Modulation of NMDA Receptor Subunits Expression by Concanavalin A.

The processes of N-methyl-D-aspartate (NMDA) receptor subunits expression were examined in cortical neurons and rat brain in order to investigate how the concanavalin A (Con A) modulates neuronal cells. Con A modulated the expression of NMDA receptor subunits in cultured cortical cells. Con A augmented the level of intracellular Ca(2+) by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). We determined whether activation of AMPA receptors was involved in the regulation of NMDA receptor expression with Con A by blocking the desensitization of AMPA receptors. The results showed that AMPA receptor antagonists suppressed NMDA receptor subunits expression in Con A-treated cortical neuronal cells. PMA elevated the expression of NMDA receptor subunits, while PKC inhibitor and tyrosine kinases inhibitor suppressed the expression of NMDA receptor subunits. Furthermore, it was shown that NMDA receptor subunits expression was modulated in a region-specific manner after the sustained microinfusion of Con A into the cerebroventricle of the rat brain. Collectively, it could be presumed that the AMPA receptor activation was involved in Con A-induced modulation of NMDA receptor subunits expression.

Anti-Inflammatory activities of licorice extract and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in BV2 cells and mice liver.

This study provides the scientific basis for the anti-inflammatory effects of licorice extract in a t-BHP (tert-butyl hydrogen peroxide)-induced liver damage model and the effects of its ingredients, glycyrrhizic acid (GA), liquiritin (LQ) and liquiritigenin (LG), in a lipopolysaccharide (LPS)-stimulated microglial cell model. The GA, LQ and LG inhibited the LPS-stimulated elevation of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interleukin (IL)-6 in BV2 (mouse brain microglia) cells. Furthermore, licorice extract inhibited the expression levels of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) in the livers of t-BHP-treated mice models. This result suggested that mechanistic-based evidence substantiating the traditional claims of licorice extract and its three bioactive components can be applied for the treatment of inflammation-related disorders, such as oxidative liver damage and inflammation diseases.

Sulforaphane attenuates obesity by inhibiting adipogenesis and activating the AMPK pathway in obese mice.

Obesity is associated with metabolic disorders. Sulforaphane, an isothiocyanate, inhibits adipogenesis and the occurrence of cardiovascular disease. In this study, we investigated whether sulforaphane could prevent high-fat diet (HFD)-induced obesity in C57BL/6N mice. Mice were fed a normal diet (ND), HFD or HFD plus 0.1% sulforaphane (SFN) for 6 weeks. Food efficiency ratios and body weight were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN attenuated HFD-induced visceral adiposity, adipocyte hypertrophy and fat accumulation in the liver. Serum total cholesterol and leptin, and liver triglyceride levels were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin in the adipose tissue of HFD-SFN mice and increased adiponectin expression. Phosphorylation of AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase in the adipose tissue of HFD-SFN-fed mice was elevated, and HMG-CoA reductase expression was decreased compared with HFD-fed mice. Thus, these results suggest that SFN may induce antiobesity activity by inhibiting adipogenesis through down-regulation of PPARγ and C/EBPα and by suppressing lipogenesis through activation of the AMPK pathway.

Cellular mechanisms of the cytotoxic effects of the zearalenone metabolites α-zearalenol and ß-zearalenol on RAW264.7 macrophages.

