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The effects of the Cichorium intybus root extract (Cii) on alcohol-induced liver disease were investigated using Chang liver cells and male Sprague Dawley rats. Silymarin, a liver-protective agent, was used as a positive control. In cell experiments, after 24 h of treatment with the extract, no cytotoxicity was noted, and death by alcohol was avoided. Migration of Chang liver cells increased after exposure to the extract at a concentration of 400 μg/mL. In animal experiments, alcohol was injected into 6-week-old rats for 1, 3, and 50 days. Oral administration of the drug was performed 30 min before alcohol administration. The control was treated with distilled water, and the drug groups were administered EtOH (40% EtOH + 2.5 mL/kg), EtOH + Cii L (low concentration, 2 mg/kg), EtOH + Cii H (high concentration, 10 mg/kg), or EtOH + silymarin (100 mg/kg). Increased liver weight was observed in the alcohol group, as were increased blood-alcohol concentration and liver damage indicators (glutamic oxalacetic transaminase (GOT), glutamic pyruvate transaminase (GPT), and triglycerides (TG)), decreased alcoholysis enzymes (ADH and ALDH), and increased CYP2E1. In the Cii treatment group, liver weight, blood-alcohol concentration, liver damage indicators (GOT, GPT, and TG), and CYP2E1 were decreased, while alcoholysis enzymes (ADH and ALDH) were increased. The degree of histopathological liver damage was compared visually and by staining with hematoxylin and eosin and oil red O. These results indicated that ingestion of Cii inhibited alcohol-induced liver damage, indicating Cii as a useful treatment for alcohol-induced liver injury.

Cymbidiumhas known antibacterial and antiedema activity and has been used as an ingredient in cosmetics and fragrances. The effects ofCymbidiumethanol extract (CYM) on allergic response and the underlying mechanisms of action have not been reported. Therefore, the purpose of this study was to determine the effect of CYM on allergic responses. Topical application of CYM was effective against immunoglobulin E (IgE)/dinitrophenyl-conjugated bovine serum albumin- (DNP-BSA-) induced degranulation of RBL-2H3 cells and anaphylaxis in ICR mice. An allergic dermatitis-like mouse model was used to evaluate the therapeutic potential of CYMin vivo.Continuous application of 2,4-dinitrochlorobenzene (DNCB) not only induced dermatitis in ICR mice but also aggravated the skin lesioning. However, the application of CYM decreased skin lesion severity, scratching behavior, and IgE levels. In addition, CYM downregulated the expression of the proinflammatory cytokines interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-)α. Studies of signal transduction pathways showed that CYM suppressed the phosphorylation of spleen tyrosine kinase (Syk), an upstream molecule. It also inhibited the phosphorylation of Akt, phospholipase C- (PLC-)γ, and mitogen-activated protein kinase kinase kinase (MEKK). These results indicate that CYM may be effective in preventing and reducing allergic response and may have therapeutic potential as an antiallergic agent in disorders such as atopic dermatitis.

Atopic dermatitis (AD) is a chronic, allergic, and inflammatory skin disease associated with eczema and dermatitis symptoms. Our previous studies have reported that eriodictyol extract inhibits immunoglobulin E (IgE)/Ag-induced type I hypersensitivity by suppressing the activation of proinflammatory cytokines, such as interleukin-4 (IL-4), and the expression of ceramide kinase. In this study, we investigated the inhibitory effect of eriodictyol on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in ICR mice. Treatment with 2 mg/mL eriodictyol for DNCB-induced AD-like skin lesions in ICR mice improved scratching behavior and skin severity score. Histological analysis demonstrated that thickening of the skin lesions were significantly reduced in the eriodictyol-treated group. Also, eriodictyol suppressed the DNCB-mediated elevation of IgE serum levels. These results suggest that eriodictyol may be a potential therapeutic resource for AD and an adjunctive agent to control itchiness in AD.

Pterocarpus indicus Willd has been widely used as a traditional medicine to treat edema, cancer, and hyperlipidemia, but its antiallergic properties and underlying mechanisms have not yet been studied. The purpose of this study was to evaluate the antiallergic activity of Pterocarpus indicus Willd water extract (PIW) using activated mast cells and an atopic dermatitis (AD)-like mouse model. PIW decreased IgE/Ag-induced mast cell degranulation and the phosphorylation of Syk and downstream signaling molecules such as PLC-γ, Akt, Erk 1/2, JNK compared to stimulated mast cells. In DNCB-induced AD-like mice, PIW reduced IgE level in serum, as well as AD-associated scratching behavior and skin severity score. These results indicate that PIW inhibits the allergic response by reducing mast cell activation and may have clinical potential as an antiallergic agent for disorders such as AD.

