Cyanidin-3-glucoside suppresses Th2 cytokines and GATA-3 transcription factor in EL-4 T cells.

Allergic disease is dominated by Th2 immune responses. Interleukin (IL)-4 and IL-13, representative Th2 cytokines, play pivotal roles in the pathogenic activation of the Th2 immune response. In this study, we found that cyanidin-3-glucoside chloride (C3G), an anthocyanin suppressed IL-4 and IL-13 produced in activated EL-4 T cells but not Th1 cytokines including IL-2, interferon-γ, or IL-12. IL-4 and IL-13 mRNA levels and luciferase activation in cells transiently transfected with IL-4 and IL-13 promoter reporter plasmids were significantly inhibited by C3G, suggesting that suppression might be, at least in part, regulated at the transcriptional level. Data from western blot and reverse transcription-polymerase chain reaction analyses of transcription factors involved in cytokine expression suggested that expression of GATA-3, but not T-bet, was downregulated in the nucleus by C3G. Taken together, our data indicate that C3G may has potential as an anti-allergic agent suppressing Th2 activation by downregulating Th2 cytokines and the GATA3 transcription factor in allergies.

Eicosapentaenoic acid and docosahexaenoic acid suppress Th2 cytokine expression in RBL-2H3 basophilic leukemia cells.

It is known that the intake of omega-3 fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), is beneficial for preventing and/or treating allergic diseases. The pathogenesis of allergic diseases is associated with overactivation of Th2-skewed immunity. Basophils generate large amounts of Th2 cytokines such as interleukin (IL)-4 and IL-13, which are critically involved in allergic inflammation. We investigated how EPA and DHA affect Th2 cytokine expression in phorbol 12-myristate 13-acetate- and ionomycin (PI)-activated RBL-2H3 basophilic leukemia cells. EPA and DHA induced a dramatic decrease in the production of IL-4 and IL-13 and their transcription in a dose-dependent manner. Luciferase assays of RBL-2H3 cells stably expressing Il4 and Il13 promoter-reporter plasmids demonstrated a significant suppression of PI-induced promoter activation. Analysis of certain transcription factors revealed that nuclear expression of c-Fos and the mRNA expression were suppressed by EPA and DHA. Furthermore, they significantly inhibited the nuclear expression and translocation of nuclear factor of activated T cells (NF-AT)1. In contrast, the expression levels of nuclear factor kappa-B (NF-κB), GATA-binding proteins (GATAs), and CCAAT/enhancer binding protein alpha (C/EBPα) were not significantly affected by EPA and DHA. Phosphorylation of extracellular signal-related kinase was inhibited by EPA and DHA, and phosphorylation of p38 mitogen-activated protein kinase was decreased by DHA, but not by EPA. Taken together, our data suggest that EPA and DHA may suppress Th2-skewed allergic immune responses by inhibiting the expression of basophilic IL-4 and IL-13.

Pheophytin a and chlorophyll a identified from environmentally friendly cultivation of green pepper enhance interleukin-2 and interferon-γ in Peyer's patches ex vivo.

The oral consumption of capsicum has been reported to increase interleukin (IL)-2 and interferon (IFN)-γ production in Peyer's patches (PP); however, the active components responsible for these effects have not been completely identified. The beneficial biological effects of green peppers cultivated under environmentally friendly farming conditions (ECP), without the use of chemical pesticides, have rarely been compared with those of green peppers cultivated under conventional farming conditions (CCP). Oral administration of ECP extract significantly induced the production of IL-2 and IFN-γ in concanavalin A-treated cells from PP ex vivo; their levels were much higher than those in the CCP extract-treated group. A comparative analysis of the HPLC profiles indicated a 1.7-fold increase of a peak, named EF-1, at 415 nm in the ECP extract. The major component of EF-1 was identified as pheophytin a, which is a chlorophyll a molecule lacking a central Mg(2+) ion, as determined from NMR data. Intake of pheophytin a and chlorophyll a significantly increased IL-2 and IFN-γ production, and the percentage of IL-2- and IFN-γ-producing CD4+ T-cells in PP. Taken together, our data suggest that ECPs produce a higher content of pheophytin a than CCPs, and pheophytin a and chlorophyll a are immune-modulating components in green vegetables.

Suppressive effects of fructus of Magnolia denudata on IL-4 and IL-13 expression in T cells.