Zearalenone (ZEN) and its metabolites are commonly found in many food commodities and are known to cause reproductive disorders and genotoxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). Although many studies have demonstrated the cytotoxic effects of these metabolites, the mechanisms by which α-ZOL or ß-ZOL mediates their cytotoxic effects appear to differ according to cell type and the exposed toxins. We evaluated the toxicity of α-ZOL and ß-ZOL on RAW264.7 macrophages and investigated the underlying mechanisms. ß-ZOL not only more strongly reduced the viability of cells than did α-ZOL, but it also induced cell death mainly by apoptosis rather than necrosis. The ZEN metabolites induced loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Use of an inhibitor specific to c-Jun N-terminal kinase (JNK), p38 kinase or p53, but not pan-caspase or caspase-8, decreased the toxin-induced generation of reactive oxygen species (ROS) and also attenuated the α-ZOL- or ß-ZOL-induced decrease of cell viability. Antioxidative enzyme or compounds such as catalase, acteoside, and (E)-1-(3,4-dihydroxyphenethyl)-3-(4-hydroxystyryl)urea suppressed the ZEN metabolite-mediated reduction of cell viability. Further, knockdown of AIF via siRNA transfection diminished the ZEN metabolite-induced cell death. Collectively, these results suggest that the activation of p53, JNK or p38 kinase by ZEN metabolites is the main upstream signal required for the mitochondrial alteration of Bcl-2/Bax signaling pathways and intracellular ROS generation, while MMP loss and nuclear translocation of AIF are the critical downstream events for ZEN metabolite-mediated apoptosis in macrophages.

Anti-platelet activity of diacetylated obovatol through regulating cyclooxygenase and lipoxygenase activities.

Obovatol has been reported biological activities such as muscle relaxative, anti-gastric ulcer, anti-allergic and anti-bacterial activities. The present study was undertaken to investigate the effect of diacetylated obovatol, an obovatol derivative, on rabbit platelet aggregation, and their possible molecular mechanisms. Effects of diacetylated obovatol on platelet activation including aggregation and serotonin secretion were examined. In addition, we investigated the effect of diacetylated obovatol on archidonic acid and metabolites liberation and intracellular calcium mobilization. Diacetylated obovatol concentration-dependently inhibited the washed rabbit platelet aggregation induced by collagen and arachidonic acid, suggesting that diacetylated obovatol may selectively inhibits collagen- and arachidonic acid-mediated signal transduction. In accordance with these results, diacetylated obovatol showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization and serotonin secretion. However, diacetylated obovatol did not inhibit arachidonic acid liberation; on the other hand, diacetylated obovatol inhibited the formation of arachidonic acid metabolites such as thromboxane A(2), prostaglandin D(2) and 12-HETE through interfering with cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. The results demonstrated that diacetylated obovatol has antiplatelet activities through inhibition of COX-1 and LOX activities.

Histologic effects of intentional-socket-assisted orthodontic movement in rabbits.

OBJECTIVE: This study aimed to evaluate the effect of an intentionally created socket on bone remodeling with orthodontic tooth movement in rabbits. METHODS: Eighteen male rabbits weighing 3.8 - 4.25 kg were used. An 8-mm deep and 2-mm wide socket was drilled in the bone 1 mm mesial to the right mandibular first premolar. The left first premolar was extracted to serve as an extraction socket. A traction force of 100 cN was applied to the right first premolar and left second premolar. Sections were obtained at the middle third of the moving tooth for both the drilled and extraction sockets and evaluated with hematoxylin and eosin staining and immunohistochemical analyses. The amount of tooth movement and tartrate-resistant acid phosphatase (TRAP)-positive cell count were compared between the 2 groups using the Mann-Whitney U test. RESULTS: At week 2, the distance of tooth movement was significantly higher in the intentional socket group (p < 0.05) than in the extraction socket group. The number of TRAP-positive cells decreased in week 2 but increased in week 3 (p < 0.05). However, there were no significant differences between the groups. Furthermore, results of transforming growth factor (TGF)-ß staining revealed no significant differences. CONCLUSIONS: The intentional socket group showed greater distance of tooth movement than did the extraction socket group at week 2. Osteoclast counts and results of immunohistochemical analyses suggested elevated bone remodeling in both the groups. Thus, osteotomy may be an effective modality for enhancing tooth movement in orthodontic treatment.

JJK694, a synthesized obovatol derivative, inhibits platelet activation by suppressing cyclooxygenase and lipoxygenase activities.

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, anti-platelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative, on rabbit platelet activation and its molecular mechanisms. JJK694 significantly inhibited washed rabbit platelet aggregation and serotonin secretion induced by collagen and arachidonic acid, but had little effect on thrombin- or U46619-induced aggregation. These results suggest that JJK694 selectively inhibits collagen- and arachidonic acid-mediated signaling. JJK694 also showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization, but it had no effect on arachidonic acid liberation. On the other hand, it significantly inhibited the formation of arachidonic acid metabolites, including thromboxane A(2) (TXA(2)), prostaglandin D(2), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by suppression of cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. These results indicate that JJK694 hasanti-platelet activities through inhibition of arachidonic acid metabolite production by suppression of COX-1 and LOX activities.