Angiogenesis, neovascularization from pre-existing vessels, is a key step in tumor growth and metastasis, and anti-angiogenic agents that can interfere with these essential steps of cancer development are a promising strategy for human cancer treatment. In this study, we characterized the anti-angiogenic effects of Coptis japonica Makino extract (CJME) and its mechanism of action. CJME significantly inhibited the proliferation, migration, and invasion of vascular endothelial growth factor (VEGF)-stimulated HUVECs. Furthermore, CJME suppressed VEGF-induced tube formation in vitro and VEGF-induced microvessel sprouting ex vivo. According to our study, CJME blocked VEGF-induced cell cycle transition in G1. CJME decreased expression of cell cycle-regulated proteins, including Cyclin D, Cyclin E, Cdk2, and Cdk4 in response to VEGF. Taken together, the results of our study indicate that CJME suppresses VEGF-induced angiogenic events such as proliferation, migration, and tube formation via cell cycle arrest in G1.

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.

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.

Inflammation is a host defense mechanism that is activated in response to harmful substances or pathogens. However, an excessive inflammatory response is a problem in itself. Macrophages secrete inflammatory mediators such as nitric oxide (NO) or cytokines through various pathways such as the nuclear factor kappa B (NF-κB)-activated pathway after recognizing pathogen-like lipopolysaccharides (LPSs). In this study, anti-inflammatory effects of Eupatorium chinensis var. simplicifolium (EUC) extracts were investigated using LPS-stimulated RAW 264.7 macrophages. The EUC root extract significantly reduced NO production, inducible nitric oxide synthase (iNOS) expression, and cyclooxygenase-2 expression in a concentration-dependent manner. In addition, the EUC root extract reduced phosphorylation of mitogen-activated protein kinases and protein kinase B, which is upstream of NF-κB. The EUC root extract also reduced the degradation of inhibitory kappa B. These results indicate that EUC root extract exerts anti-inflammatory effects, which are mediated by inhibition of iNOS expression and the NF-κB pathway.

Mast cells are the principal effector cells involved in the allergic response, through the release of histamine. We investigated the effect of eriodictyol, derived from the painted maple and yerba santa, on mast cell degranulation and on an allergic response in an animal model. We also investigated its effect on the expression of the ceramide kinase (CERK) involved in calcium-dependent degranulation, and on ceramide activation by multiple cytokines. Eriodictyol suppressed the release of beta-hexosaminidase, a marker of degranulation, and the expression of interleukin (IL)-4 mRNA. It inhibited the expression of CERK mRNA, reduced the ceramide concentration in antigen-stimulated mast cells, and suppressed the passive cutaneous anaphylaxis (PCA) reaction in mice in a dose-dependent manner. These results suggest that eriodictyol can inhibit mast cell degranulation through inhibition of ceramide kinase, and that it might potentially serve as an anti-allergic agent.

Allergic response is associated with mast cells, through the release of arachidonic acid, proinflammatory cytokines, and histamine. We investigated the effect of Platycodon grandiflorum including platycodin D (PG-Platycodin D) on allergic responses in an animal model and on mast cell degranulation. PG-Platycodin D suppressed the release of β-hexosaminidase, a type of marker associated with degranulation. PG-Platycodin D efficiently inhibited the passive cutaneous anaphylaxis (PCA) reaction in ICR mice. In addition, molecular analysis demonstrated that PG-Platycodin D inhibited mRNA expression of both IL-3. These results suggest that PG-Platycodin D can inhibit mast cell degranulation through the inhibition of IL-3 mRNA expression, and that PG-Platycodin D may potentially serve as an anti-allergic agent.

