Desalted Salicornia europaea extract attenuated vascular neointima formation by inhibiting the MAPK pathway-mediated migration and proliferation in vascular smooth muscle cells.

Salicornia europaea L. (SE) has been used as folk medicine for the treatment of various diseases such as obesity, diabetes, and cancer. However, its effects on atherosclerotic events in vascular smooth muscle cells (VSMCs) remain unknown. The present study explored the effects of the ethyl acetate fraction of desalted SE hot water extract (SEWEAF) on atherosclerotic responses (especially migration and proliferation) in VSMCs and vascular neointima formation. Treatment with the SEWEAF significantly suppressed the platelet-derived growth factor (PDGF)-BB-induced VSMC migration and proliferation as well the phosphorylation of mitogen-activated protein kinases (MAPKs) such as the p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2. Moreover, oral administration of the SEWEAF resulted in the attenuation of neointima formation in balloon-injured rat carotid arteries. Additionally, HPLC analysis showed that the major components in the two subfractions of the SEWEAF were five phenolic acids and four flavonols. In the SEWEAF components, for which atherosclerosis-linked responses in VSMCs have not been known, p-coumaric acid, quercetin-3-ß-d-glucoside, and isorhamnetin-3-ß-d-glucoside inhibited both PDGF-BB-induced migration and proliferation and isorhamnetin attenuated only PDGF-BB-stimulated VSMC proliferation. These results suggest that the SEWEAF may suppress PDGF-BB-induced VSMC migration by downregulating the phosphorylation of p38 MAPK and ERK1/2, thus leading to the reduction of neointimal hyperplasia during vascular remodeling. Therefore, the desalted SE extract, SEWEAF may be a potential ingredient for dietary supplements or nutraceuticals to ameliorate and/or prevent vascular remodeling-related disorders.

Cardioprotective effect of KR-33889, a novel PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cells and isolated rat hearts.

Oxidative stress plays a critical role in cardiac injury during ischemia/reperfusion (I/R). Despite a potent cardioprotective activity of KR-33889, a novel poly (ADP-ribose) polymerase inhibitor, its underlying mechanism remains unresolved. This study was designed to investigate the protective effects of KR-33889 against oxidative stress-induced apoptosis in rat cardiomyocytes H9c2 cells and isolated rat hearts. H2O2 caused severe injury to H9c2 cells, mainly due to apoptosis, as revealed by TUNEL assay. However, KR-33889 pretreatment significantly attenuated H2O2-induced apoptosis of H9c2 cells, which was accompanied by decrease in expression of both cleaved caspase-3 and Bax and increase in Bcl-2 expression and the ratio of Bcl-2/Bax. KR-33889 also significantly enhanced the expression of anti-oxidant enzymes including heme oxygenase-1, Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase, thereby inhibiting production of intracellular ROS. Furthermore, KR-33889 reversed H2O2-induced decrease in phosphorylation of Akt, GSK-3ß, ERK1/2, p38 MAPK, and SAPK/JNK during most H2O2 exposure time. In globally ischemic rat hearts, KR-33889 inhibited both I/R-induced decrease in cardiac contractility and apoptosis by increasing Bcl-2, decreasing both cleaved caspase-3 and Bax expression, and enhancing expression of anti-oxidant enzymes. Taken together, these results suggest that KR-33889 may have therapeutic potential to prevent I/R-induced heart injury in ischemic heart diseases mainly by reducing oxidative stress-mediated myocardial apoptosis.

The Hot-Water Extract of Smilacis Chinae Rhizome Suppresses 2,4-Dinitrochlorobenzene and House Dust Mite-Induced Atopic Dermatitis-Like Skin Lesions in Mice.

Smilacis Chinae Rhizome (SCR) has been used as an oriental folk medicine for various biological activities. However, its effect on atopic dermatitis (AD) remains undetermined to date. We assessed the effect of orally administered hot-water extract of SCR on AD-like skin lesions in mice and its underlying mechanisms. AD-like murine model was prepared by repeated alternate application of house dust mite (Dermatophagoides farinae) extract (DFE) and 2,4-dinitrochlorobenzene (DNCB) for 4 weeks, topically to the ears. Daily oral administration of SCR for 3 and 4 weeks significantly reduced inflammatory ear thickening, with the effect being enhanced at the earlier start and longer period of administration. This effect was accompanied by a significant decrease in both Th2 and Th1 serum antibodies (total IgE, DFE-specific IgE, and IgG2a). Histological analysis showed that SCR markedly decreased the epidermal/dermal ear thickening and the dermal infiltration of inflammatory cells. Furthermore, SCR suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-17, IL-18, TSLP, and IFN-γ genes in the ear tissue. Taken together, our observations demonstrate that chronic oral administration of SCR exerts beneficial effect in mouse AD model, suggesting that SCR has the therapeutic potential as an orally active treatment of AD by modulating both Th1 and Th2 responses.