Rhamnetin-induced suppression of clonal expansion during early stage of adipogenesis.

Adipocyte differentiation plays a pivotal role in the progression of obesity which is a major risk factor for several diseases such as diabetes, hypertension and coronary heart disease. In this study, the inhibitory effect of rhamnetin, a flavonoid compound, on adipogenesis in 3T3-L1 cells was investigated. Rhamnetin decreased the accumulation of lipid droplets, and inhibited the elevation of triglyceride content in the adipocytes (IC(50) = 17.3 µM). The expressions of PPARγ, C/EBPα, and perilipin, adipocyte differentiation markers, were significantly reduced by rhamnetin. Triglyceride biosynthesis and clonal expansion of adipocytes were completely inhibited during the early stage by rhamnetin. Additionally, rhamnetin significantly decreased the expression of C/EBPß, an early stage marker. Our results indicate that suppression of clonal expansion during the early stage of adipogenesis by rhamnetin may be associated with inhibition of the C/EBPß, C/EBPα, and PPARγ pathways.

Continuous presence of H2O2 induces mitochondrial-mediated, MAPK- and caspase-independent growth inhibition and cytotoxicity in human gingival fibroblasts.

The continuous generation of reactive oxygen species (ROS) is one of the most important events that occur during periodontal inflammation. Hydrogen peroxide (H(2)O(2)) is widely used in dental clinics. Many investigators have tried to elucidate the exact effect of H(2)O(2) on human gingival fibroblasts (HGFs). These studies have shown that H(2)O(2) induces growth inhibition and apoptosis in cells. However, the mechanisms involved in H(2)O(2)-induced cell death in HGFs are not completely understood. In this study, we examine how continuously generated H(2)O(2) affects the viability and proliferation of HGFs using glucose oxidase (GO). We also explored the mechanisms by which the continuous presence of H(2)O(2) induces cell death. GO treatment not only inhibited HGF growth and proliferation, but it also induced cell death in HGFs without typical apoptotic features such as nuclear DNA laddering. This GO-mediated cytotoxicity was proportional to the levels of intracellular ROS that were generated, rather than proportional to changes of cellular antioxidant activities. GO treatment also resulted in the loss of mitochondrial membrane potential and the relocation of mitochondrial apoptogenic factors. There was also an acute and severe depletion of cellular ATP levels. However, none of the pharmacological inhibitors specific for mitogen-activated protein kinases (MAPKs) or pancaspase prevented GO-induced cell death. Treatment with either catalase or acteoside significantly attenuated the GO-mediated cytotoxicity in the HGFs, thereby suggesting a protective effect of antioxidants against ROS-mediated gingival damage. Here we demonstrate that continuously generated H(2)O(2) not only inhibits the viability and proliferation of HGFs, but also causes pyknotic/necrotic cell death through mitochondrial stress-mediated, MAPK- and caspase-independent pathways.

Continuously generated H2O2 stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts.

Numerous studies have shown that hydrogen peroxide (H(2)O(2)) inhibits proliferation and osteoblastic differentiation in bone-like cells. Human periodontal ligament fibroblasts (PLF) are capable of differentiating into osteoblasts and are exposed to oxidative stress during periodontal inflammation. However, the cellular responses of PLF to H(2)O(2) have not been identified. In this study, we examined how H(2)O(2) affects the viability and proliferation of PLF by exposing the cells to glucose oxidase (GO) or direct addition of H(2)O(2). We also explored the effects of GO on the osteoblastic differentiation of PLF and the mechanisms involved. The viability and proliferation in PLF were increased with the addition of 10 mU/ml GO but not by volumes greater than 15 mU/ml or by H(2)O(2) itself. GO-stimulated DNA synthesis was correlated with the increase in cyclin E protein levels in the cells. Osteoblastic differentiation of PLF was also augmented by combined treatment with GO, as evidenced by the increases in alkaline phosphatase activity, mineralization, collagen synthesis, and osteocalcin content in the cells. The inductions of runt-related transcription factor 2 and osterix mRNA and proteins were further increased in PLF incubated in combination with GO compared to those in untreated cells. These results demonstrate that the continuous presence of H(2)O(2) stimulates the proliferation of PLF and augments their potential to differentiate into osteoblasts through the up-regulation of bone-specific transcription factors. Collectively, we suggest that H(2)O(2) may elicit the functions of PLF in maintaining the dimensions of the periodontal ligament and in mediating a balanced metabolism in alveolar bone.