Ethnopharmacological evidence Agrimonia pilosa Ledeb (Rosaceae, AP) has long been used as a traditional medicine in Korea and other Asian countries to treat various diseases. Aim of the study In the present study, the anti-inflammatory and anti-allergic effects of AP extract in in vitro cell lines and in vivo mouse model of inflammation and the molecular mechanisms involved were reported. Materials and methods Using Raw 264.7 murine macrophages the effects of methanol extract of AP in lipopolysaccharide (LPS)-induced production of inflammatory mediators were measured. Further IgE-DNP-induced interleukin (IL)-4 production and degranulation in RBL-2H3 rat basophilic cell lines was also estimated. To investigate the anti-asthmatic effect of AP in vivo , airway inflammation in ovalbumin (OVA)-induced mouse model was used. Results AP attenuated the production of inflammatory mediators such as NO, PGE 2 and pro-inflammatory cytokines in LPS-induced Raw 264.7 cells. Further, AP inhibited IL-4 production and degranulation in IgE-DNP-induced RBL-2H3 cells. Furthermore, AP attenuated the infiltration of immune cells into lung, cytokines production in broncho-alveolar lavage fluid (BALF) and airway-hyperresponsiveness (AHR) on OVA-induced mouse model of inflammation. Conclusion Our results showed that AP attenuated the activation of macrophages, basophils, and inhibited the OVA-induced airway inflammation. The molecular mechanisms leading to AP's potent anti-inflammatory and anti-allergic effects might be through regulation of TRIF-dependent and Syk-PLCγ/AKT signaling pathways, suggesting that AP may provide a valuable therapeutic strategy in treating various inflammatory diseases including asthma.

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.

Mast cells are major effector cells associated with allergic responses. They are activated through the release of histamine, arachidonic acid, and proinflammatory cytokines. We investigated the effect of nodakenin, derived from the roots of Angelica gigas Nakai, on mast cell degranulation and on an allergic response in an animal model. We also investigated the effect of nodakenin on expression of multiple cytokines. Nodakenin suppressed the release of β-hexosaminidase, a marker of degranulation, as well as the expression of interleukin IL-4 and TNF-α mRNA. Nodakenin inhibited the passive cutaneous anaphylaxis (PCA) reaction in ICR mice in a dose-dependent manner. These results suggest that nodakenin can inhibit mast cell degranulation through the inhibition of IL-4 and TNF-α mRNA expression, and that nodakenin may potentially serve as an anti-allergic agent.

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [(3)H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

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.

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 μM) 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 G 1 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.

The proliferation of vascular smooth muscle cells (VSMCs) induced by injury to the intima of arteries is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis. Uncaria rhynchophylla is traditional Chinese herb that has been applied to the treatment of convulsive disorders, such as epilepsy, in China. In the present study, we examined whether corynoxeine exerts inhibitory effects on platelet-derived growth factor (PDGF)-BB-induced rat aortic VSMC proliferation and the possible mechanism of such effects. Pre-treatment of VSMCs with corynoxeine (5-50 microM) for 24 h resulted in significant decreases in cell number without any cytotoxicity; the inhibition percentages were 25.0+/-12.5, 63.0+/-27.5 and 88.0+/-12.5% at 5, 20 and 50 microM, respectively. Also, corynoxeine significantly inhibited the 50 ng/ml PDGF-BB-induced DNA synthesis of VSMCs in a concentration-dependent manner without any cytotoxicity; the inhibitions were 32.8+/-11.0, 51.8+/-8.0 and 76.9+/-7.4% at concentrations of 5, 20 and 50 microM, respectively. Pre-incubation of VSMCs with corynoxeine significantly inhibited PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas corynoxeine had no effects on mitogen-activated protein kinase (MAPK/ERK)-activating kinase 1 and 2 (MEK1/2), Akt, or phospholipase C (PLC)gamma1 activation or on PDGF receptor beta (PDGF-Rbeta) phosphorylation. These results suggest that corynoxeine is a potent ERK1/2 inhibitor of key PDGF-BB-induced VSMC proliferation and may be useful in the prevention and treatment of vascular diseases and restenosis after angioplasty.