The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3ß and ERK1/2 signaling pathway.

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3ß and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3ß and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3ß and ERK1/2 signaling pathways.

Cardioprotective effects of rhamnetin in H9c2 cardiomyoblast cells under H2O2-induced apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Many studies have emphasized that flavonoids, found in various fruits, vegetables, and seeds, as well as tea and red wine, have potential health-promoting and disease-preventing effects. Rhamnetin is a flavonoid that exhibits antioxidant capabilities. However, little is known about its effect on cardiac myocytes under oxidative stress and the underlying mechanisms. MATERIALS AND METHODS: H9c2 cardiomyoblast cells were subjected to H2O2, to study the protective effect of rhamnetin on cell viability, apoptosis, and ROS production. Signaling proteins related to apoptosis, survival, and redox were analyzed by Western blot. Furthermore, the mRNA expressions of SIRTs were tested by real time-polymerase chain reaction (PCR). RESULTS: We investigated the protective effects of rhamnetin against H2O2-induced apoptosis in H9c2 cardiomyoblasts. Rhamnetin protected cells against H2O2-induced cell death without any cytotoxicity, as determined by the XTT assay, LDH assay, TUNEL assay, Hoechst 33342 assay, and Western blot analysis of apoptosis-related proteins. Rhamnetin also enhanced the expression of catalase and Mn-SOD, thereby inhibiting production of intracellular ROS. Furthermore, rhamnetin recovered the H2O2-induced decrease in phosphorylation of Akt/GSK-3ß and MAPKs (ERK1/2, p38 MAPK, and JNK) and pretreatment with their inhibitors, attenuating the rhamnetin-induced cytoprotective effect. Further studies with real time-PCR and a sirtuin inhibitor showed that cardioprotection by rhamnetin occurred through induction of SIRT3 and SIRT4. CONCLUSIONS: Taken together, these results suggest that rhamnetin may have novel therapeutic potential to protect the heart from ischemia-related injury.

Cordycepin, 3'-deoxyadenosine, prevents rat hearts from ischemia/reperfusion injury via activation of Akt/GSK-3ß/p70S6K signaling pathway and HO-1 expression.

Cordycepin (3'-deoxyadenosine) isolated from Cordyceps militaris, a species of the fungal genus Cordyceps, has been shown to exhibit many pharmacological functions, such as anticancer, anti-inflammatory, and antioxidant activities. In this study, we investigated the preventive role of cordycepin in ischemic/reperfusion (I/R) injury of isolated rat hearts and anesthetized rats. After Sprague-Dawley rats received cordycepin (3, 10, and 30 mg/kg) or control (0.5 % carboxyl methylcellulose) orally once a day for a week, hearts were isolated and mounted on Langendorff heart perfusion system. Isolated hearts were perfused with Krebs-Henseleit buffer for 15-min pre-ischemic stabilization period and subjected to 30-min global ischemia and 30-min reperfusion. Cordycepin administration (10 mg/kg, p.o.) significantly increased left ventricular developed pressure during the reperfusion period compared to that in the control group, but without any effect on coronary flow. Cordycepin (10 mg/kg, p.o.) significantly increased the phosphorylation of Akt/GSK-3ß/p70S6K pathways, which are known to modulate multiple survival pathways. In addition, cordycepin decreased Bax and cleaved caspase-3 expression while increasing Bcl-2 expression, Bcl-2/Bax ratio, and heme oxygenase (HO-1) expression in isolated rat hearts. In anesthetized rats subjected to 30 min occlusion of left anterior descending coronary artery/2.5-h reperfusion, cordycepin (1, 3, and 10 mg/kg, i.v.) administered 15 min before the onset of ischemia dose-dependently decreased the infarct size in left ventricle. In conclusion, cordycepin could be an attractive therapeutic candidate with oral activity against I/R-associated heart diseases such as myocardial infarction.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 µM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5-2 µM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway.

5-AIQ inhibits H2O2-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3ß signaling pathway in H9c2 cardiomyocytes.