Magnolia species have been used for the treatment of allergic diseases in Asia as folk medicine; however, the cellular and molecular mechanisms of its anti-allergic effects have rarely been investigated. In this study, we demonstrated that a methanolic extract of the fructus of Magnolia denudata has suppressive effects on Th2 cytokine production such as IL-4 and IL-13, but not IFN-γ and IL-17, produced by both phorbol 12-myristate 13-acetate/ionomycin (PI)- and CD3/CD28-stimulated EL-4 T cells. Moreover, the mRNA expression of Th2 cytokines was significantly inhibited, and luciferase activity in cells transiently transfected with IL-4 or IL-13 promoter reporter plasmids was suppressed by M. denudata, indicating that M. denudata may regulate these expression at the transcriptional level. Western blot analysis for transcription factors involved in the cytokine gene expression indicated that the activation of c-Jun was significantly downregulated in the nucleus of cells, while the activations of nuclear factor of activated T cells, nuclear factor kappa B and c-Fos, were not affected. Furthermore, the mRNA expression and nuclear translocation of GATA-binding protein 3, a key transcriptional factor for Th2 commitment and Th2 cytokine expression, but not T-bet and RORγt, were dramatically downregulated by M. denudata. Treatment with M. denudata suppressed the phosphorylation of p38 mitogen-activated protein kinase; however, the PI-induced phosphorylation of extracellular signal-related kinase and c-Jun N-terminal kinase was unaffected. Taken together, our study indicated that M. denudata inhibited IL-4 and IL-13 expression, possibly through regulation of p38 mitogen-activated protein kinase phosphorylation and selective transcription factors, such as GATA-3 and c-Jun, in EL-4 T cells.

Set, a putative oncogene, as a biomarker for prenatal exposure to bisphenol A.

BACKGROUND: Bisphenol A (BPA), an endocrine disrupting chemical, has been suspected to pose carcinogenic risks. However, likely mechanisms are obscure and there are difficulties to estimating its real significance for cancer development. METHODS: We therefore studied BPA-induced proteomic alterations in immune organs of ICR mice offspring that were prenatally exposed to BPA (15 and 300 mg/L of drinking water). We performed 2D-gel analyses of samples, considering differences in spleen, exposure levels, sex, and ages. RESULTS: From proteomic analyses, we found various proteins were up- or down-regulated by BPA. Among them, SET, a putative oncogene and inhibitor of phosphatase 2A, was significantly down- regulated in a BPA dose-dependent manner. We also confirmed down-regulation of SET in western blot and real time PCR analyses. From gene network analysis, SET is predicted to communicate with other genes including CYP17, which is involved in biosynthesis and metabolism of sex-hormones. CONCLUSIONS: This study provided evidence that SET can be applied as a new biomarker for prenatal BPA exposure and suggests a potential new mechanism of action in that BPA may disrupt CYP17 via SET.

Inhibition of pentylenetetrazole-induced seizure in mice by using a 4 Hz magnetic field: a comparative study with a 60 Hz magnetic field.

We investigated the comparative effects of 4 and 60 Hz magnetic fields on pentylenetetrazole (PTZ)-induced seizure in mice. For this study, we measured the latent time to seizure, seizure duration, and lethality induced by PTZ in mice exposed to 4 and 60 Hz magnetic fields (MF) for 30 min. Compared to sham-exposed controls, the latent time to tail twitching and seizure in the 4 Hz MF group was significantly decreased while the latent time to seizure in the 60 Hz MF group was significantly increased. The seizure duration in the 4 Hz MF group was significantly decreased while that in the 60 Hz MF group was significantly increased. More importantly, while the mice exposed to a 60 Hz MF experienced significantly increased lethality after seizure convulsion, those exposed to a 4 Hz MF showed no lethality, with a shortening of the duration of seizure. This beneficial effect of a 4 Hz MF on seizure has the same implication as the anti-oxidative effects of a 4 Hz MF observed in our previous work. The results of our current and previous works indicate that a 4 Hz MF may be used as a therapeutic physical agent for the treatment of oxidative stress-induced diseases, including seizure, with or without chemical drugs.

Inhibitory effects of OD 78 [3-(4-bromo-phenoxy)-4,5-dihydroxybenzoic acid-methyl ester] on the proliferation and migration of TNF-α-induced rat aortic smooth muscle cells.

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the formation and progression of intimal thickening in early-phase atherosclerosis and in restenosis after vascular injury. Tumor necrosis factor-α (TNF-α) is released from macrophages in atherosclerotic lesions and from neointimal vascular smooth muscle cells after balloon-injury. Obovatol, a major biphenolic component isolated from the Magnolia obovata leaf, is known to have anti-inflammatory and antitumor activities. The goal of this study was to examine the cardioprotective effects of the obovatol derivative OD 78 on the TNF-α-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs). The antiproliferative effects of OD 78 on RASMCs were examined by cell counting and [(3)H]-thymidine incorporation assays. Treatment of cells with 1-4 µM OD 78 inhibited the proliferation and DNA synthesis of TNF-α-stimulated RASMCs in a concentration-dependent manner, without cytotoxicity. Treatment with OD 78 inhibited TNF-α-mediated p38 phosphorylation, but did not change the activation of extracellular signal-regulated kinase or c-Jun N-terminal kinase. Furthermore, treatment with OD 78 decreased TNF-α-induced levels of cyclin E, cyclin D1, CDK2, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein, but not the CDK4 expression level. Also, OD 78 inhibits the migration of TNF-α-induced RASMC in transwells. OD 78 treatment strongly decreased matrix metalloproteinase-9 (MMP-9) expression in a dose-dependent manner, but the MMP-2 expression was unchanged. These results show that OD 78 may be developed as a potential antiproliferative agent for the treatment of angioplasty restenosis and atherosclerosis.