Mycotoxin zearalenone induces AIF- and ROS-mediated cell death through p53- and MAPK-dependent signaling pathways in RAW264.7 macrophages.

Zearalenone (ZEN) is commonly found in many food commodities and is known to cause reproductive disorders and genotoxic effects. However, the mode of ZEN-induced cell death of macrophages and the mechanisms by which ZEN causes cytotoxicity remain unclear. The present study shows that ZEN treatment reduces viability of RAW264.7 cells in a dose-dependent manner. ZEN causes predominantly necrotic and late apoptotic cell death. ZEN treatment also results in the loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Pre-treatment of the cells with either z-VAD-fmk or z-IETD-fmk does not attenuate ZEN-mediated cell death, whereas catalase suppresses the ZEN-induced decrease in viability in RAW264.7 cells. Treating the cells with c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or p53 inhibitor prevented ZEN-mediated changes, such as MMP loss, cellular reactive oxygen species (ROS) increase, and cell death. JNK or p38 MAPK inhibitor inhibited mitochondrial alterations of Bcl-2 and Bax proteins with attendant decreases in cellular ROS levels. Knockdown of AIF via siRNA transfection also diminished ZEN-induced cell death. Further, adenosine triphosphate was markedly depleted in the ZEN-exposed cells. Collectively, these results suggest that ZEN induces cytotoxicity in RAW264.7 cells via AIF- and ROS-mediated signaling, in which the activations of p53 and JNK/p38 play a key role.

Chemoprevention of a flavonoid fraction from Rhus verniciflua Stokes on aflatoxin B1-induced hepatic damage in mice.

Since aflatoxin B(1) (AFB(1))-mediated hepatic damage is related to the production of AFB(1)-8,9-epoxide and reactive oxygen species, bioactive compounds having antioxidant potentials are suggested to be capable of reducing AFB(1)-induced toxicity. We previously purified a mixture of flavonoids that we named RCMF (Rhus verniciflua Stokes chloroform-methanol fraction), from a traditional Korean food additive and herbal medicine. RCMF exhibited various biological effects, including antioxidant and antitumor activities. In this study, we examined whether RCMF protects against AFB(1)-induced liver injury using in vitro and in vivo systems. Pretreatment of HepG2 cells with RCMF significantly reduced AFB(1)-stimulated production of ROS and malondialdehyde (MDA) to the control levels. RCMF also prevented the reduction in HepG2 cell viability caused by AFB(1). Oral administration of RCMF to mice significantly suppressed an AFB(1)-induced increase in serum levels of alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase. It also prevented MDA formation and blocked decreases in glutathione levels and superoxide dismutase activities in the livers of AFB(1)-treated mice. In addition, RCMF supplementation prevented an AFB(1) -induced decrease in serum titers of IgA and IgG1. Collectively, these results suggest that RCMF attenuates AFB(1)-mediated damage to the liver, and that this effect is at least partially related to the restoration of antioxidant defense systems and an increase in AFB(1)-GSH conjugate formation.

Inhibitory effect of fenofibrate on neointima hyperplasia via G(0)/G(1) arrest of cell proliferation.