Diet is one of the most important factors that influence the risks for cardiovascular diseases. Genistein, an isoflavone found in soy, may benefit the cardiovascular system. Here, we investigated the effect of genistein on platelet-derived growth factor (PDGF)-BB–induced proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). Genistein significantly inhibited 25 ng/ml PDGF-BB–induced RASMC proliferation and [3H]-thymidine incorporation into DNA at 10, 20, and 40 μM. In accordance with these findings, genistein blocked the PDGF-BB–inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Western blot analysis showed that genistein not only inhibited phosphorylation of retinoblastoma protein (pRb) and expression of cyclin E, cyclin-dependent kinase (CDK) 2, and proliferating cell nuclear antigen (PCNA) protein, but also inhibited downregulation of cyclin-dependent kinase inhibitor (CKI) p27kip1. However, genistein did not affect p21cip1, CDK4, and cyclin D1 expression or early signal transduction through PDGF beta-receptor, extracellular signal-regulated kinases 1/2 (ERK1/2), Akt, and phospholipase C (PLC) γ1 phosphorylation. These results suggest that genistein inhibits PDGF-BB–induced RASMC proliferation via G0/G1 arrest in association with induction of p27kip1, which may contribute to the beneficial effects of genistein on the cardiovascular system. Keywords:: genistein, rat aortic smooth muscle cell, cell cycle, p27kip1, cardiovascular disease

Epothilone D (Epo-D) is a paclitaxel-like microtubule-stabilizing agent that was isolated from the myxobacterium Sorangium cellulosum. Although Epo-D can inhibit proliferation in multiple tumor cell lines, the effect of Epo-D on neointimal hyperplasia after angioplasty has not been reported. The aim of the present study was to investigate the effects of Epo-D on neointimal hyperplasia using an in vivo rat carotid artery injury model. We demonstrated that local Epo-D treatment significantly reduced neointimal hyperplasia after in vivo rat carotid artery injury, and Epo-D potently inhibited DNA synthesis, cell cycle progression and cell proliferation after FBS- and PDGF-BB-stimulation; PDGF-BB has been identified as the most potent growth factor for stimulating the proliferation of activated rat aortic smooth muscle cells (RASMCs). To clarify the specific effects of Epo-D on cell cycle machinery, we examined its effects on cyclin-dependent kinase (CDK)2, CDK4, cyclin E, p27, and retinoblastoma (Rb) proteins as cell cycle-related proteins in cellular lysates from PDGF-BB-stimulated RASMCs. Epo-D treatment significantly decreased the level of CDK2 protein, but did not change the levels of CDK4 and cyclin E proteins. Furthermore, Epo-D inhibited the phosphorylation of Rb, a key regulator of the G1 to S phase transition in the cell cycle. These findings suggest that Epo-D may regulate the cell cycle G1-checkpoint proteins as its major molecular mechanism for inhibiting neointimal hyperplasia after in vivo rat carotid artery injury.

Platelet derived growth factor (PDGF)-BB is one of the most potent vascular smooth muscle cell (VSMC) proliferative factors, and abnormal VSMC proliferation by PDGF-BB plays an important role in the development and progression of atherosclerosis. The aim of this study was to assess the effect of NQ304 [2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone], a newly synthesized 1,4-naphthoquinone derivative, on the proliferation of PDGF-BB-stimulated rat aortic VSMCs. Antiproliferative effects of NQ304 on rat aortic VSMCs were examined by direct cell counting and by using [(3)H] thymidine incorporation assays. It was found that NQ304 potently the growth of VSMCs. Preincubation with NQ304 (1-10 microM) significantly inhibited proliferation and DNA synthesis of 50 ng/ml PDGF-BB-stimulated rat aortic VSMCs in a concentration-dependent manner. In addition, we investigated the mechanism of proliferation suppression by NQ304 in PDGF-BB-stimulated rat aortic VSMCs, and found that PDGF-BB-stimulated immediate-early gene expression (c-fos), activator protein (AP)-1 activation, extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and Akt kinase were significantly inhibited by NQ304. An examination of the suppressive effects of NQ304 on PDGF-BB-stimulated VSMC cycle progression showed that NQ304 (10 microM) induced the G1 phase arrest of PDGF-BB-stimulated cell cycle progression by elevating p21(cip1) mRNA expression. These findings suggest that the inhibitory effects of NQ304 on DNA synthesis, proliferation, and cell cycle progression on PDGF-BB-stimulated VSMCs are mediated via the downregulations of AP-1 activation and c-fos expression achieved in turn via the suppressions of the phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2 signaling pathways.

The abnormal proliferation of aortic vascular smooth muscle cells (VSMCs) plays a central role in the pathogenesis of atherosclerosis and restenosis after angioplasty and possibly also in the development of hypertension. The present study was designed to examine the inhibitory effects and the mechanism of luteolin 7-glucoside (L7G) on the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs. L7G significantly inhibited the PDGF-BB-induced proliferation and the DNA synthesis of the VSMCs in a concentration-dependent manner. Pre-incubation of the VSMCs with L7G significantly inhibited the PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and the phospholipase C (PLC)-gamma1 activation. However, L7G had almost no affect on the phosphorylation of PDGF-beta receptor tyrosine kinase, which was induced by PDGF-BB. These results suggest that L7G inhibits the PDGF-BB-induced proliferation of VSMCs via the blocking of PLC-gamma1, Akt, and ERK1/2 phosphorylation.

