Brain-Derived Neurotrophic Factor Up-Regulation by the Methanol Extract of Foxtail Millet in Human Peripheral Cells.

Brain-derived neurotrophic factor (BDNF) plays important roles in synaptic plasticity and neuronal differentiation. The neurotrophic hypothesis of depression, which suggests that reduced BDNF in the hippocampus underlies depression, has attracted increasing attention. Stress, a major cause of depression, leads to decreased BDNF levels, and administration of BDNF into the hippocampus shows an antidepressant effect. BDNF is synthesized in peripheral tissues as well as in the brain. Since BDNF crosses the blood-brain barrier, intake of food ingredients that elevate BDNF in peripheral tissues may be useful for the prevention and treatment of depression. However, no screening method for BDNF up-regulators in peripheral tissues has been reported. In this study, we revealed that ACHN human kidney adenocarcinoma cells secreted BDNF. In addition, we demonstrated that the methanol extract of foxtail millet up-regulated BDNF levels in ACHN cells. Our results indicate that ACHN cells could be useful in the screening for peripheral-BDNF up-regulators, and that foxtail millet may have the potential to elevate BDNF levels in peripheral tissues.

Decreased Plasma Octanoylated Ghrelin Levels in Mice by Oleanolic Acid.

Ghrelin is a stomach-derived peptide hormone with an appetite-stimulating effect. Octanoylation on the serine-3 residue of ghrelin by ghrelin O-acyl transferase (GOAT) is essential for its orexigenic effect. Mature octanoylated ghrelin is generated by the C-terminal cleavage of octanoylated proghrelin via prohormone convertases (furin, PC1/3, or PC2). We previously established an AGS-GHRL8 cell line that produces octanoylated ghrelin in the presence of octanoic acid, and found that oleanolic acid suppresses octanoylated ghrelin production in AGS-GHRL8 cells. Here, we investigated the effects of oleanolic acid in C57BL/6J mice fed a standard, high-fat, or high-glucose diet. Oral administration of oleanolic acid for seven days (20 or 40 mg/kg) reduced plasma octanoylated ghrelin levels and body weight gain in the standard diet-fed mice but not in other two diet-fed mice. There were no significant differences in ghrelin, GOAT, furin, PC1/3, and PC2 gene expression levels between the vehicle- and oleanolic acid-treated mice fed a standard diet. Octanoyl-CoA is a substrate for ghrelin octanoylation by GOAT. We found that oleanolic acid did not affect octanoyl-CoA production in vitro. Hence, the inhibitory effect of oleanolic acid on octanoylated ghrelin production may not be related to the decrease in octanoyl-CoA. The results of this study may provide valuable knowledge for the development of anti-obesity agents with an inhibitory effect on octanoylated ghrelin production.

Inhibitory Effect of (-)-Epigallocatechin-3-O-gallate on Octanoylated Ghrelin Levels in Vitro and in Vivo.

Ghrelin is an orexigenic peptide hormone produced in the stomach. The major active form is octanoylated ghrelin, which is modified with an n-octanoic acid at the serine-3 residue. Inhibition of octanoylated ghrelin production is useful for the prevention and improvement of obesity. We previously developed a cell-based assay system employing a ghrelin-expressing cell line, AGS-GHRL8, and found various compounds that decreased octanoylated ghrelin levels using this system. (-)-Epigallocatechin-3-O-gallate (EGCG) is a bioactive catechin in green tea and reportedly has an anti-obesity effect; however, it remains unclear whether EGCG inhibits octanoylated ghrelin production. Therefore, in this study, we investigated the effect of EGCG on octanoylated ghrelin levels in AGS-GHRL8 cells and C57BL/6J mice. EGCG significantly reduced the octanoylated ghrelin level in AGS-GHRL8 cells. In mice, three days of treatment with TEAVIGO®, which contains 97.69% EGCG, lowered the plasma octanoylated ghrelin level by 40% from that in control mice. In addition, TEAVIGO® reduced the mRNA expression of ghrelin and prohormone convertase 1/3, an enzyme responsible for the processing of proghrelin to mature ghrelin, in the mouse stomach, suggesting that the reduced expression of these genes may contribute to the inhibition of octanoylated ghrelin production. These results suggest a decrease in the octanoylated ghrelin level to be involved in the anti-obesity effect of EGCG, which thus has potential for the development of anti-obesity agents with ghrelin-lowering effect.