We have previously reported that fenofibrate displayed a potent antithrombotic effect by the inhibition of platelet aggregation. The present study was designed to investigate the effects of fenofibrate on the neointimal hyperplasia and its possible molecular mechanism. Neointimal hyperplasia was measured in balloon-inflated-induced vascular injury model of male Sprague-Dawley rats and cell proliferation was measured in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Fenofibrate-treated group showed a significant reduction in neointimal formation (0.07±0.04mm(2)) from the control (0.13±0.04mm(2)). Fenofibrate significantly inhibited platelet-derived growth factor (PDGF)-BB-induced cell counting and [(3)H]-thymidine incorporation into DNA. Fenofibrate suppressed the PDGF-BB-inducible progression through G(0)/G(1) to S phase of cell cycle. Moreover, fenofibrate inhibited not only phosphorylation of retinoblastoma (Rb) protein and expression of cyclin D/E, CDK 2/4 and proliferating cell nuclear antigen (PCNA) proteins but also mitogen-activated protein kinase (MAPK) signaling pathways such as ERK 1/2, p38 and JNK phosphorylation. In conclusion, the present study demonstrates that fenofibrate significantly inhibits neointimal formation via G(0)/G(1) arrest of PDGF-BB-induced cell proliferation in association with the inhibition of MAPK, which resulted in the downregulation of expressions of cyclin D/E, CDK 2/4 and PCNA proteins, suggesting that fenofibrate may be useful for individuals with a high risk of thrombotic or cardiovascular diseases.

(E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea inhibits proliferation of MCF-7 cells through G1 cell cycle arrest and mitochondria-mediated apoptosis.

Growing interest in the beneficial effects of antioxidants has inspired the synthesis of new phenolic acid phenethyl ureas (PAPUs) with enhanced antioxidant potential. We have previously shown the capacity of one PAPU compound, (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea (PAPU1), to induce caspase-dependent apoptosis in melanoma cells. In the present study, we examined the anti-proliferative effects of PAPU compounds on MCF-7 human breast cancer cells and determined the molecular mechanisms involved. Treatment with PAPU compounds inhibited predominantly proliferation in these cells, where the PAPU1 was the most efficient form. Flow cytometric analysis showed that PAPU1 blocked cell cycle progression in the G(0)/G(1) phase, and reduced the proportion of cells in G(2)/M phase. This was related to the inhibition of cell cycle regulatory factors, including cyclin D/E and cyclin-dependent kinase (CDK) 2/4, through induction of p21(Cip1). PAPU1 also induced the mitochondrial-mediated and caspase-dependent apoptosis in MCF-7 cells. This was evidenced by cellular changes in the levels of Bcl-2 and Bax, loss of the mitochondrial membrane potential, release of cytochrome c into the cytosol, and caspase-9 activation. Collectively, our results suggest that G(1) cell cycle regulatory proteins and mitochondrial pathways are the crucial targets of PAPU1 in the chemoprevention of breast cancer cells.

Inhibitory effects of BST406, a newly synthesized benzylideneacetophenone derivative, on abnormal vascular smooth muscle cell proliferation.

Benzylideneacetophenone analogues are known to have several significant biological activities, including antiinflammatory, antitumor, antibacterial, antiviral, and gastric-protective activities. However, the antiproliferative effects of benzylideneacetophenone analogues on vascular smooth muscle cells (VSMCs) are unknown. The aim of this study was to elucidate the antiproliferative effects and molecular mechanism of BST406, a newly synthesized benzylideneacetophenone derivative, on platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. BST406 inhibited [(3)H]-thymidine incorporation into DNA in VSMCs following treatment with PDGFBB 25 ng/ml. PDGF-BB-stimulated DNA synthesis was significantly reduced. Moreover, pretreatment with BST406 (0-10microM) suppressed the proliferation of PDGF-BB-stimulated cells in a concentration-dependent manner. We also investigated the mechanism of the antiproliferative effects of BST406 in PDGF-BB-stimulated VSMCs. In Western blot analysis, PDGF-BB-stimulated (25 ng/ml) phospholipase-C (PLC)gamma1 and Akt phosphorylation was inhibited by BST406 (0-10microM). However, BST406 did not inhibit the PDGF-receptor beta-chain (PDGF-Rbeta) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation induced by PDGF-BB. To confirm that the inhibitory effects of BST406 are mediated through the inhibition of PLCgamma1 or Akt, the effects of inhibitors on cell viability were examined. U73122 completely inhibited PDGF-BB-induced proliferation of VSMCs. However, LY294002 10microM had no significant effects on PDGF-BB-induced proliferation. These findings suggest that the inhibitory effects of BST406 on the proliferation of PDGF-BB-stimulated VSMCs are mediated by suppression of the PLCgamma1 signaling pathways. Our observations may explain, in part, the mechanistic basis for the prevention of cardiovascular disease (such as atherosclerosis and restenosis after coronary angioplasty) by BST406.