Luteolin occurs as glycosylated forms in celery, green pepper, perilla leaf and camomile tea, and has been shown to possess antimutagenic, antitumorigenic, antioxidant and antiinflammatory properties. In this study, we have investigated the antiproliferable effect and its mechanism of luteolin on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (VSMCs). Luteolin significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of rat aortic VSMCs in a concentration-dependent manner. In addition, flow cytometry analysis of DNA content revealed blocking of the PDGF-BB-inducible cell cycle progression by luteolin. Pre-incubation of rat aortic VSMCs with luteolin significantly inhibited the PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and phospholipase C (PLC)-gamma1 activation as well as c-fos gene expression. Consisted with these findings, luteolin inhibited PDGF-Rbeta phosphorylation induced by PDGF-BB in a concentration-dependent manner. These results suggest that the inhibitory effect of luteolin on the PDGF-BB-induced proliferation of rat aortic VSMCs may be mediated by blocking phosphorylation of PDGF-Rbeta.

The inhibitory effects of apigenin on the growth factor-induced proliferative responses, and expression of mitogen-activated protein (MAP) kinase and its downstream c-fos in rat aortic vascular smooth muscle cells (VSMCs) were investigated. Apigenin significantly inhibited both 5 % fetal bovine serum (FBS)- and 50 ng/mL platelet derived growth factor-BB (PDGF-BB)-induced proliferation on primary cultured rat VSMCs in a concentration-dependent manner. In addition, apigenin resulted in a significant inhibition of the FBS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and expression of c-fos mRNA. These results suggest that apigenin inhibits FBS- and PDGF-BB-induced VSMC proliferation, and its activity may be mediated, at least in part, by down regulation of ERK 1/2 and its downstream c-fos mRNA.

Nodakenin, derived from the roots of Angelica gigas Nakai, is an important natural resource and medicinal material with anti-allergic and anti- inflammatory activities. We have previously shown that nodakenin inhibits IgE/Ag-induced degranulation in mast cells. In this study, we investigated the inhibitory effect of nodakenin on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)- like skin lesions in ICR mice. Scratching behavior, skin severity score, blood IgE level, and skin thickness were improved in DNCB-induced AD-like ICR mice. Our results showed that nodakenin suppressed the increase of AD-like skin lesions in ICR mice. These results suggest that nodakenin may be a potential therapeutic resource for AD as well as an adjunctive agent to control itching associated with AD.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an essential functional role in the pathogenesis of vascular disorders, such as atherosclerosis, restenosis, and neointimal hyperplasia. In this study, we examined the effects of meso-dihydroguaiaretic acid (MDGA) on platelet-derived growth factor (PDGF)-BB-induced proliferation and the molecular basis of its underlying mechanism of action in rat aortic VSMCs. Incubation of resting VSMCs with MDGA for 24 h significantly diminished PDGF-BB-induced DNA synthesis in a dose-dependent manner. We also examined the effects of MDGA on PDGF-BB signal transduction. Pre-treatment of VSMCs with MDGA inhibited PDGF-BB-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and C-Jun N-terminal kinase (JNK). MDGA also effectively inhibited phosphorylation of Akt, phospholipase C gamma 1 (PLCγ1), and PDGF receptor beta (PDGFRβ). These results indicate that MDGA may inhibit proliferation of VSMCs by suppressing autophosphorylation of PDGFRβ, and may be useful in the treatment of VSMC-associated vascular disease such as atherosclerosis, restenosis, and neointimal hyperplasia after angioplasty.

Cardiovascular disease is associated with a multitude of pathophysiologic conditions, including vascular smooth muscle cell (VSMC) proliferation in response to vessel injury. Diethylstilbestrol (DES) was previously prescribed for at-risk pregnancies to prevent abortion, miscarriage, and premature labor. Our aim in this study was to elucidate the effects and molecular mechanism of DES on proliferation and cell cycle progression of platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. Treating the cells with DES (1-7 μM) dramatically inhibited cell proliferation in a dose-dependent manner without any cytotoxic effects. In addition, DES blocked cell cycle progression from PDGF-BB-stimulated cells, which we found was related to down-regulation of the cell cycle regulatory factors, cyclin D1, and cyclin E. Our data demonstrate that DES inhibits rat aortic VSMC proliferation and cell cycle progression through regulation of cell cycle-related proteins. Therefore, our observations may explain, in part, the mechanistic basis underlying the therapeutic effects of DES in cardiovascular disease.