Bioactive Triterpenes from the Root of Salvia miltiorrhiza Bunge.

Danshen (Salvia miltiorrhiza) is a well-known medicinal herb in the oriental medicine. The current study on bioactive triterpenoid in the root of S. miltiorrhiza led to the isolation of a new highly hydroxylated ursane-type triterpene, urs-12-ene-2α,3ß,7ß,16α-tetraol (1) and five known ones including 2ß-hydroxypomolic acid (2), maslinic acid (3), asiatic acid (4), ursolic acid (5), and oleanolic acid (6). Their structures were elucidated on the basis of extensive spectroscopic analyses and comparison with literature data. The antiproliferative testing against HL-60 cells revealed that the new compound 1 and ursolic acid (5) showed weak and moderate activities with IC50 values of 42.2 and 11.7 µM. In addition, compounds 1-3 showed inhibitory effect on ghrelin activity. Copyright © 2017 John Wiley & Sons, Ltd.

Triterpenes suppress octanoylated ghrelin production in ghrelin-expressing human gastric carcinoma cells.

Ghrelin is an appetite-stimulating peptide hormone with an octanoyl modification at serine 3 that is essential for its orexigenic effect. Ghrelin O-acyltransferase (GOAT) is the enzyme that catalyzes ghrelin acylation using fatty acyl-coenzyme A as a substrate. We previously developed an assay system based on the AGS-GHRL8 cell line that produces octanoylated ghrelin in the presence of octanoic acid, and demonstrated that some fatty acids suppressed octanoylated ghrelin production. Recent studies have reported that triterpenes have anti-obesity effect. Since such triterpenes, like fatty acids, have a carboxyl group, we speculated that they can suppress octanoylated ghrelin production. To test this hypothesis, we investigated the effect of triterpenes on octanoylated ghrelin production. Asiatic acid, corosolic acid, glycyrrhetinic acid, oleanolic acid and ursolic acid suppressed octanoylated ghrelin levels in AGS-GHRL8 cells without decreasing transcript expression of GOAT or furin, a protease required for ghrelin maturation. ß-amyrin had no effect on octanoylated ghrelin level, which was only slightly inhibited by uvaol; the fact that both these triterpenes lack a carboxyl group indicates that this group is important for suppressing octanoylated ghrelin production. These results suggest that triterpenes may have the potential as obesity-preventing agents with suppressive effect on octanoylated ghrelin production.

Inhibitory Effect of Oleic Acid on Octanoylated Ghrelin Production.

Ghrelin is a growth hormone-releasing peptide that also displays orexigenic activity. Since serine-3 acylation with octanoylate (octanoylation) is essential for the orexigenic activity of ghrelin, suppression of octanoylation could lead to amelioration or prevention of obesity. To enable the exploration of inhibitors of octanoylated ghrelin production, we developed a cell-based assay system using AGS-GHRL8 cells, in which octanoylated ghrelin concentration increases in the presence of octanoic acid. Using this assay system, we investigated whether fatty acids contained in foods or oils, such as acetic acid, stearic acid, oleic acid, linoleic acid, and α-linolenic acid, have inhibitory effects on octanoylated ghrelin production. Acetic acid did not suppress the increase in octanoylated ghrelin production in AGS-GHRL8 cells, which was induced by the addition of octanoic acid. However, stearic acid, oleic acid, linoleic acid, and α-linolenic acid significantly suppressed octanoylated ghrelin production, with the effect of oleic acid being the strongest. Additionally, oleic acid decreased the serum concentration of octanoylated ghrelin in mice. The serum concentration of des-acyl ghrelin (without acyl modification) was also decreased, but the decrease was smaller than that of octanoylated ghrelin. Decreased octanoylated ghrelin production likely resulted from post-translational ghrelin processing, as there were no significant differences in gene expression in the stomach between oleic acid-treated mice and controls. These results suggest that oleic acid is a potential inhibitor of octanoylated ghrelin production and that our assay system is a valuable tool for screening compounds with suppressive effects on octanoylated ghrelin production.

Generation of an anti-Dabigatran Monoclonal Antibody and Its Use in a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Serum Dabigatran.