Inonotus obliquus extracts suppress antigen-specific IgE production through the modulation of Th1/Th2 cytokines in ovalbumin-sensitized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaga mushroom (Inonotus obliquus, IO) has been used as a folk remedy for cancer, digestive system diseases, and other illnesses in Russia and Eastern Europe. AIM OF THE STUDY: In the present study, we investigated the immunomodulating effects of IO through in vivo and ex vivo studies. MATERIALS AND METHODS: Serum immunoglobulins (IgE, IgG(1), and IgG(2a)) and cytokines (interleukin (IL)-4, interferon (IFN)-γ, and IL-2) were measured in concanavalin A (ConA)-stimulated splenocytes and CD4(+) T cells. The nitric oxide (NO) secretion of lipopolysaccharide (LPS)-stimulated peritoneal macrophages was also measured after oral administration of 50, 100, or 200 mg kg(-1) d(-1) IO hot water extract (IOE) to ovalbumin (OVA)-sensitized BALB/c mice. RESULTS: We found that the OVA-induced increase in serum IgE and IgG(2a) was significantly suppressed when IOE was orally administered after the second immunization with OVA. ConA stimulation in spleen cells isolated from OVA-sensitized mice treated with 100 mg kg(-1) IOE resulted in a 25.2% decrease in IL-4 production and a 102.4% increase in IFN-γ, compared to the controls. Moreover, IL-4, IFN-γ, and IL-2 were significantly reduced after ConA stimulation in isolated CD4(+)T cells. We also determined that IOE inhibits the secretion of NO from LPS-stimulated peritoneal macrophages ex vivo. CONCLUSIONS: We suggest that IO modulates immune responses through secretion of Th1/Th2 cytokines in immune cells and regulates antigen-specific antibody production.

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.

Suppressive effects of T-412, a flavone on interleukin-4 production in T cells.

Interleukin (IL)-4 has been suggested as a molecular therapeutic target to prevent and/or treat various allergic diseases and several flavonoids have been suggested as anti-allergic agents suppressing IL-4 production. In an effort to find novel candidates for anti-allergic agents from natural sources, we screened several flavonoids affecting on IL-4 production. In this study, we showed that 7,8,4'-trihydroxyflavone (T-412) significantly decreased IL-4 production both in phorbol 12-myristate 13-acetate (PMA) and ionomycin (PI)-activated EL-4 T cells and concanavalin A (ConA)-activated murine CD4(+) T cells in a dose- and time-dependent manner. The PI-induced increase of IL-4 mRNA expression was dramatically suppressed by T-412 at 6 h, indicating the suppression is regulated at transcriptional level. T-412 also significantly inhibited IL-4 gene promoter activity in EL-4 T cells transiently transfected with luciferase reporter plasmid containing IL-4 promoter (pGL4.14-IL-4). Western blot analysis of the transcription factors revealed that T-412 suppressed the nuclear expression of nuclear factor of activated T cells (NF-AT)c1, c-Jun and c-Maf, but not c-Fos and nuclear factor kappa B (NF-kappaB). Our data suggested that T-412 might have potential as a candidate for anti-allergic agent having suppressive effects on IL-4 production in activated T cells by controlling the transcription of IL-4.

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-beta-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.

Antiproliferative action of cudraflavone B, isolated from Cudrania tricuspidata, through the downregulation of pRb phosphorylation in aortic smooth muscle cell proliferation signaling.