The extracellular signal-regulated kinases/mitogen activated protein kinase (ERK/MAPK) and nuclear factor-κB (NF-κB) pathways are critical for cell survival and proliferation. Alpinumisoflavone (AIF), isolated from the African medicinal plant Erythrina lysistemon, is a member of the isoflavone group. In this report, we demonstrated that AIF treatment induces cell death of human lung tumor cells. Incubation of lung tumor cells with AIF increased the sub-G1 population and caspase 3/7 activity, suggesting that the cell death is caused by apoptosis. To identify the signaling pathway involved in the tumor cell death, we examined the modulation of transcriptional activity using various reporter constructs and found that AIF significantly deregulated both the ERK/MAPK and NF-κB pathways. Western blot analysis with antibodies to MAP/ERK kinase (MEK) and ERK showed that AIF dephosphorylates both MEK and ERK. Alpinumisoflavone also repressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells by inhibiting NF-κB-dependent transcription. Therefore, the cell death induced by AIF may be via repressing both the ERK/MAPK and NF-κB pathways.

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.

Macrophage-derived nitric oxide (NO) plays an important role in protection against microbial infection in immune responses. Overproduction of NO by inducible nitric synthase (iNOS) is known to be closely correlated with the pathology of a variety of diseases and inflammations. In this study, we investigated the inhibitory effect of polyethylene glycol coated gold nanoparticles (GNP) on NO production and its molecular mechanism in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. It was found that GNP inhibited LPS-induced NO production and iNOS expression in RAW264.7 cells. Furthermore, GNP suppressed LPS-induced activation of NF-κB through the inhibition of Akt activity. GNP also inhibited LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) via down-regulation of interferon-beta (IFN-β) expression. Our results suggest that GNP inhibits NO production and iNOS expression through blocking the activation of NF-κB and STAT1 in LPS-stimulated RAW264.7 cells.

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC50 values of 0.71 ± 0.2, 0.82 ± 0.3, and 0.45 ± 0.1 µM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A2 receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA2 formation, but had no effect on the PGD2 production, indicating an inhibitory effect on TXA2 synthase, which was supported by a TXA2 synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA2 formation converted from PGH2. On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA2 synthase activity and TXA2 receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12. Keywords:: antithrombotic agent, platelet aggregation, thromboxane A2

Indoledione derivatives have pronounced biological effects, i.e., cytotoxic activities against cancer cell lines and antifungal and antibacterial activities. The present study was designed to investigate the effects of YSK2821, a newly synthesized indoledione derivative, on platelet-derived growth factor (PDGF-BB)-induced vascular smooth muscle cell (VSMC) proliferation, as well as the molecular mechanisms of the anti-proliferative effects of YSK2821 in VSMCs. We found that YSK2821 caused the accumulation of cells in the G1 phase of the cell cycle and inhibited [3H]-thymidine incorporation. We demonstrated that YSK2821 remarkably decreased Akt kinase phosphorylation as the mechanism by which YSK2821 suppressed cell signal transduction events in VSMC proliferation. Furthermore, in terms of the effects of YSK2821 on cell cycle-related proteins, YSK2821 enhanced the expression of the cyclin-dependent protein kinase (CDK) inhibitor p27 and down-regulated CDK2 and cyclin E expression, but did not affect CDK4 and cyclin D1 expression. YSK2821 also inhibited the phosphorylation of Rb, a key regulator in the cell cycle. These results indicate that YSK2821, a newly synthesized indoledione derivative, may inhibit VSMC proliferation via a phosphatidylinositol (PI)-3 kinase-dependent pathway, and thus shed light on a novel role for YSK2821 as a potential preventive regulator of cardiovascular disease.