BACKGROUND: Dabigatran (DT) is a direct thrombin inhibitor used to prevent venous and arterial thromboembolism due to atrial fibrillation. DT is the active form of the commercially available prodrug DT etexilate. Although DT has many clinical advantages over warfarin, it increases the incidence of bleeding in patients with renal dysfunction. Circulating levels of DT are increased in such patients because it is mainly eliminated by renal excretion. Therapeutic drug monitoring may therefore help to prevent adverse DT effects, but no method for measuring circulating DT levels has been reported, except for an analysis by liquid chromatography-tandem mass spectrometry. This study sought to develop a novel enzyme-linked immunosorbent assay (ELISA) to measure DT concentrations. METHODS: Mice were immunized with a DT-keyhole limpet hemocyanin conjugate to generate an anti-DT antibody. Immunized mouse splenocytes and myeloma cells (SP2/0) were fused to obtain an anti-DT monoclonal antibody (DT-mAb). DT-mAb and DT solutions were added to microplate wells coated with a DT-human serum albumin conjugate. DT concentrations were determined based on the principles of ELISA. RESULTS: DT-mAb was successfully purified from a hybridoma, and the competitive ELISA developed using this DT-mAb could evaluate DT concentrations ranging from 7.8 to 125 ng/mL. The ELISA signal was not linear using DT-spiked serum; however, it was linear when serum ultrafiltrate was used. Weak cross-reactivity with DT etexilate was detected, but no cross-reactivity was observed with other structurally related drugs or drugs commonly used for the treatment of atrial fibrillation. CONCLUSIONS: The developed competitive ELISA is a valuable and specific tool to analyze free DT in serum ultrafiltrate for therapeutic drug monitoring and pharmacokinetic studies.

Nutritional Effect of Oral Supplement Enriched in ω-3 Fatty Acids, Arginine, RNA on Immune Response and Leukocyte-platelet Aggregate Formation in Patients Undergoing Cardiac Surgery.

The aim of the present study was to investigate the influence of a supplement enriched in ω-3 fatty acids on immune responses and platelet-leukocyte complex formation in patients undergoing cardiac surgery. Patients in the supplement group (n = 7) took a supplement enriched in ω-3 fatty acids (Impact(®)) in addition to a hospital diet for five successive days before surgery; those in the control group (n = 7) took only hospital diet and did not take Impact(®). Blood samples in both groups were collected at same time points. Before surgery, samples were collected five days before surgery, at the start of supplementation (baseline), and the end of supplementation (postoperative day (POD)-0). After surgery, samples were collected on POD-1 and POD-7. The expression of human leukocyte antigen (HLA)-DR, the ratio of CD4-/CD8-positive cells, the production of interferon (IFN)-γ by CD4-positive cells, plasma levels of cytokines, and leukocyte-platelet aggregates were measured. Before surgery (POD-0), the supplement caused significant increases in HLA-DR expression, CD4/CD8 ratio, and plasma levels of IFN-γ; these levels were significantly higher compared to those in the control group (P < 0.05, respectively). After surgery (POD-1), all values dramatically decreased in comparison with those of POD-0; however, the values in the supplement group were significantly higher compared to their respective markers in the control group (P < 0.05, respectively). Significant differences of HLA-DR expression and CD4/CD8 ratio persisted through POD-7. Before surgery (POD-0), plasma levels of interleukin (IL)-10 in the supplement group decreased significantly compared with those in the control group (P < 0.05). After surgery (POD-1), plasma levels of IL-10 in both the control and supplement groups increased; these levels in the supplement group were significantly lower than those in the control group (P < 0.05). Significant decreases in the percentage of leukocyte-platelet aggregates were found after supplementation; the difference between the supplement and the control groups was found on POD-0 and POD-1 (P < 0.05, respectively). In conclusion, the dietary supplement increased HLA-DR expression, the CD4/CD8 ratio, and the production of IFN-γ by CD4-positive cells; conversely, the levels of IL-10 and the formation of leukocyte-platelet aggregates before and after surgery were suppressed. These beneficial effects may decrease the incidence of complications after surgery.

Factors involved in the cisplatin resistance of KCP­4 human epidermoid carcinoma cells.