Poly(adenosine 5'-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H2O2-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H2O2-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H2O2-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3ß (GSK-3ß), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3ß activation. In addition, inhibiting the Akt/GSK-3ß pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H2O2-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3ß pathway.

Differential regulation of low and high voltage-activated calcium channels in neonatal rat myocytes following chronic PKA modulation.

The biophysical properties of voltage-dependent cardiac calcium channels (VDCC) can be modulated by protein kinases. In this study, we investigate whether long-term treatment with protein kinase A (PKA) modulators alters the VDCC activity in neonatal ventricular myocytes. Using whole-cell patch-clamp recordings, we found an increase in high-voltage activated (HVA) current density and a corresponding decrease in low-voltage activated (LVA) current density in neonatal rat ventricular myocytes up to 6 days in culture. Long-term exposure to 8Br-cAMP, a PKA stimulator, increased the HVA current density at 7 and 24 hours. In contrast, H89, a PKA inhibitor, caused a biphasic change in the HVA, an initial reduction at 7 hours exposure followed by an increase up to 4 days. In addition, H89 caused a sustained increase in LVA currents from 7 hours to 4 days. These findings suggest that chronic exposure to H89 changes LVA and HVA calcium current activities in cardiac myocytes. PKA is a key target of ß-adrenoceptor activiation, thus, our findings suggest long-term repeated use of ß-adrenergic drugs may induce unexpected functional alteration of VDCCs.

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1ß-stimulated human alveolar epithelial cells.

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1ß. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.

Cardioprotective effects of the novel Na+/H+ exchanger-1 inhibitor KR-32560 in a perfused rat heart model of global ischemia and reperfusion: Involvement of the Akt-GSK-3beta cell survival pathway and antioxidant enzyme.

To investigate the cardioprotective effects and mechanism of action of KR-32560 {[5-(2-methoxy-5-fluorophenyl)furan-2-ylcarbonyl]guanidine}, a newly synthesized NHE-1 inhibitor, we evaluated the effects of KR-32560 on cardiac function in a rat model of ischemia/reperfusion (I/R)-induced heart injury as well as the role antioxidant enzymes and pro-survival proteins play these observed effects. In isolated rat hearts subjected to 25 min of global ischemia followed by 30 min of reperfusion, KR-32560 (3 and 10 microM) significantly reversed the I/Rinduced decrease in left ventricular developed pressure and increase in left ventricular enddiastolic pressure. In rat hearts reperfused for 30 min, KR-32560 (10 microM) significantly decreased the malondialdehyde content while increasing the activities of both glutathione peroxidase and catalase, two important antioxidant enzymes. Western blotting analysis of left ventricles subjected to I/R showed that KR-32560 significantly increased phosphorylation of both Akt and GSK-3beta in a dose-dependent manner, with no effect on the phosphorylation of eNOS. These results suggest that KR-32560 exerts potent cardioprotective effects against I/Rinduced rat heart injury and that its mechanism involves antioxidant enzymes and the Akt-GSK-3beta cell survival pathway.

Oral administration of curcumin suppresses production of matrix metalloproteinase (MMP)-1 and MMP-3 to ameliorate collagen-induced arthritis: inhibition of the PKCdelta/JNK/c-Jun pathway.

We investigated whether oral administration of curcumin suppressed type II collagen-induced arthritis (CIA) in mice and its effect and mechanism on matrix metalloproteinase (MMP)-1 and MMP-3 production in CIA mice, RA fibroblast-like synoviocytes (FLS), and chondrocytes. CIA in mice was suppressed by oral administration of curcumin in a dose-dependent manner. Macroscopic observations were confirmed by histological examinations. Histological changes including infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw sections were extensively suppressed by curcumin. The histological scores were consistent with clinical arthritis indexes. Production of MMP-1 and MMP-3 were inhibited by curcumin in CIA hind paw sections and tumor necrosis factor (TNF)-alpha-stimulated FLS and chondrocytes in a dose-dependent manner. As for the mechanism, curcumin inhibited activating phosphorylation of protein kinase Cdelta (PKCdelta) in CIA, FLS, and chondrocytes. Curcumin also suppressed the JNK and c-Jun activation in those cells. This study suggests that the suppression of MMP-1 and MMP-3 production by curcumin in CIA is mediated through the inhibition of PKCdelta and the JNK/c-Jun signaling pathway.