Studies previously reported that melittin, a major bioactive component of bee venom, inhibits vascular smooth muscle cell (VSMC) proliferation through suppression of nuclear factor (NF)-kappaB and Akt activation and through enhancement of proapoptotic protein expression. In this study, the effects of melittin were investigated on the tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta receptor (Rbeta) and its downstream intracellular signal transduction. When combined with PDGF-Rbeta inhibitor, melittin exhibited a synergic inhibitory effect on PDGF-BB-induced rat aortic VSMC proliferation. In addition, melittin inhibited PDGF-Rbeta phosphorylation in a concentration-dependent manner. Accordingly, the downstream signal transduction of PDGF-Rbeta, such as ERK1/2, Akt, and PLCgamma1 phosphorylation, was also inhibited by melittin in the same manner. These findings suggest that, in addition to suppressing NF-kappaB activation, the antiproliferative effect of melittin in VSMC may be mediated, at least in part, by the inhibition of PDGF-Rbeta tyrosine phosphorylation and its downstream intracellular signal transduction.

Hyperproliferation of platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (VSMC) is a hallmark of atherosclerosis and related vascular disorders. In the previous study, we reported that KTJ740 [2-chloro-3-(4-(ethylcarboxy)-phenyl)-amino-1,4-naphthoquinone], a newly synthesized vitamin K derivative, has potent antithrombotic effects in mice and antiplatelet activity in vitro and ex vivo. In the present study, we have tested that KTJ740 could inhibit PDGF-BB-stimulated VSMC proliferation. We have examined the potential inhibitory effect of this compound on rat aortic smooth muscle cells (RASMCs). Our results show that this compound significantly inhibits PDGF-BB-stimulated RASMC number and DNA synthesis in a concentration-dependent manner. Furthermore, we have examined its effect on cell cycle progression by flow cytometry. KTJ740 treatment resulted in a significant arrest in cell cycle progression of RASMCs induced by PDGF-BB, and this effect was achieved by suppressing activation of PDGF-beta receptor (PDGF-Rbeta) tyrosine kinase pathway. These results suggest that a possibility of KTJ740 can be a potential agent to control vascular disorders and its antiproliferative mechanism may be mediated through PDGF-Rbeta tyrosine kinase-dependent signaling pathway.

Platelet derived growth factor (PDGF)-BB is one of the most potent vascular smooth muscle cell (VSMC) proliferative factors, and abnormal VSMC proliferation by PDGF-BB plays an important role in the development and progression of cardiovascular problems, including restenosis after coronary angioplasty and atherosclerosis. Previous phytochemical studies on the stems or root barks of Cudrania tricuspidata (Moraceae) resulted in the isolation of various isoprenylated xanthones and flavonoids, some of which have anti-cancer, hepatoprotective, anti-inflammatory and anti-oxidant activities. In the present study, we investigated the antiproliferative effect of cudratricusxanthone A isolated from the root bark of C. tricuspidata and its underlying mechanism in VSMCs. Antiproliferative effects of cudratricusxanthone A on VSMCs were examined by direct cell counting and [3H]-thymidine incorporation assays. Cudratricusxanthone A inhibited [3H]-thymidine incorporations into DNA in VSMCs that occurred in response to treatment with 50 ng/ml PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced to 86.1, 80.2, 64.2 and 25.1% at concentrations of 0.1, 1, 2 and 3 microM, respectively. Moreover, pre-treatment with cudratricusxanthone A (0.1-3 microM) suppressed this PDGF-BB-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 11.1, 22.7, 51.3 and 81.5% at the concentrations of 0.1, 1, 2 and 3 microM, respectively. We also investigated the mechanism of antiproliferative effects by cudratricusxanthone A in PDGF-BB-stimulated VSMCs. In Western blot analysis, 50 ng/ml PDGF-BB-stimulated phospholipase C (PLC)gamma1, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations were inhibited by cudratricusxanthone A (0.1-3 microM). Consisted with these findings, cudratricusxanthone A inhibited PDGF-receptor beta chain (PDGF-Rbeta) phosphorylation induced by PDGF-BB in a concentration-dependent manner. These findings suggest that the inhibitory effects of cudratricusxanthone A on DNA synthesis and proliferation by PDGF-BB-stimulated VSMCs are mediated by the suppressions of the PDGF-Rbeta and its downstream signaling pathways. Our observation may explain in part mechanistic basis for the prevention of cardiovascular diseases, such as atherosclerosis and restenosis after coronary angioplasty by cudratricusxanthone A.