KCP-4 is a cisplatin-resistant cell line established from human epidermoid carcinoma KB-3-1 cells. Although our previous study revealed that one of the mechanisms for cisplatin resistance in KCP-4 cells is the activation of NF-κB, its high resistance is considered to be induced by multiple mechanisms. In the present study, we explored other factors involved in the development of cisplatin resistance in KCP-4 cells. Since it has been reported that an unknown efflux pump exports cisplatin from KCP-4 cells in an ATP-dependent manner, we examined 48 types of ATP-binding cassette proteins as candidate cisplatin efflux transporters. The mRNA expression levels of ABCA1, ABCA3, ABCA7 and ABCB10 in KCP-4 cells were higher when compared to those in KB-3-1 cells. These expression levels in cisplatin-sensitive revertant KCP-4 cells (KCP-4R cells), were reduced in parallel with the sensitivity of these cells to cisplatin and their intracellular accumulation of cisplatin. Next, we investigated the occurrence of mutations in p53 in KCP-4 cells. We found a heterozygous missense mutation at codon 72 (p.Pro72Arg) in p53 of both KCP-4 and KB-3-1 cells, but the protein expression level of p53 in KCP-4 cells was higher when compared to that in KB-3-1. These results suggest that ABCA1, ABCA3, ABCA7 and ABCB10 are candidate genes for the cisplatin efflux transporter that is involved in the cisplatin resistance of KCP-4 cells, and that the mutation at codon 72 of p53 may contribute to the development of cisplatin resistance.

Establishment of a gastric cell-based assay system for exploring inhibitors of octanoylated ghrelin production.

Ghrelin, a gastric hormone, is a growth hormone-releasing peptide. Its serine-3 acylation with octanoic acid is essential for its orexigenic activity, and therefore, inhibition of the acylation of ghrelin may help in decreasing appetite and preventing obesity. This study aimed to establish a human gastric cell-based assay system to evaluate candidate inhibitors of octanoylated ghrelin production. In human gastric carcinoma AGS cells, obligatory factors for the posttranslational modification of ghrelin, such as certain prohormone convertases responsible for processing of proghrelin to the mature ghrelin and the enzyme-catalyzing acyl-modification of ghrelin, were well expressed, but ghrelin was expressed at low levels. Accordingly, we transfected a ghrelin-expressing vector into AGS cells and isolated a stable ghrelin-expressing cell line (AGS-GHRL8). AGS-GHRL8 cells secreted octanoylated ghrelin in accordance with the concentrations of octanoic acid in the culture medium. Given that ingested heptanoic acid is used for the acyl-modification of ghrelin, we evaluated whether heptanoic acid inhibits production of octanoylated ghrelin in AGS-GHRL8 cells. Butyric acid was used as a control. Indeed, heptanoic acid predictably decreased the secretion of octanoylated ghrelin, whereas butyric acid did not. The AGS-GHRL8 line established in this study will facilitate the screening of inhibitors of octanoylated ghrelin production.

Anti-Proliferative Activities and Apoptosis Induction by Triterpenes Derived from Eriobotrya japonica in Human Leukemia Cell Lines.

Eriobotrya japonica leaf is a traditional herbal medicine that contains numerous triterpenes, which have various pharmacological properties. In this study, we investigated the anti-proliferative activity of four triterpenes derived from E. japonica, including corosolic acid (CA), ursolic acid (UA), maslinic acid (MA) and oleanolic acid (OA), in human leukemia cell lines. CA showed the strongest anti-proliferative activity in all of the leukemia cell lines tested, but not in normal human skin fibroblast cell lines. To determine the mechanism underlying the anti-proliferative effect of CA, we examined the effect of CA on molecular events known as apoptosis induction. CA induced chromatin condensation, DNA fragmentation, sub-G(1) phase DNA, activation of caspase-3, -8 and -9 and the cleavage of PARP in HL-60. CA also activated Bid and Bax, leading to the loss of mitochondrial membrane potential (∆ψ(m)) and cytochrome c release into the cytosol, whereas Bcl-2 and Bcl-xL were unaffected by CA. These results suggest that CA has an anti-proliferative effect on leukemia cells via the induction of apoptosis mediated by mitochondrial dysfunction and caspase activation. CA may be a potential chemotherapeutic agent for the treatment of human leukemia.