Cudrania tricuspidata has been proposed to possess anti-inflammatory, antioxidant, hepatoprotective, and antitumor activities. Although cudraflavone B, isolated from the root bark of C. tricuspidata, has a variety of pharmacological effects, its effects on rat aortic smooth muscle cells (RASMCs) are unclear. In the present study, cudraflavone B was found to inhibit cell proliferation and DNA synthesis in cultured RASMCs. Pretreatment with cudraflavone B (0.1-4 microM) suppressed platelet-derived growth factor-BB (PDGF-BB)-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 19.7%, 36.4%, 52.3%, and 99.1% at concentrations of 0.1, 1, 2, and 4 microM, respectively. Moreover, cudraflavone B inhibited [H]-thymidine incorporation into DNA in RASMCs in response to 25 ng/mL PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced by 15.9%, 31.7%, 43.1%, and 78.2% at concentrations of 0.1, 1, 2, and 4 muM, respectively. Thus, cudraflavone B blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Furthermore, PDGF-BB-induced phosphorylation of retinoblastoma protein (pRb), the hyperphosphorylation of which is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by cudraflavone B. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDKs), we investigated the expression of CDK2, CDK4, cyclin E, and cyclin D1 and the CDK inhibitors p21 and p27. Treatment with cudraflavone B downregulated the cyclins and CDKs and upregulated the expression of p21 and p27, a CDK inhibitor. These findings suggest that cudraflavone B inhibits RASMC proliferation via the induction of p21 and p27 expression and subsequent cell cycle arrest with reduction of pRb phosphorylation at the G1-S phase.

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

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

Corynoxeine isolated from the hook of Uncaria rhynchophylla inhibits rat aortic vascular smooth muscle cell proliferation through the blocking of extracellular signal regulated kinase 1/2 phosphorylation.

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.

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 T(H)2 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 B(4) (LTB(4)) 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.

4 Hz magnetic field decreases oxidative stress in mouse brain: a chemiluminescence study.

We investigated the effects of delta and theta brain wave frequency magnetic fields (3, 4, and 5) on mouse brain by detecting photonic oxidative stress makers; spontaneous photon emission (SPE) and lucigenin and tert-butyl hydroperoxide (TBHP) induced chemiluminescences (CL). For this purpose, Balb/C mice were exposed to 3, 4, and 5 Hz magnetic fields (MF) at 0.7 mT for 3 h, respectively. After that we monitored SPE and lucigenin and TBHP-induced CL of the homogenates of mice brains. There was a significant decrease in SPE in the 4 Hz MF-exposed group. Lucigenin-induced CL was also significantly decreased only in the 4 Hz MF-exposed group. TBHP-induced CL was also distinctively decreased by all frequencies, 3, 4, and 5 Hz MF exposures. These results showed that oxidative stress in a mouse brain was decreased by 4 Hz MF. We suggest that the application of 4 Hz MF will contribute to magnetic field therapy.

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.

Downregulation of peritoneal macrophage activity in mice exposed to bisphenol A during pregnancy and lactation.

Bisphenol A (BPA) is an environmental endocrine disrupter that is known to be transferred to the fetus via the placenta and to the neonate via milk. In this study, we investigated BPA-induced alterations of the activities of murine peritoneal macrophages in dams and 7 week old offspring of dams exposed to BPA from gestational day 7 until lactation on day 21 after delivery, i.e. 34-36 days. BPA was administered in drinking water at three doses, 15, 75, and 300 mg/L. Dams were sacrificed 21 days after delivery and offspring at the age of 7 weeks. Peritoneal macrophages were cultured in the presence of LPS or LPS plus IFN-gamma for 2 or 4 days. We found that nitric oxide (NO) production by maternal macrophages was significantly decreased in a BPA-dose dependent manner. However, while a significant reduction of NO production by macrophages in the offspring was observed at BPA concentrations of 75 mg/L and 300 mg/L in drinking water, this effect was not seen at the lowest concentration of 15 mg/L. Similar inhibition of tumor necrosis factor-alpha (TNF-alpha) production was observed with macrophages from both BPA-exposed dams and offspring. Thus, our results suggest that exposure to BPA during gestation and lactation induces downregulation of the activities of macrophages in both dams and offspring.

Comparison study of the extraction methods of paraquat in post-mortem human blood samples.

The most important step for Paraquat analysis in post-mortem human blood (PMB) is its extraction from the specimens, as Paraquat is insoluble in organic solvents due to its ionic form. The most common extraction method, solid phase extraction (SPE), has been used for the extraction of Paraquat from PMB. However, SPE procedures are somewhat time-consuming, and resulted in unsatisfactory recovery in our laboratory. Therefore, SPE procedures, with five extraction solvents for the liquid-liquid extraction (LLE) of paraquat in PMB, were compared using HPLC, and the chloroform-ethanol (7:3, v/v) solvent mixture was found to be the most effective. The recoveries of Paraquat using the 7:3 solvent mixture in human whole blood samples, which were already spiked with paraquat standards (1.05, 2.10 and 4.21 microg/mL) averaged 98.20, 105.71 and 99.40%, but the recoveries from the SPE were about 74.29, 78.50 and 80.10%, respectively. Linearity was obtained for the range of Paraquat standards, with a correlation coefficient; r2 > 0.999. The limit of detection (LOD, with S/N > or =3) and limit of quantitation (LOQ, with S/N > or =10) were 0.01 and 0.05 microg/mL, respectively. The extraction method was successfully applied to seven real post-mortem cases involving paraquat poisoning.