KR-31761, a novel K+(ATP)-channel opener, exerts cardioprotective effects by opening both mitochondrial K+(ATP) and Sarcolemmal K+(ATP) channels in rat models of ischemia/reperfusion-induced heart injury.

The cardioprotective effects of KR-31761, a newly synthesized K+(ATP) opener, were evaluated in rat models of ischemia/reperfusion (I/R) heart injury. In isolated rat hearts subjected to 30-min global ischemia/30-min reperfusion, KR-31761 perfused prior to ischemia significantly increased both the left ventricular developed pressure (% of predrug LVDP: 17.8, 45.1, 54.2, and 62.6 for the control, 1 microM, 3 microM, and 10 microM, respectively) and double product (DP: heart rate x LVDP; % of predrug DP: 17.5, 44.9, 56.2, and 64.5 for the control, 1 microM, 3 microM, and 10 microM, respectively) at 30-min reperfusion while decreasing the left ventricular end-diastolic pressure (LVEDP). KR-31761 (10 microM) significantly increased the time to contracture during the ischemic period, whereas it concentration-dependently decreased the lactate dehydrogenase release during reperfusion. All these parameters were significantly reversed by 5-hydroxydecanoate (5-HD, 100 microM) and glyburide (1 microM), selective and nonselective blockers of the mitochondrial K+(ATP) (mitoK+(ATP)) channel and K+(ATP) channel, respectively. In anesthetized rats subjected to 30-min occlusion of left anterior descending coronary artery/2.5-h reperfusion, KR-31761 administered 15 min before the onset of ischemia significantly decreased the infarct size (72.2%, 55.1%, and 47.1% for the control, 0.3 mg/kg, i.v., and 1.0 mg/kg, i.v., respectively); and these effects were completely and almost completely abolished by 5-HD (10 mg/kg, i.v.) and HMR-1098, a selective blocker of sarcolemmal K+(ATP) (sarcK+(ATP)) channel (6 mg/kg, i.v.) administered 5 min prior to KR-31761 (72.3% and 67.9%, respectively). KR-31761 only slightly relaxed methoxamine-precontracted rat aorta (IC50: > 30.0 microM). These results suggest that KR-31761 exerts potent cardioprotective effects through the opening of both mitoK+(ATP) and sarcK+(ATP) channels in rat hearts with a minimal vasorelaxant effect.

Mast cell-mediated allergic response is suppressed by Sophorae flos: inhibition of SRC-family kinase.

Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC(50) values of approximately 31.6 microg/mL (RBL-2H3 mast cells) and approximately 47.8 microg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-alpha and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.

KR-31762, a novel KATP channel opener, exerts cardioprotective effects by opening SarcKATP channels in rat models of ischemia/reperfusion-induced heart injury.

The cardioprotective effects of KR-31762, a newly synthesized K+(ATP) opener, were evaluated in rat models of ischemia/reperfusion (I/R) heart injury. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, KR-31762 (3 and 10 microM) significantly increased the left ventricular developed pressure (LVDP) and double product (heart rate x LVDP) after 30-min reperfusion in a concentration-dependent manner, while decreasing the left ventricular end-diastolic pressure (LVEDP). KR-31762 also significantly increased the time to contracture (TTC) during ischemic period (20.0, 22.4 and 26.4 min for control, 3 and 10 microM, respectively), while decreasing the release of lactate dehydrogenase (LDH) from the heart during 30 min reperfusion (30.4, 14.3 and 19.7 U/g heart weight, respectively). All these parameters except LDH release were reversed by glyburide (1 microM), a nonselective blocker of K+(ATP) channel, but not by 5-hydroxydecanoate, a selective blocker of mitoK+(ATP) channel. In anesthetized rats subjected to 45-min occlusion of left anterior descending coronary artery followed by 90-min reperfusion, KR-31762 significantly decreased the infarct size (60.8, 40.5 and 37.8% for control, 0.3 and 1.0 mg/kg, iv, respectively). KR-31762 slightly relaxed the isolated rat aorta precontracted with methoxamine (IC(50): 23.5 microM). These results suggest that KR-31762 exerts potent cardioprotective effects through the opening of sarcolemmal K(ATP) channel in rat hearts with the minimal vasorelaxant effects.

15,16-dihydrotanshinone I, a major component from Salvia miltiorrhiza Bunge (Dansham), inhibits rabbit platelet aggregation by suppressing intracellular calcium mobilization.