Involvement of NF-κB activation in the cisplatin resistance of human epidermoid carcinoma KCP-4 cells.

cis-Diamminedichloroplatinum II (cisplatin) is one of the most potent antitumor agents for the treatment of various types of cancer. In spite of its therapeutic usefulness, the intrinsic resistance acquired under continuous treatment limits its benefit in cancer therapy. KCP-4, a cisplatin-resistant cell line, was derived from human epidermoid carcinoma KB-3-1 cells. Since the accumulation of cisplatin in KCP-4 cells is markedly reduced by the presence of an efflux pump, this pump is thought to be related to cisplatin resistance of the KCP-4 cells. However, given that KCP-4 cells are tremendously resistant to cisplatin compared with KB-3-1 cells, it is possible that another mechanism exists. The aim of this study was to investigate whether the activation of nuclear factor-kappa B (NF-κB) contributes to the cisplatin resistance of KCP-4 cells. We used the level of translocated NF-κB into the nucleus, determined by immunoblot analysis, as the indicator of NF-κB activation. The activation level of NF-κB was higher in KCP-4 cells than in KB-3-1 cells. KCP-4 cells were treated with a combination of cisplatin and curcumin, an inhibitor of NF-κB activation, and the cell viabilities were subsequently determined by the MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. In the presence of 10 µmol/l curcumin, we found that the sensitivity of KCP-4 cells to 100 and 300 µmol/l cisplatin was augmented. Additionally, curcumin reduced the activation levels of NF-κB in KCP-4 cells, and suppressed the expression levels of Bcl-2, Bcl-xL and survivin, which are apoptosis-related proteins regulated by NF-κB. Our results suggest that the high cisplatin resistance of KCP-4 cells compared with KB-3-1 cells results from multiple mechanisms other than increased cisplatin efflux, including the activation of NF-κB.

Eriobotryae folium extract suppresses LPS-induced iNOS and COX-2 expression by inhibition of NF-kappaB and MAPK activation in murine macrophages.

Eriobotryae folium (EF), the dried leaves of Eriobotrya japonica (Thunb.) Lindl. has been traditionally used to treat various diseases such as chronic bronchitis, cough, inflammation, skin diseases, and diabetes. In this study, we examined the effects of Eriobotryae folium extract (EFE) on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2(PGE2) in RAW264 murine macrophage cells. EFE suppressed LPS-induced NO and PGE2 production in a dose-dependent manner. Consistent with these observations, EFE reduced the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both protein and mRNA levels. Furthermore, EFE significantly inhibited LPS-induced NF-kappaB binding activity, which was associated with the inhibition of IkappaB-alpha degradation. EFE also attenuated LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK). These results suggest that the anti-inflammatory properties of EF might result from inhibition of iNOS and COX-2 expression through the downregulation of NF-kappaB activation and MAPK phosphorylation in LPS-stimulated RAW264 cells.

Enhanced platelet responsiveness due to chilling and its relation to CD40 ligand level and platelet-leukocyte aggregate formation.

To investigate platelet responsiveness during cold storage of whole blood, we examined platelet aggregation, expression of CD40 ligand (CD40L) on platelets, the plasma levels of soluble form of CD40L (sCD40L) as well as platelet-leukocyte aggregates. Flow cytometry analysis was performed to investigate platelet-leukocyte aggregate formation using antibodies against CD42b and CD45 and platelet activation using antibodies against P-selectin and PAC-1. Blood samples were collected from healthy volunteers, patients with cardiovascular diseases, or both. In the healthy volunteers' blood samples stored at 4 degrees C for 6 h, platelet aggregation in response to 1 micromol/l ADP was enhanced, and released levels of soluble form of P-selectin and thromboxane B2 in response to 1 micromol/l ADP markedly increased. In the samples stored at 4 degrees C for 6 h but not stimulated by any agonists, CD40L expression on the platelets was increased, and plasma levels of sCD40L were also elevated. Under the same condition, the increase in simultaneous expression of CD45 and CD42b was observed. In patients with cardiovascular diseases, the platelet aggregability, coexpression of P-selectin and PAC-1, expression of CD40L on platelets and both CD45-bound and CD42b-bound subsets were all comparable to those of healthy volunteers' samples stored at 4 degrees C for 6 h. Plasma levels of sCD40L in patients were higher than those in healthy volunteers' control. Taken together, storage of whole blood at 4 degrees C for 6 h caused platelet activation comparable to that of patients with cardiovascular diseases, and enhanced platelet activity in such patients may be involved in increased risk for thromboembolic events.

Contactin-associated protein (Caspr) 2 interacts with carboxypeptidase E in the CNS.

