-inflammatory transcription factors nuclear factor kappa B (NF-kappaB), activator protein-1 (AP-1), and cAMP-response element-binding protein (CREB). In this respect, the six-membered ring compounds AA and BA showed equal potency, whereas the five-membered ring compounds, IAA, were effective only when used at higher concentrations. Furthermore, with regard to the mechanism of NF-kappaB suppression, we excluded a possible role for glucocorticoid receptor alpha (GRalpha), peroxisome proliferators-activated receptor alpha/gamma (PPARalpha or PPARgamma), nuclear receptors (NRs) that are also known to inhibit inflammation by directly interfering with the activity of pro-inflammatory transcription factors. Interestingly, combining hop acids and selective agonists for GRalpha, PPARalpha, or PPARgamma resulted in additive inhibition of NF-kappaB activity after TNF treatment, which may open up new avenues for combinatorial anti-inflammatory strategies with fewer side effects. Finally, systemic administration of HBA efficiently inhibited acute local inflammation in vivo.

Oleic acid-induced ADRP expression requires both AP-1 and PPAR response elements, and is reduced by Pycnogenol through mRNA degradation in NMuLi liver cells.

Fatty acids stimulate lipid accumulation in parallel with increased expression of adipose differentiation-related protein (ADRP) in liver cells. Although it is generally considered that the fatty acid effect on ADRP expression is mediated by peroxisome proliferator-activated receptors (PPARs), we identified here an additional molecular mechanism using the NMuLi mouse liver nonparenchymal cell line, which expresses PPARgamma and delta but not alpha. Oleic acid (OA) and specific ligands for PPARgamma and -delta stimulated ADRP expression as well as the -2,090-bp ADRP promoter activity which encompasses the PPAR response element (PPRE) adjacent to an Ets/activator protein (AP)-1 site. When the AP-1 site was mutated, OA failed to stimulate the activity despite the presence of the PPRE, whereas ligands for PPARgamma and -delta did stimulate it and so did a PPARalpha ligand under the coexpression of PPARalpha. DNA binding of AP-1 was stimulated by OA but not by PPAR ligands. Because we previously demonstrated that Pycnogenol (PYC), a French maritime pine bark extract, suppressed ADRP expression in macrophages partly by suppression of AP-1 activity, we tested the effect of PYC on NMuLi cells. PYC reduced the OA-induced ADRP expression along with suppression of lipid droplet formation. However, PYC neither suppressed the OA-stimulated ADRP promoter activity nor DNA binding of AP-1 but, instead, reduced the ADRP mRNA half-life. All these results indicate that the effect of OA on ADRP expression requires AP-1 as well as PPRE, and PYC suppresses the ADRP expression in part by facilitating mRNA degradation. PYC, a widely used dietary supplement, could be beneficial for the prevention of excessive lipid accumulation such as hepatic steatosis.

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells.

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages.

8-Prenylkaempferol is a prenylflavonoid isolated from the roots of Sophora flavescens, a Chinese herb with anti-inflammatory properties. However whether 8-prenylkaempferol itself displayed an anti-inflammatory activity remained unclear. In this study, we evaluated the effect of 8-prenylkaempferol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. 8-Prenylkaempferol inhibited significantly LPS-induced NO production through suppressing inducible NO synthase (iNOS) expression at both protein and mRNA levels but failed to affect sodium nitroprusside-triggered NO production, iNOS enzyme activity, and cell viability. Further investigation of the mechanisms revealed that 8-prenylkaempferol inhibited LPS-induced c-Jun phosphorylation (a major component of activator protein-1, AP-1), but did not attenuate IkB-alpha degradation nor NF-kappaB nuclear translocation. Cellular signaling analysis using mitogen-activating protein kinase (MAPK) inhibitors including 2'-amino-3'-methoxyflavone (PD98059, MEK1/2 inhibitor), 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580, p38 kinase inhibitor) and anthra[1-9-cd]pyrazol-6(2H)-one (SP600125, c-Jun N-terminal kinase inhibitor) demonstrated that extracellular signal-regulated kinase1/2 (ERK1/2), p38 and JNK all participated in LPS-stimulated iNOS expression and NO production, but 8-prenylkaempferol interfered selectively with JNK phosphorylation. On the other hand, LPS-induced c-Jun phosphorylation was attenuated in the presence of SP600125. We suggested that interfering with JNK-mediated c-Jun phosphorylation and thus blocking AP-1 activation might contribute to the suppression effects of 8-prenylkaempferol on iNOS. These findings provided the first molecular basis that 8-prenylkaempferol is an effective agent for attenuating pro-inflammatory NO induction.

Retinoic acid induces apoptosis by a non-classical mechanism of ERK1/2 activation.

