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Hypoxic microenvironment and dysregulated endoplasmic reticulum stress/unfolded protein response (UPR) system are considered important factors that promote cancer progression. Although osthole extracted from Cnidium monnieri(Fructus Cnidii) has been confirmed to exhibit an anticancer activity in various cancers, the effects of osthole in hypoxic colon cancer cells have not been explored. Therefore, the aim of this study was to examine whether osthole has an inhibitory effect on hypoxic colon cancer HCT116 cells and further investigate the underlying molecular mechanisms. Treatment with osthole significantly attenuated the cell viability, proliferation, and migration in hypoxic HCT116 cells. Osthole also activated UPR signaling such as phospho-eukaryotic initiation factor 2 alpha (EIF2[Formula: see text]/ATF4/CHOP/DR5 cascade accompanied by upregulation of pro-apoptotic proteins. Moreover, the tubule-like formation of human umbilical vein endothelial cells, the secretion of vascular endothelial growth factor A, and the expression and activity of hypoxia-inducible factor-1[Formula: see text] (HIF-1[Formula: see text] in hypoxic HCT116 cells were markedly suppressed by osthole. However, suppressing EIF2[Formula: see text] phosphorylation with salubrinal or ISRIB markedly reversed the effects of osthole on the expressions of pro-apoptotic proteins and HIF-1[Formula: see text]. Co-treatment of hypoxic HCT116 cells with osthole greatly increased the sensitivity to cisplatin and the expressions of phospho-EIF2[Formula: see text] and cleaved caspase 3. Collectively, the inhibitory effect of osthole in hypoxic HCT116 cells may be associated with EIF2[Formula: see text] phosphorylation-mediated apoptosis and translational repression of HIF-1[Formula: see text]. Taken together, osthole may be a potential agent in the treatment of colon cancer.

Hypoxic microenvironment and dysregulated endoplasmic reticulum stress/unfolded protein response (UPR) system are considered important factors that promote cancer progression. Although osthole extracted from

Objective In the present study, we investigated the effects of dextromethorphan (DM) and its metabolites, including dextrorphan (LK2), 3-methoxymorphinan (LK3), and 3-hydroxymorphinan (LK4), on platelet aggregation in vitro and the inflammatory pain caused by carrageenan in rats, and their underlying mechanisms. Materials and methods Rabbit platelets were pretreated with DM or its metabolites to assess their effects on platelet aggregation and related target mediators. In addition, the analgesic activity and the underlying mechanisms of DM and LK3 were investigated in a carrageenan-evoked thermal hyperalgesia rat model. Results The inhibitory potency of DM and its metabolites on platelet aggregation induced by arachidonic acid or collagen was LK3> DM > LK4>> LK2 as demonstrated by the half-maximal inhibitory concentration values. Moreover, the mechanisms of the antiplatelet effect of DM and LK3 may involve the inhibition of intracellular calcium mobilization, expression of platelet surface glycoprotein IIb/IIIa, the formation of thromboxane B2, and elevation of platelet membrane fluidity. DM and LK3 also exhibited analgesic effects on carrageenan-evoked thermal hyperalgesia by suppressing the production of pro-inflammatory cytokines, nitric oxide, prostaglandin E2, and neutrophil infiltration in inflammatory sites. Conclusion DM and its metabolites, especially LK3, exhibit both antiplatelet and analgesic effects, and may, therefore, potentially ameliorate platelet hyperactivity and inflammatory-related diseases.

Skeletal muscle atrophy, the most characteristic feature of cancer cachexia, often occurs in patients with cancer undergoing chemotherapy. Antrodia cinnamomea (AC) a widely used edible medical fungus, exhibits hepatoprotective, anti-inflammatory and anticancer activities. In this study, we investigated whether combined treatment with the ethonolic extract of AC ameliorates cachexia symptoms, especially muscle wasting, in lung tumor-bearing mice treated with chemotherapy. Our results revealed that gemcitabine and cisplatin-induced severe body weight loss and skeletal muscle atrophy in the mice with cancer were greatly attenuated after AC extract administration. The protection may be attributed to the inhibition of skeletal muscle proteolysis by suppressing myostatin and activin release, muscle wasting-related FoxO3/MuRF-1/MAFbx signaling, proteasomal enzyme activity, and pro-inflammatory cytokine production. A significant decrease in insulin-like growth factor 1 (IGF-1) expression and formation was observed in the atrophying muscle of the conventional chemotherapy treatment group (CGC), and this decrease was markedly reversed by AC treatment. Additionally, the anorexia, intestinal injury and dysfunction that occurred in the CGC group were mitigated by AC extract. Taken together, these results demonstrated that the AC extract has a protective effect against chemotherapy-induced muscle atrophy mainly by attenuating muscle proteolysis, pro-inflammatory cytokine production, and anorexia, and activating IGF-1-dependent protein synthesis.