To identify proteins interacting with the intracellular domain of the neural cell adhesion molecule contactin-associated protein 2 (Caspr2), yeast two-hybrid screening was performed. We identified carboxypeptidase E (CPE) as a Caspr2-interacting candidate protein. Glutathione S-transferase pull-down and immunoprecipitation analyses indicated that Caspr2 was associated with CPE in vitro and in vivo. Both Caspr2 and CPE were expressed predominantly in the CNS. Immunohistochemical analyses revealed that both Caspr2- and CPE-like immunoreactivities were found to co-localize in the apical dendrites and cell bodies of rat cortical neurons. In subcellular localization analysis, Caspr2- and CPE-like immunoreactivities were co-migrated in the fractions of Golgi/ER. Additionally, in COS-7 cells co-transfected with CPE and Caspr2 cDNAs, Caspr2- and CPE-immunoreactivities were co-localized in both Golgi and membrane, whereas it was only observed in Golgi of either COS-7 cell transfected with CPE or Caspr2 cDNA alone. It is known that the membrane-bound form of CPE functions as a sorting receptor of prohormones in the trans-Golgi network. Taken together, our data suggest that CPE may be a key molecule to regulate Caspr2 trafficking to the cell membrane.

The small heat shock protein alphaB-crystallin inhibits differentiation-induced caspase 3 activation and myogenic differentiation.

Myoblasts respond to growth factor deprivation either by diffentiation into multinucleated myotubes or by undergoing apoptosis. The induction of apoptosis and differentiation in myogenic lineage may use overlapping cellular mechanisms. Here we demonstrate that the expression of the small heat shock protein alphaB-crystallin as well as MyoD and myogenin is induced during myogenic differentiation in C2C12 cells, and these inductions occur at an early stage in the differentiation in vitro. To investigate the effect of alphaB-crystallin on myogenic differentiation and apoptosis, C2C12 cells were infected with adenovirus vector bearing full-length alphaB-crystallin cDNA. Overexpression of alphaB-crystallin in C2C12 cells suppressed differentiation-induced apoptosis and activation of caspase 3, and also decreased the expression of MyoD and myogenin during myogenic differentiation of C2C12 cells induced by the differentiation medium. Our findings suggest that stress such as growth factor deprivation plays an important role in triggering apoptosis associated with myogenic differentiation and alphaB-crystallin suppressed the differentiation, apoptosis and caspase 3 activity.

Inhibitory effects of edaravone on the production of tumor necrosis factor-alpha in the isolated heart undergoing ischemia and reperfusion.

We evaluated the effects of edaravone, a hydroxyl radical scavenging agent, on the production of tumor necrosis factor-alpha (TNF-alpha) in myocardium, and the release of TNF-alpha and P-selectin from myocardium after ischemia-reperfusion injury in isolated Langendorff-perfused rat hearts. Cardiodynamic function at stable points during perfusion and 5, 15, 30, and 60 min after the initiation of reperfusion was evaluated by left ventricular developed pressure, rate of increase in left ventricular pressure and rate of decrease in ventricular pressure, coronary flow, and heart rate. At 60 min after the initiation of reperfusion, myocardial infarct size was estimated microscopically using triphenyltetrazolium chloride staining, and expression of TNF-alpha in myocardium was detected by Western blot and immunohistochemistry. At the same time points as the measurement of cardiodynamic function, TNF-alpha and the soluble form of P-selectin in coronary effluent were measured by enzyme immunoassay. At all time points during reperfusion, edaravone markedly improved cardiodynamic function and reduced myocardial infarct size in comparison to the control. In myocardium in the control, TNF-alpha was detected in the endothelial cells and other cells bearing some resemblance to interstitial cells and monocyte cells. Edaravone suppressed this cytokine expression in the corresponding sites. P-selectin as well as TNF-alpha was found in the coronary effluent of the control, and edaravone significantly decreased soluble P-selectin levels in comparison to the control (P < 0.01). Edaravone might have protective effects on cardiac function through reduction of infarct size via decrease of production of TNF-alpha in myocardium induced by ischemia-reperfusion injury and through reduction of the release of adhesion molecules such as P-selectin from vascular endothelial cells.

Effects of atorvastatin and aspirin combined therapy on inflammatory responses in patients undergoing coronary artery bypass grafting.

