Myoung-Yun Pyo

Pterostilbene, a natural dimethylated analog of resveratrol, inhibits rat aortic vascular smooth muscle cell proliferation by blocking Akt-dependent pathway.

Vascular smooth muscle cells (VSMCs) are the main cellular component in the arterial wall, and abnormal proliferation of VSMCs plays a central role in the pathogenesis of atherosclerosis and restenosis after angioplasty, and possibly in the development of hypertension. Pterostilbene, a natural dimethylated analog of resveratrol, is known to have diverse pharmacological activities including anti-cancer, anti-inflammation and anti-oxidant activities. The present study was designed to investigate the effects of pterostilbene on platelet-derived growth factor (PDGF)-BB-induced VSMCs proliferation as well as the molecular mechanisms of the antiproliferative effects. The cell growth of VSMCs was determined by cell counting and [(3)H]thymidine incorporation assays. Pterostilbene significantly inhibited the DNA synthesis and proliferation of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. The inhibition percentages of pterostilbene at 1, 3 and 5microM to VSMCs proliferation were 68.5, 80.7 and 94.6%, respectively. The DNA synthesis of pterostilbene at 1, 3 and 5microM in VSMCs was inhibited by 47.4, 76.7 and 100%, respectively. Pterostilbene inhibited the PDGF-BB-stimulated phosphorylation of Akt kinase. However, pterostilbene did not change the expression of extracellular signal-related kinase (ERK) 1/2, PLCgamma1, phosphatidylinositol (PI)3 kinase and PDGF-Rbeta phosphorylation. In addition, pterostilbene down-regulated the cell cycle-related proteins including the expression of cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, retinoblastoma (Rb) proteins and proliferative cell nuclear antigen (PCNA). These findings suggest that the inhibition of pterostilbene to the cell proliferation and DNA synthesis of PDGF-BB-stimulated VSMCs may be mediated by the suppression of Akt kinase. Furthermore, pterostilbene may be a potential anti-proliferative agent for the treatment of atherosclerosis and angioplasty restenosis.

Comparative estrogenic effects of p-nonylphenol by 3-day uterotrophic assay and female pubertal onset assay.

Nonylphenol (NP) is widely used as a component of detergents, paints, pesticides, and many other formulated products. Several studies have demonstrated that NP is estrogenic in fish, avian, and mammalian cells. NP also competitively inhibits the binding of 17 beta-estradiol (E2) to the estrogen receptor (ER). However, there are relatively few in vivo data related to this issue in mammals. The aim of this study was to investigate the estrogenic activity of NP in animal models. We performed a 3-day uterotrophic assay using immature female rats for comparison with other endpoints of Tier I screening including vaginal opening (VO) in prepubertal intact female rats. For the uterotrophic assay, diethylstilbestrol (DES) (0.2 and 1.0 microg/kg) and p-NP (10, 25, 50, 100, and 200 mg/kg) were administered subcutaneously to immature Sprague-Dawley female rats for 3 consecutive days (postnatal days (PND) 20, 21, and 22). For the female pubertal onset assay, DES (0.2, 1.0, and 5.0 microg/kg) and p-NP (10, 50, and 100 mg/kg) were administered daily by oral gavage from 21 days of age for 20 days. In the uterotrophic assay, statistically significant increases in uterine wet weight were observed at doses of 100 and 200 mg/kg p-NP. DES (0.2 and 1.0 microg/kg) also significantly increased uterine weight compared to the vehicle control. In the female pubertal onset assay, the age of VO was advanced following oral exposure to DES (1.0 and 5.0 microg/kg) and p-NP (50 and 100 mg/kg). Estrous cyclicity was monitored in prepubertal rats from the day of VO to the day of necropsy. Irregular estrous cycles were observed in the groups treated with DES (5.0 microg/kg) and p-NP (50 and 100 mg/kg). High-dose DES (5.0 microg/kg) produced a persistent estrus state, whereas p-NP (50 and 100 mg/kg) increased the number of days in diestrus. Serum thyroxine (T(4)) concentrations were decreased in a dose-dependent manner by DES and p-NP treatment. A significant decrease in serum T(4) level was observed at high-dose DES (5.0 microg/kg) and p-NP (100 mg/kg). Serum TSH level was significantly increased by DES (5.0 microg/kg) treatment. Statistically significant decreases in ovarian weight were observed in female rats treated with DES (5.0 microg/kg) and p-NP (100 mg/kg). Our data demonstrate that p-NP can accelerate the onset of puberty and alter estrous cyclicity in prepubertal female rats at oral doses lower than the subcutaneous doses typically used in the uterotrophic assay. We therefore suggest that the female pubertal onset assay may be used as a sensitive testing method to detect environmental agents with weak estrogenic activity, but requires further research.