The protective effects of paclitaxel on platelet aggregation through the inhibition of thromboxane A2 synthase.

Paclitaxel is an anticancer drug used in the treatment of ovarian, breast, head and neck, lung, and prostate cancer. We investigated the antiplatelet activity of paclitaxel in vitro as well as a possible antiplatelet mechanism. Paclitaxel inhibited washed rabbit platelet aggregation induced by collagen in a concentration dependent manner, with an IC(50) of 59.7 +/- 3.5. However, it had little effect on platelet aggregation mediated by arachidonic acid, U46619, a thromboxane (TX) A(2) mimic, or thrombin, suggesting that paclitaxel may strongly inhibit collagen mediated signal transduction. In accordance with these findings, paclitaxel blocked collagen induced cytosolic calcium mobilization, arachidonic acid liberation, and serotonin secretion. In addition, it inhibited arachidonic acid mediated platelet aggregation by about 37% by interfering with TXA(2) synthase as measured by the formation of arachidonic acid mediated TXA(2) and prostaglandin D(2), as well as cyclooxygenase-1 and TXA(2) synthase activity assays. Taken together, these results point to a cellular mechanism for the antiplatelet activity of paclitaxel through the inhibition of TXA(2) synthase and cytosolic calcium mobilization. This may contribute to the beneficial effects of paclitaxel on the cardiovascular system.

A phenolic acid phenethyl urea compound inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines in cell culture.

We previously used the Curtius rearrangement to synthesize various phenolic acid phenethyl urea compounds from phenolic acids and demonstrated their beneficial anti-oxidant and anti-cancer effects. Here, we investigated the effects of one of these synthetic compounds, (E)-1-(3,4-dihydroxystyryl)-3-(4-hydroxyphenethyl)urea (DSHP-U), on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and cytokine secretion in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. DSHP-U suppressed LPS-induced NO production and iNOS expression at a concentration of 50 microM and inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. Inhibitors of phosphorylated (p)-ERK and p-p38, but not of p-JNK, reduced LPS-stimulated NO production. DSHP-U also prevented the nuclear translocation of the Rel A (p65) subunit and DNA-NF-kappaB binding by suppressing IkappaBalpha phosphorylation and by the degradation of IkappaBalpha in LPS-stimulated cells. Furthermore, DSHP-U decreased the production of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 in LPS-treated macrophages. However, the LPS-stimulated expression of LPS receptors, such as Toll-like receptor 4, myeloid differentiation factor-2, and CD14, was unchanged after DSHP-U treatment at significantly high levels. Our data suggest that DSHP-U blocks NO and inflammatory cytokine production in LPS-stimulated macrophages and that these effects are mainly mediated through the inhibition of the ERK/p38- and NF-kappaB signaling pathways.

JY0691, a newly synthesized obovatol derivative, inhibits cell cycle progression of rat aortic smooth muscle cells through up-regulation of p21(cip1).