Oleanolic acid (3beta-hydroxy-olea-12-en-28-oic), a pentacyclic triterpenoid, exists widely in the plant kingdom and has a wide variety of pharmacological effects such as antitumor, antifungal, insecticidal, hepatoprotective and anti-HIV activities. This paper reports that oleanolic acid induces the aggregation of rabbit platelets, a mechanism was also investigated. Oleanolic acid at concentrations of 25, 50, 100 and 200 microM induced the aggregation of washed rabbit platelets in a concentration-dependent manner. Pretreating the platelets with U73122, a phospholipase C (PLC) inhibitor, blocked the oleanolic acid induced-aggregation, whereas acetylsalicylic acid (ASA), a cyclooxygenase (COX) inhibitor, had no effect. In addition, the effect of oleanolic acid on serotonin secretion, which is a marker for dense granule secretion, was determined. Oleanolic acid induced serotonin secretion in a similar concentration-dependent manner as observed with platelet aggregation. Pretreating the platelets with U73122 blocked the oleanolic acid-induced serotonin secretion and cytosolic calcium mobilization. Overall, these results suggest that oleanolic acid can induce platelet aggregation, which may be mediated by the stimulation of PLC-mediated cytosolic calcium mobilization.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury model and examined the potential effects of epothilone B on cardiovascular disease. We found that epothilone B potently prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly inhibited 5% fetal bovine serum (FBS)- and 50 ng/ml platelet-derived growth factor (PDGF)-BB-induced proliferation and cell cycle progression in rat aortic VSMCs. Furthermore, FBS and PDGF-BB induced the activations of extracellular signal-regulated kinases 1 and 2, Akt, phospholipase C gamma 1, and PDGF-receptor beta chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant decrease in the level of cyclin-dependent protein kinase (CDK) 2, whereas it caused no change in the levels of cyclin E and down-regulated the phosphorylation of retinoblastoma, which plays a critical role in cell cycle regulation. Furthermore, levels of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of VSMCs. These findings suggest that epothilone B can inhibit neointimal formation via the cell cycle arrest by the regulation of the cell cycle-related proteins in VSMCs.

Ceramide kinase (CERK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P) and is known to be activated by calcium. Although several groups have examined the functions of CERK and its product C1P, the functions of C1P and CERK are not understood. We studied the RBL-2H3 cell line, a widely used model for mast cells, and found that CERK and C1P are required for activation of the degranulation process in mast cells. We found that C1P formation was enhanced during activation induced by IgE/antigen or by Ca(2+) ionophore A23187. The formation of C1P required the intracellular elevation of Ca(2+). We generated RBL-2H3 cells that stably express CERK, and when these cells were treated with A23187, a concomitant C1P formation was observed and degranulation increased 4-fold, compared with mock transfectants. The cell-permeable N-acetylsphingosine (C(2)-ceramide), a poor substrate of CERK, inhibited both the formation of C1P and degranulation, indicating that C1P formation was necessary for degranulation. Exogenous introduction of CERK into permeabilized RBL-2H3 cells caused degranulation. We identified a cytosolic localization of CERK that provides exposure to cytosolic Ca(2+). Taken together, these results indicate that C1P formation is a necessary step in the degranulation pathway in RBL-2H3 cells.

Tetrandrine (TET) is a well known naturally occurred nonspecific Ca(2+) channel blocker. It has long been used for the treatment of arrhythmia, hypertension, and occlusive cardiovascular disorders. The objective of the present study was to investigate the effect of TET on the proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). TET significantly inhibited both 10% fetal bovine serum (FBS) and 50 ng/ml platelet-derived growth factor (PDGF)-BB-induced proliferation, [(3) H] ]thymidine incorporation into DNA, and p42/p44 mitogen-activated protein kinase (ERK1/2) phosphorylation at the concentration of 1.0 and 5.0 microM. Flow cytometry analysis of DNA content in synchronized cells revealed blocking of the FBS-inducible cell cycle progression by TET. In accordance with these findings, TET 5 microM caused a 48% decrease in the early elevation of c-fos expression induced after 10% FBS addition. Furthermore, in contrast to its distinguishable higher potency of Ca(2+) antagonistic activity, verapamil showed lower potent antiproliferative activities than TET. These results suggest that TET can exert antiproliferative effects against mitogenic stimuli for RASMCs in vitro by a mechanism that involves the MAPK pathway, altering cell cycle progression, and the inhibitory action cannot be limited to its Ca(2+) modulation.