Antiplatelet activity of β-carboline alkaloids from Perganum harmala : A possible mechanism through inhibiting PLCγ2 phosphorylation

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

Proteomic Biomarkers for Prenatal Bisphenol A-Exposure in Mouse Immune Organs

Many investigators have encountered difficulty in clarifying the risks of exposure to bisphenol A (BPA), an endocrine disrupting chemical in epidemiological studies or animal experiments. In the present study, we developed biomarkers of BPA‐induced proteomic alterations in immune organs of mouse offspring that were prenatally exposed to BPA (15 and 300 mg/L of drinking water; they were exposed to 8.9 ± 1.8 mg of BPA/kg/day and 171.1 ± 16.8 mg of BPA/kg/day, respectively) that were evaluated in terms of sex, age, and BPA‐exposure levels. We performed 2D‐gel analyses of samples from various tissues (thymus and spleen), exposure levels, sex, and ages (3‐ and 7‐week‐old) (N = 48), and found seven proteins that were altered in a BPA dose‐dependent manner. Among them, we further studiedapo‐AI, DPPIII, and VAT1, which are suspected to be associated with endocrine disorders. By performing Western blots, we confirmed BPA upregulation of all three proteins. Moreover, the apo‐AI mRNA levels were increased in a BPA dose‐dependent manner in 3‐ and 7‐week‐old female mice. Females and young offspring were somewhat more sensitive to protein alterations than others. Our study, which is based on proteome analyses, suggests that apo‐AI, DPPIII, and VAT represent protein biomarkers for BPA and provide useful mechanistic clues for BPA‐induced endocrine disruption. Environ. Mol. Mutagen., 2008.

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

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

Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-β-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca2+ influx, phospholipase C activation, and Ca2+ release from internal storage in a G protein-independent and protein kinase C-independent manner.

Bisphenol A-induced downregulation of murine macrophage activities in vitro and ex vivo

Bisphenol A (BPA) is known to have detrimental effects on the reproductive system, but the toxicity of BPA on immune responses has not been systematically investigated. We investigated the effects of BPA exposure on the activities of murine peritoneal macrophages through evaluation of BPA-induced alteration of nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) synthesis, and expression of co-stimulatory molecules B7. Macrophages were examined ex vivo from mice orally treated with various doses of BPA for 5 consecutive days per week for 4 weeks followed by culture for 2 or 4 days in the presence of lipopolysaccharides (LPS). Macrophages from naive mice were also stimulated with LPS ± BPA for 2 or 4 days. NO production was decreased with the in vitro exposure to 1, 10 and 100μM BPA. NO production was lower in the BPA-exposed mice than the control mice with all doses. In vitro, BPA suppressed TNF-α secretion with significant reduction at 10 and 100μM BPA. Similar findings were observed with the macrophages from the BPA-exposed mice. This study provides the substantial evidence on BPA-induced alteration in macrophage activity.