This study was conducted to compare the effects of atorvastatin plus aspirin combined therapy on inflammatory responses, endothelial cell function, and blood coagulation system in patients undergoing coronary artery bypass grafting (CABG) to aspirin monotherapy. The patients were randomized into atorvastatin plus aspirin combined therapy group and aspirin monotherapy group. Reduced total cholesterol in the combined therapy group was found in a short term of medication for 14 days. On postoperative day (POD)-14, inhibitory effects of the combined therapy on whole blood aggregation as well as platelet activation assessed by flow cytometry were stronger than those of the monotherapy. Furthermore, cytokine, cytokine receptors, c-reactive protein and alpha1-acid glycoprotein in the combined therapy group were down-regulated on POD-14. At the same time, circulating levels of thromboxane A(2), vascular endothelial growth factor and thrombin-antithrombin III complex as well as P-selectin, L-selectin and intercellular adhesion molecule-1 were down-regulated, while E-selectin and transforming growth factor-beta1 was up-regulated. Atorvastatin plus aspirin combined therapy may improve inflammatory responses, accelerated platelet function, vascular endothelial cell function, blood coagulation system at the early stage such as 14th day after CABG. In conclusion, atorvastatin and aspirin combined therapy may bring beneficial effects to the patient after CABG.

Influence of preoperative administration of omega-3 fatty acid-enriched supplement on inflammatory and immune responses in patients undergoing major surgery for cancer.

OBJECTIVE: Polyunsaturated fatty acid supplementation may produce beneficial effects after surgery. We investigated the influence of preoperative administration of a supplement rich in arginine, omega-3 fatty acids, and RNA, Impact (Japan), on inflammatory and immune responses in patients undergoing major surgery for cancer. METHODS: Patients in the supplement group (n = 12) received 1 L/d of Impact (Japan) for 5 d before surgery, and those in the control group (n = 14) received an ordinary diet without Impact (Japan) before surgery. Plasma levels of omega-3 and omega-6 fatty acids, thromboxane B(2), prostaglandin E(2), inflammatory markers, nutritional markers, cytokines, and cytokine receptors were obtained 5 d before the operation at the starting point of supplementation in the supplement group. Samples were collected on postoperative days (PODs) 0, 1, 3, and 7. RESULTS: After taking the supplement, significant increases in omega-3 fatty acids and rapid turnover proteins were found the day after ending supplementation (POD-0), whereas thromboxane B(2) levels and the ratio of omega-6 fatty acids to omega-3 fatty acids were significantly lower than before supplementation (P < 0.001). On POD-0 only, inflammatory markers and cytokine receptors in the supplement group showed low levels in comparison with the control group (P < 0.05). On POD-1 and POD-3, remarkable decreases in polymorphonuclear leukocyte-elastase and interleukin-8 in the supplement group were observed. CONCLUSION: Our findings suggest that oral administration of a supplement rich in omega-3 fatty acids for 5 d before surgery may improve not only preoperative nutritional status but also preoperative and postoperative inflammatory and immune responses in patients who have cancer.

Evaluation of anti-platelet aggregatory effects of aspirin, cilostazol and ramatroban on platelet-rich plasma and whole blood.

To compare property in anti-platelet effects of aspirin (a cyclooxygenase inhibitor), cilostazol (a phosphodiesterase III inhibitor) and ramatroban (a specific thromboxane A2 receptor antagonist), we measured human platelet-rich plasma (PRP) aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid, and whole blood (WB) aggregation induced by ADP. The release of P-selectin, transforming growth factor-beta 1, and the formation of thromboxane A2 in response to agonists were also investigated. Inhibitory effects of 100 micromol/l aspirin, 10 micromol/l cilostazol and 1 micromol/l ramatroban on 5 micromol/l ADP-induced PRP aggregation were similar. However, aspirin strongly inhibited thromboxane A2 formation in response to 5 micromol/l ADP compared with other drugs. Inhibitory effects of 10 micromol/l cilostazol on PRP aggregation and the release of molecules were quite similar in responsiveness induced by the three agonists. Aspirin and cilostazol inhibited platelet aggregation in a concentration-dependent, non-linear fashion, while ramatroban inhibited linearly with increasing concentration. Anti-platelet effects of drugs having different pharmacological mechanisms were demonstrated clearly by measuring PRP aggregation induced by the three agonists, and by measuring WB aggregation that most probably reflects not only platelet-platelet interactions, but also platelet-leukocyte interactions, as well as the release of intraplatelet molecules.