Hwa Jin Park

The mechanism of anti-platelet activity of davallialactone: Involvement of intracellular calcium ions, extracellular signal-regulated kinase 2 and p38 mitogen-activated protein kinase

This study was designed to investigate the effect of davallialactone, which was isolated from the mushroom Inonotus xeranticus, on platelet aggregation induced by collagen, thrombin and ADP. We found that davallialactone dose-dependently inhibited platelet aggregation that was stimulated either by collagen (2.5 microg/ml), a potent ligand of integrin alpha2beta1 and glycoprotein VI, or by thrombin (0.1U/ml), a potent agonist of the protease-activated receptors (PARs) PAR1 and PAR3. In addition, davallialactone inhibited platelet aggregation induced by ADP, an agonist of P2Y receptor. To understand the mechanism of anti-platelet activity, we determined whether davallialactone affected the downstream signaling in collagen-activated platelets. Using the fura-2/AM fluorometric assay, we found that davallialactone dose-dependently inhibited intracellular calcium concentration levels ([Ca2+]i). Moreover, davallialactone inhibited the phosphorylation of extracellular signal-regulated protein kinase (ERK)-2 and p38 mitogen-activated protein kinase (MAPK), in a dose-dependent manner. The tyrosine phosphorylation of 60 and 85kDa proteins, which were activated by collagen, were differentially inhibited by davallialactone. Taken together, these data suggest that davallialactone may have potential anti-platelet aggregation activity via suppression of intracellular downstream signaling pathways.

Evaluation of antioxidant, antinociceptive, and anti‐inflammatory activities of ethanol extracts from Aloe saponaria Haw.

Aloe species are traditionally prescribed for hypertension, burning, and rheumatoid arthritis. To elucidate the mechanism of the antihypertensive and anti‐inflammatory activities of this herb, the ethanol fraction from A. saponaria Haw. was evaluated for antioxidative activity using xanthine‐xanthine oxidase (XO) assay, 2,2‐Diphenyl‐lpicrylhydrazyl radical (DPPH) assay, lipopolysaccharide (LPS)‐induced nitric oxide (NO) production in RAW 264.7 cell, and antinociceptive activity using a tail‐flick assay and hind paw pressure assay in cisplatin‐treated hyperalgesic rats. The ethanol fraction displayed potent antioxidative activities in XO assay. In addition, ethanol fractions showed potent scavenging effects in DPPH assay. We next examined whether ethanol fractions showed anti‐inflammatory activities. Ethanol fractions significantly suppressed NO production from LPS‐activated RAW264.7 cells. As expected, ethanol fractions dose‐dependently inhibited the messenger RNA expression of inducible NO synthase (iNOS). Moreover, ethanol fractions potently suppressed the expression of cycloxygenase (COX)‐2 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), which are stimulated by LPS in RAW264.7 cells. In addition, ethanol fractions significantly blocked cisplatin‐induced hyperalgesia using tail‐flick assay and hind paw pressure test in rats. Taken altogether, ethanol extracts of aloe may be useful as a functional food or as a drug against reactive oxygen species (ROS) mediated diseases. Copyright

Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: Critical roles of ERK2 and cAMP

Ginsenoside Rg3, a single ginseng saponin, is known to be a major anti‐platelet component of protopanaxadiol that is isolated from Korean red ginseng. In this study, we investigated whether dihydroginsenoside Rg3, a stable chemical derivative of ginsenoside Rg3, also demonstrated anti‐platelet activity. Dihydroginsenoside Rg3 inhibited thrombin‐induced platelet aggregation in a concentration‐dependent manner with an IC50 (concentration producing 50% inhibition) of 18.8 ± 0.4 μM. Ginsenoside Rg3 inhibited platelet aggregation which was induced by thrombin (0.1 U mL−1) with an IC50 of 40.2 ± 0.9 μM. We next determined whether dihydroginsenoside Rg3 affected different types of ligand‐induced platelet aggregation. We found that dihydroginsenoside Rg3 inhibited collagen‐induced platelet aggregation with an IC50 of 20.0 ± 0.9 μM. To elucidate the inhibitory mechanism of dihydroginsenoside Rg3 on aggregation, we analysed its downstream signalling pathway. It was interesting to note that dihydroginsenoside Rg3 elevated cyclic AMP production in resting platelets, but did not affect cyclic GMP production. In addition, we found that dihydroginsenoside Rg3 potently suppressed phosphorylation of extracellular signal‐regulated kinase 2 (ERK2), which was stimulated by collagen (2.5 μg mL−1), but not of p38 mitogen‐activated protein kinase. Taken together, our results indicate that dihydroginsenoside Rg3 potently inhibited platelet aggregation via the modulation of downstream signalling components such as cAMP and ERK2.