Radix Salviae miltiorrhiza (RSM, 'Dansham' in Korea, 'Danshen' in Chinese), the root of Salviae miltiorrhiza Bunge (Labiate) has been used as Chinese fork medicine for the treatment of cardiovascular diseases such as angina pectoris, coronary heart disease, myocardial infarction, and hypertension. In the present study, we evaluated the inhibitory effects of 15,16-Dihydrotanshinone I, one of the major ingredients of Salvia miltiorrhiza Bunge, on platelet aggregation, with elucidation of its mechanisms of action. 15,16-Dihydrotanshinone I concentration-dependently inhibited collagen-induced aggregation of rabbit washed platelets with IC50 of 8.7+/-5.6 microM, the potency being about seven-fold greater than EGCG, an active Green tea catechin component (IC50: 56.6+/-48.7 microM). 15,16-Dihydrotanshinone I significantly inhibited the intracellular calcium ([Ca2+]i) mobilization in a concentration-dependent manner. 15,16-dihdydrotanshinone I also significantly suppressed collagen (50 microg/mL)-induced liberation of [3H]Arachidonic acid from [3H]Arachidonic acid-incorporated rabbit platelet. In addition, 15,16-Dihydrotanshinone I at 50 microM slightly but significantly inhibited collagen-induced production of thromboxane B2. These results indicate that 15,16-Dihydrotanshinone I exert potent anti-platelet activity via suppression of [Ca2+]i mobilization and arachidonic acid liberation.

Spleen tyrosine kinase participates in Src-mediated migration and proliferation by PDGF-BB in rat aortic smooth muscle cells.

Tyrosine kinases, Src and spleen tyrosine kinase (Syk), play crucial roles in cell responses to platelet-derived growth factor (PDGF) and may have their functional interactions. In this study, we focused on investigating the roles of Syk in the regulation of Src signaling in PDGF-mediated vascular cell responses. Migration, proliferation, and activity of kinases were determined in rat aortic smooth muscle cells (RASMCs). PDGF-BB (10 ng/mL) induced the migration and proliferation of RASMCs, which were significantly inhibited by PP2 (10 microM) and piceatannol (30 microM), inhibitors of Src and Syk, respectively. The phosphorylation of Syk induced by PDGF-BB was abolished by PP2. PDGF-BB increased the co-association of the PDGFbeta-receptor and the kinases, Src or Syk, and its maximal binding to Src was achieved in a shorter time than that to Syk. PDGF-BB stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2, which was inhibited by PP2 and piceatannol. PDGF-BB-induced proliferation and migration were inhibited by SB203580 (30 microM) and PD98059 (30 microM), inhibitors of p38 MAPK and ERK1/2, respectively. These results imply that Syk is regulated by Src kinase, which participates in migration and proliferation in response to PDGF-BB in RASMCs.

Cardioprotective effects of BMS-180448, a prototype mitoK(ATP) channel opener, and the role of salvage kinases, in the rat model of global ischemia and reperfusion heart injury.

To investigate the involvement of reperfusion-induced salvage kinases (RISK) as possible signaling molecules for the cardioprotective effects of BMS-180448, a prototype mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel opener, we measured its cardioprotective effects in a rat model of ischemia/reperfusion (I/R) heart injury, together with western blotting analysis of five different signaling proteins. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, BMS-180448 (1, 3 and 10 microM) significantly increased reperfusion left ventricular developed pressure (LVDP) and 30-min reperfusion double product (heart rate x LVDP) in a concentration-dependent manner, while decreasing left ventricular end-diastolic pressure (LVEDP) throughout reperfusion period in a concentration-dependent manner. SDS-PAGE/western blotting analysis of left ventricle reperfused for 30 min revealed that BMS-180448 significantly decreased phospho-GSK3beta at high concentration, whereas it tended to increase slightly phospho-eNOS and phospho-p70S6K with concentration. However, BMS-180448 had no effect on phospho-Akt and phospho-Bad. These results suggest that the cardioprotective effects of BMS-180448 against I/R heart injury may result from direct activation of mitoK(ATP) channel in cardiomyocytes, with the minimal role of RISK pathway in the activation of this channel and the cardioprotective effects of BMS-180448.

Effects of KTJ740, a novel antithrombotic agent, on platelet-derived growth factor-induced rat aortic smooth muscle cell proliferation and cell cycle progression.

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.

Oleanolic acid, a pentacyclic triterpenoid, induces rabbit platelet aggregation through a phospholipase C-calcium dependent signaling pathway.

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.