Even though RA is involved in differentiation and apoptosis of normal and cancer cells, being sometimes used as adjuvant in chemotherapy, its mechanisms of action involve multiple overlapping pathways that still remain unclear. Recent studies point out that RA exerts rapid and non-genomic effects, which are independent of RAR/RXR-mediated gene transcription. In this work, we reported that RA treatment for 24 h decreases cell viability, induces apoptosis dependent on caspase-3 activation, and activates the transcription factor AP-1 in cultured Sertoli cells. Moreover, RA induced a rapid and non-classical stimulation of ERK1/2. ERK1/2 activation was mediated by MEK1/2, and the protein synthesis inhibitor cycloheximide did not alter the pattern of RA-induced ERK1/2 phosphorylation. Pharmacological inhibition of MEK1/2-ERK1/2 pathway with UO126 blocked caspase-3 activation, decreased AP-1 binding to DNA and inhibited apoptosis. Overall, our data suggest that a rapid and non-genomic effect of RA upon MEK1/2-ERK1/2 pathway leads to caspase-3 activation and caspase-3-dependent apoptosis in cultured Sertoli cells. The non-canonical RA signaling presented in this work evokes new perspectives of RA action, which may play an important role in mediating early biological effects of RA modulating cell death in normal and tumor cells.

Suppression of tissue necrosis factor-alpha or hydrogen peroxide-activated primary human T lymphocytes by Ginkgo biloba extract through down-regulation of activator protein-1 signal transduction.

PURPOSE: It was unknown whether Ginkgo biloba extract has regulatory effects on human T lymphocytes activated by tissue necrosis factor-alpha (TNF-alpha), which has an important role on the progression of inflammatory atherosclerotic plaques. We evaluated the effects of G. biloba extract on activated human peripheral T lymphocytes, which were isolated from human whole blood. METHODS: The human T lymphocytes were treated with 25-100 microg G. biloba extract for 2h first. Then they were activated by TNF-alpha and H(2)O(2) to investigate the modulatory effects of G. biloba extract on human T lymphocytes. Electrophoretic mobility shift assay, Western blot (Immunoblot) analysis and immunoprecipitation kinase assays were used. RESULTS: The inhibition of activated human T lymphocyte specifically correlated with the down-regulation of AP-1 DNA-binding activities. G. biloba extract was unique in its ability to inhibit the activation of c-Jun NH2-terminal protein kinase. CONCLUSIONS: G. biloba extract might have its novel therapeutic effects on inflammation-based atherosclerotic diseases.

Inhibitory effects of thunberginols A and B isolated from Hydrangeae Dulcis Folium on mRNA expression of cytokines and on activation of activator protein-1 in RBL-2H3 cells.

Previously, thunberginols A and B from the processed leaves of Hydrangeae macrophylla var. thunbergii (Hydrangea dulcis folium) substantially inhibited the degranulation caused by antigen and calcium ionophore A23187, and the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-4 by antigen in RBL-2H3 cells. In the present study, we examined the effect of thunberginol B on the expression of mRNA of several cytokines [ILs-2, 3, 4 and 13, TNF-alpha and granulocyte/macrophage-colony stimulating factor (GM-CSF)] and effects of thunberginols A and B on activator protein (AP)-1 composed of c-jun and c-fos, which is essential for the expression of the cytokine mRNA, in RBL-2H3 cells. Thunberginol B inhibited up-regulated genes of all cytokines, and thunberginols A and B (30 microM) inhibited the phosphorylation of c-jun and expression of c-fos mRNA and phosphorylation of extracellular signal-regulated kinases (ERK1/2). In addition, the profile of gene expression by thunberginol B was similar to that by luteolin, a natural flavone with a potent anti-allergic effect.

Macrophage paraoxonase 2 (PON2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via PPAR gamma and AP-1 pathway activation.

Paraoxonase 2 (PON2), a member of the paraoxonase gene family, was shown to protect macrophages against oxidative stress. Pomegranate juice (PJ), which contains potent polyphenol anti-oxidants, exhibits similar effects. We questioned possible association between PJ polyphenolics, macrophage oxidative stress, and cellular PON2 expression, in relation to the activation of specific PON2 transcription factors. Incubation of J774A.1 macrophages with PJ (0-50 microM of total polyphenols) dose-dependently increased expression (mRNA, protein) and activity and reduced macrophage oxidative status. These effects could be attributed to the PJ unique polyphenols, punicalagin and gallic acid. PJ polyphenol-induced up-regulation of PON2 was inhibited by 40% upon using the PPAR gamma inhibitor GW9662 (50 microM). Accordingly, the PPAR gamma ligand, rosiglitazone, dose-dependently stimulated macrophage PON2 expression, by up to 80%. Inhibition of AP-1 activation with SP600125, attenuated PJ-induced up-regulation of PON2 by 40%. Similarly, incubation of macrophages with PJ polyphenols in the presence of GW9662 or SP600125, significantly reduced their capacity to protect the cells against oxidative stress. We conclude that the anti-oxidative characteristics of PJ unique phenolics punicalagin and gallic acid could be related, at least in part, to their stimulatory effect on macrophage PON2 expression, a phenomenon which was shown to be associated with activation of the transcription factors PAPR gamma and AP-1.

Diallyl disulfide and diallyl trisulfide up-regulate the expression of the pi class of glutathione S-transferase via an AP-1-dependent pathway.

Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I.

Ethanol prevents development of destructive arthritis.

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by (i) down-regulation of leukocyte migration and (ii) up-regulation of testosterone secretion, with the latter leading to decreased NF-kappaB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.