Surfactin C inhibits platelet aggregation

This study was designed to investigate the effect of surfactin C, which is derived from Bacillus subtilis, on platelet aggregation and homotypic leucocyte aggregation. Surfactin C strongly and dose‐dependently inhibited platelet aggregation, which was stimulated both by thrombin (0.1 U mL−1), a potent agonist that activates the G protein‐coupled protease receptor, and by collagen (5 μg mL−1), a potent ligand that activates αIIbβ3 with IC50 values (concentration inhibiting platelet aggregation by 50%) of 10.9 and 17.0 μM, respectively. Moreover, surfactin C significantly suppressed the intracellular Ca2+ mobilization in thrombin‐activated platelets. Surfactin C, however, did not affect various integrin‐mediated U937 cell aggregation, implying that the anti‐platelet activity of surfactin C was not due to its detergent effect but by its action on the downstream signalling pathway. Therefore, the results suggest that surfactin C may have a beneficial therapeutic effect on aberrant platelet aggregation‐mediated cardiovascular diseases.

Cordycepin (3‘-deoxyadenosine) inhibits human platelet aggregation induced by U46619, a TXA2 analogue

Cordycepin (3′‐deoxyadenosine), which comes from Cordyceps militaris, the Chinese medicinal fungal genus Cordyceps, is known to have anti‐tumour activity. In this study, we investigated the novel effect of cordycepin on human platelet aggregation that was induced by U46619, a thromboxane A2 (TXA2) analogue. TXA2 is an aggregation‐inducing autacoidal molecule that is produced in various agonist‐activated platelets. Cordycepin completely inhibited U46619‐induced platelet aggregation and simultaneously reduced cytosolic free Ca2+ ([Ca2+]i), which was increased by U46619 (5 μM) up to 66%. Furthermore, the U46619‐stimulated phosphorylation of Ca2+‐dependent proteins (20 kDa of a myosin light chain and 47 kDa of pleckstrin) was strongly inhibited by cordycepin. These results suggest that cordycepin may have a beneficial effect on autacoidal TXA2‐mediated thrombotic diseases by inhibiting TXA2‐induced platelet aggregation via suppression of the Ca2+ level.

Ischemia induces regulator of G protein signaling 2 (RGS2) protein upregulation and enhances apoptosis in astrocytes

Regulator of G protein signaling (RGS) family members, such as RGS2, interact with Galpha subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4-6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemia-induced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKCdelta, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKCdelta downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKCdelta mRNA and protein expressions. Ischemia-induced PKCdelta phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKCdelta knockdown, and this endogenous PKCdelta suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKCdelta and p38 MAPK pathways.

Inhibitory Effects of Epigallocatechin-3-Gallate on Microsomal Cyclooxygenase-1 Activity in Platelets

In this study, we investigated the effect of (–)-epigallocatechin-3-gallate (EGCG), a major component of green tea catechins from green tea leaves, on activities of cyclooxygenase (COX)-1 and thromboxane synthase (TXAS), thromboxane A2 (TXA2) production associated microsomal enzymes. EGCG inhibited COX-1 activity to 96.9%, and TXAS activity to 20% in platelet microsomal fraction having cytochrome c reductase (an endoplasmic reticulum marker enzyme) activity and expressing COX-1 (70 kDa) and TXAS (58 kDa) proteins. The inhibitory ratio of COX-1 to TXAS by EGCG was 4.8. These results mean that EGCG has a stronger selectivity in COX-1 inhibition than TXAS inhibition. In special, a nonsteroid anti-inflammatory drug aspirin, a COX-1 inhibitor, inhibited COX-1 activity by 11.3% at the same concentration (50 μM) as EGCG that inhibited COX-1 activity to 96.9% as compared with that of control. This suggests that EGCG has a stronger effect than that of aspirin on inhibition of COX-1 activity. Accordingly, we demonstrate that EGCG might be used as a crucial tool for a strong negative regulator of COX-1/TXA2 signaling pathway to inhibit thrombotic disease-associated platelet aggregation.