Obovatol is known to have various biological activities such as muscle relaxation, anti-gastric ulcer, anti-inflammatory, anti-allergic, and anti-bacterial activities. We examined the effects of JY0691, a newly synthesized obovatol derivative, on the viability and proliferation in rat aortic smooth muscle cells. We also determined the mechanism by which the compound induces cell cycle arrest of the cells. Treatment with JY0691 (0-3 microM) inhibited proliferation of the cells in a dose-dependent manner without any cytotoxic effect. JY0691 treatment did not decrease the levels of platelet-derived growth factor (PDGF) receptor and several protein kinases which had stimulated by exposing the cells to 25 ng/ml PDGF-BB. In contrast, the compound arrested the cell cycle progression in the G(0)/G(1) phase, which was related to the down-regulation of cell cycle regulatory factors such as cyclin D(1)/E, cyclin-dependent kinase (CDK)2/4, and proliferating cell nuclear antigen. Further, JY0691 induced cell cycle arrest in the G1 phase through up-regulation of p21(cip1), but not of p27(kip1), where p53-mediated signaling was in part involved. Our current findings suggest that JY0691 contains anti-proliferative potential on aortic smooth muscle cells.

Antithrombotic and antiplatelet activities of fenofibrate, a lipid-lowering drug.

Fenofibrate, a lipid-lowering drug, inhibits hydroxyl-methylglutaryl coenzyme A (HMG-CoA)-reductase activity, thus reducing cholesterol synthesis and increasing the clearance of circulating LDL-cholesterol via the high affinity receptor system. In addition, fenofibrate has beneficial effects such as the inhibition of tissue factor expression, antithrombotic effect and anti-inflammatory effect. The aim of this study was to investigate the effects of fenofibrate on thrombus formation in vivo and platelet activation in vitro and ex vivo. The carotid arteries of male Sprague-Dawley rats were subjected to chemical injury by FeCl(3), and then blood flow was measured with a blood flowmeter. Fenofibrate (200 and 400mg/kg/day for 1 week) delayed the time to occlusion by 61.3% (p<0.05, n=10) and 90.7% (p<0.01, n=10), respectively. Fenofibrate also significantly inhibited ex vivo platelet aggregations induced by collagen (7.5microg/ml) (p<0.01, n=11) and ADP (10microM) (p<0.01, n=11), respectively, but did not affect coagulation times following activated partial thromboplastin and prothrombin activation, indicating the antithrombotic effect was mediated by its inhibition on platelet activation rather than coagulation system. This antiplatelet activity was revealed to be mediated by the suppression of thromboxane A(2) receptor, cytosolic calcium mobilization, and cyclooxygenase (COX)-1 activity. Taken together, we demonstrate that fenofibrate can significantly inhibit artery thrombus formation in vivo, which may be due to antiplatelet activity via the inhibition of thromboxane A(2) receptor, cytosolic calcium mobilization and COX-1 activity, and the beneficial effect of fenofibrate on cardiovascular system may be also due to its modulation of platelet activation.

Antiplatelet activity of beta-carboline alkaloids from Perganum harmala: a possible mechanism through inhibiting PLCgamma2 phosphorylation.

Beta-carboline alkaloids including harmalol, harmaline, norharmane, harmol, harmine and harmane are important constituents of the medicinal plant, Perganum harmala L. (Zygophylaceae), which has been used in traditional medicine. In the present study, the antiplatelet activities of six beta-carboline alkaloid compounds were investigated in vitro. At a concentration of 200 microM, these compounds have no effect on arachidonic acid (AA)-, thrombin- and U46619 (a thromboxane A2 mimic)-stimulated platelet aggregation. On the contrary, it was revealed that collagen-induced platelet aggregation could be inhibited by these compounds with different potencies (harmane and harmine were most potent, harmol had medium potency, and harmol, norharmane, harmalol and harmaline had a weak, non significant effect), indicating a selective inhibition on collagen-mediated platelet activation. Consistently, further study revealed that collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, cytosolic calcium mobilization and arachidonic acid liberation were completely inhibited by harmane and harmine in a concentration-dependent manner, while the other compounds were only partially or not effective at all. Taken together, these results indicate that three of these six beta-carboline alkaloids can selectively affect collagen-induced platelet aggregation with different potencies; in particular, harmane and harmine were most potent, and their antiplatelet activities may be mediated by inhibiting PLCgamma2 and protein tyrosine phosphorylation with sequential suppression of cytosolic calcium mobilization and arachidonic acid liberation, indicating that harmane and harmine have a potential to be developed as a novel agent for atherothrombotic diseases.