Blockade of Atopic Dermatitis-Like Skin Lesions by DA-9102, a Natural Medicine Isolated from Actinidia arguta, in the Mg-Deficiency Induced Dermatitis Model of Hairless Rats

DA-9102 isolated from Actinidia arguta is a candidate of natural medicine currently under Phase II clinical trial for atopic dermatitis in Korea. In this study, spontaneous dermatitis was induced by magnesium deficiency in hairless rats and this system was applied to assess the suppressive effects of DA-9102 on atopic dermatitis-like skin disease. Oral administration of DA-9102 at a dose of 100 mg/kg for 16 days substantially suppressed the occurrence of spontaneous dermatitis. Eczematous skin lesions, water loss and scratching behavior were significantly decreased by DA-9102 in a dose-dependent manner. Infiltration of inflammatory cells into the skin and pathologic remodeling of the epidermis and dermis were much less than the Mg-def. group. Results from flow cytometry analysis of peripheral blood mononuclear cells indicated that DA-9102 suppressed activation of leukocytes. The decrease in the number of CD45RA+ cells was accompanied by a lower level of IgE in DA-9102 treated rats, and the reduction in the number of CD11b+ cells by DA-9102 in both periphery and skin was significant. Further, DA-9102 not only suppressed the mRNA expression of TH2 cytokines including IL-4 and IL-10 in the lymph node but it also decreased the levels of inflammatory mediators such as nitric oxide and leukotriene B4 (LTB4) in the serum. Taken together, these results suggest that DA-9102 is an orally applicable potent immune modulator capable of controlling the occurrence of atopic dermatitis-like skin disease.

Effects of repeated hair washing and a single hair dyeing on concentrations of methamphetamine and amphetamine in human hairs

The effects of repeated hair washing and a single hair dyeing on concentrations of methamphetamine (MA) and amphetamine (AM) in hair samples of MA addicts were studied. Thirty-one MA positive hair samples collected from male (n = 24, 24-51 yrs) and female abusers (n = 7, 17-46 yrs) were evaluated for MA and AM concentrations changes after repeated hair washing and a single hair dyeing. Thirty-one MA positive hair samples, no additional treatment hair sample group (NAT), were treated in vitro with liquid soap or three kinds of hair dyes which were black, brown and yellow color hair dye, respectively. Quantitation of AM and MA in hair samples was utilized GC-MS using selected ion monitoring. MA and AM concentrations in NAT were 10.41 ± 8.91 ng/mg (range 1.50-30.0 ng/mg) and 2.24 ± 2.75 ng/mg (range 0.41-12.90 ng/mg). And, their concentrations were decreased about 23.3 ± 4.5% (range 16.7-32.8%) in hair repeated washing group (WAS) and 32.6 ± 4.82 (22.2-41.9) in three kinds of a single hair dyeing groups in comparison to original concentrations of MA and AM in NAT. A statistically significant difference was found between NAT and WAS or three hair dyeing groups (p < 0.01), but not between WAS and three hair dyeing groups, and not between each hair dyeing groups with each three kinds of hair dyes (p > 0.05).

Influence of melatonin on immunotoxicity of lead

The results suggested that immunotoxicity induced by lead [Pb, as Pb(NO(3))(2)] was significantly restored or prevented by melatonin (MLT). MLT (10 or 50 mg/kg) was orally administered to ICR mice daily for 28 days, and Pb was also administered at 35 mg/kg in the same way 2 h after the administration of MLT, and the normal mice were given vehicle. Within the Pb plus MLT-treated group, the body weight gains and the relative thymus weights were significantly increased when compared with the treatment of Pb alone. The relative spleen and liver weights were increased by the treatment of Pb alone, and then restored to normal value by MLT treatment. Hemagglutination (HA) titer, plaque-forming cell response to sheep red blood cell (SRBC), and secondary IgG antibody response to BSA were significantly enhanced in the Pb plus MLT-treated mice, as opposed to when compared with the treatment of Pb alone. The mitogenic response of splenic T cell to concanavalin A and that of B cells to lipopolysaccharide was remarkably increased by MLT treatment when compared with treatment of Pb alone. Splenic CD4(+)cells were significantly increased by MLT treatment when compared with treatment of Pb alone. In case of CD8(+) cells, the slight enhancement was observed in MLT treatment. Splenic T and B cells were significantly increased by MLT treatment when compared with the treatment of Pb alone. The natural killer cell, phagocytic activity and the number of peripheral leukocytes were significantly enhanced in Pb plus MLT-treated mice when compared with the treatment of Pb alone.