Protopanaxadiol modulates LPS-induced inflammatory activity in murinemacrophage RAW264.7 cells

Protopanaxadiol (PPD) is a mixture of protopanaxadiol type saponins with a dammarane skeleton, from Korean red ginseng (Panax ginseng C.A. Meyer; Araliaceae). Korean ginseng is well-known herb to treat almost all kinds of diseases in Oriental medicine. This herb was particularly prescribed for treatment various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, and diabetes mellitus, for centuries. To understand the efficacy of ginseng against inflammatory diseases, we aimed to show anti-inflammatory activities of the PPD in murine macrophage cell line, RAW264.7 cells using nitric oxide (NO) production assay and the expressions of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, and monocyte chemotactic protein-1 (MCP-1). We found that PPD saponin significantly blocked LPS (1 μg/ml)-induced NO production in a dose-dependent manner. In addition, PPD abrogated the expressions of LPS-induced pro-inflammatory cytokines, such as IL-1β and MCP-1. Moreover, cyclooxygenase (COX)-2, a critical enzyme to produce prostaglandin E2 (PGE2), was significantly inhibited by PPD in LPS-activated RAW264.7 cells. Taken together, these results suggested that anti-inflammatory efficacy of Korean red ginseng on inflammatory diseases is, at least, due to the NO inhibitory activity and the inhibition of the expressional level of inflammatory cytokines and/or mediators.

Inhibitory Activities of Red Ginseng Acidic Polysaccharide in Platelet Aggregation

Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng (Panax ginseng C.A. Meyer), has been shown to have a variety of biological functions such as immunostimulating and anti-tumor activities. In the present study, we investigated whether RGAP inhibited ligand-induced platelet aggregation. The washed platelet-rich plasma was prepared from male SD rats with successive centrifugation. The platelets (10?/ml) were preincubated with 1 mM of CaCl₂for 2 min either in the presence or in the absence of RGAP (10 ~ 50 ㎍/ml) and were stimulated with collagen (2.5 ㎍/ml) and thrombin (0.1 U/ml). RGAP dose-dependently inhibited thrombin-induced platelet aggregation with IC 50 value of 26.2±2.0 ㎍/ml. In collagen-induced platelet aggregation, RGAP inhibited the reaction with an IC 50 value of 31.5±3.0 ㎍/ml. RGAP potently suppressed the intracellular calcium ion, which was stimulated by thrombin (0.1 U/ml). Among mitogen-activated protein kinase (MAPK) subtypes, the extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK were analyzed in the present study. RGAP inhibited the phosphorylation of ERK2 and p38 MAPK, which was activated by collagen (2.5 ㎍/ml). Finally, these results suggested that besides saponin fraction, RGAP take an important role in the preventive effect of Korean red ginseng against cardiovascular disease such as thrombosis and atherosclerosis.

RGS3 Suppresses cAMP Response Element (CRE) Activity Mediated by CB2 Cannabinoid Receptor in HEK293 Cells

RGS proteins have been identified as negative regulators of G protein signalling pathways and attenuate the activity of GPCR receptors. However, information on the regulatory effects of RGS proteins in the activity of cannabinoid receptors is limited. In this study, the role of RGS proteins on the signal transduction of the CB2 cannabinoid receptor was investigated in HEK293 cells co-transfected with CB2-receptors and plasmids encoding RGS2, RGS3, RGS4 and RGS5. Treatment of cells with WIN55, 212-2, a CB2 receptor agonist, inhibited forskolin-induced cAMP response element (CRE) activity in CB2-transfected HEK293 (CB2-HEK293) cells. This inhibitory effect of WIN 55, 212-2 on CRE activity was reversed by co-transfection of CB2-HEK293 cells with RGS3, but not with RGS2, RGS4 and RGS5. However, endogenous RGS3 protein knocked down by a small interfering siRNA targeting RGS3 gene enhanced inhibition of forskolin induced CRE activity via agonist induced CB2 receptor signal transduction. These results indicate the functional role of endogenous RGS protein in cannabinoid signaling pathways and define receptor-selective roles of endogenous RGS3 in modulating CRE transcriptional responses to agonist induced CB2 receptor activity.