Baicalein (BE) extracted from Scutellaria baicalensis Georgi is able to alleviate various cardiovascular and inflammatory diseases. However, the effects of BE on pulmonary arterial hypertension (PAH) remain unknown. Therefore, the present study aimed to examine whether BE ameliorates pneumonectomy and monocrotaline-induced PAH in rats and further investigate the underlying molecular mechanisms. Administration of BE greatly attenuated the development of PAH as evidenced by an improvement of its characteristic features, including elevation of right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Moreover, the increased protein expression of endothelin-1 (ET-1) and ETA receptor (ETAR), superoxide overproduction, and activation of Akt/ERK1/2/GSK3[Formula: see text]/[Formula: see text]-catenin pathway that occurred in the lungs of PAH rats were markedly reversed by BE treatment. Compared with the untreated PAH rats, higher expression of endothelial nitric oxide synthase (eNOS), but lower levels of inducible nitric oxide synthase and vWF were observed in BE-treated PAH rats. Collectively, treatment with BE remarkably attenuates the pathogenesis of PAH, and the protection of BE may be associated with suppressing Akt/Erk1/2/GSK3[Formula: see text]/[Formula: see text]-catenin/ET-1/ETAR signaling and preventing endothelial dysfunction. These results suggest that BE is a potential agent for treatment of PAH.

// Meng-Chuan Chen 1, * , Wen-Lin Hsu 2, 3, * , Pa-An Hwang 4 , Yen-Lin Chen 5 , Tz-Chong Chou 6, 7, 8 1 Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan 2 School of Medicine, Tzu Chi University, Hualien, Taiwan 3 Department of Radiation Oncology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan 4 Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 5 Department of Pathology, Cardinal Tien Hospital, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan 6 Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan 7 Department of Biotechnology, Asia University, Taichung, Taiwan 8 China Medical University Hospital, China Medical University, Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Tz-Chong Chou, email: tcchou48@mail.tcu.edu.tw Keywords: fucoidan, cancer cachexia, muscle atrophy, chemotherapy, inflammation Received: November 11, 2015 Accepted: May 17, 2016 Published: June 13, 2016 ABSTRACT Cancer cachexia is characterized by anorexia, skeletal muscle atrophy, and systemic inflammation. Fucoidan extracted from brown algae exhibits anti-inflammatory and anticancer activities. However, whether fucoidan ameliorates tumour and chemotherapy-induced muscle atrophy and -related cachectic symptoms remains unknown. Compared with mice with bladder cancer treated with chemotherapy alone (TGC group), those treated with a combination of low molecular weight fucoidan (LMWF) and chemotherapy drugs such as gemcitabine and cisplatin (TGCF) showed a significant reduction of body weight loss, muscle atrophy, and intestinal injury and dysfunction. Moreover, myostatin, activin A, and pro-inflammatory cytokine production, FoxO3 expression and activation, NF-κB activation, MuRF-1 and MAFbx/atrogin-1 expression, and proteasome activity in muscle were significantly decreased in the TGCF group compared with the TGC group. In addition, insulin-like growth factor 1 (IGF-1) expression and formation, and IGF-1-regulated mTOR/p70S6k/4EBP-1 protein synthesis signalling were elevated in the TGCF group compared with the TGC group. Taken together, these results suggest that LMWF is a potential agent for preventing cancer cachexia-associated muscle atrophy during chemotherapy. Furthermore, the beneficial effect of LMWF may be attributed to suppressing NF-κB-evoked inflammation, myostatin and activin A production, and subsequent muscle proteolysis, and enhancing IGF-1-dependent protein synthesis.

Fucoidan extracted from brown algae has multiple beneficial functions. In this study, we investigated the effects of low-molecular-weight fucoidan (oligo-FO) on renal fibrosis under in vitro and in vivo diabetic conditions, and its molecular mechanisms. Advanced glycation product (AGE)-stimulated rat renal proximal tubular epithelial cells (NRK-52E) and diabetic mice induced by high-fat diet and intraperitoneal injection of streptozotocin and nicotinamide were used. Oligo-FO treatment significantly inhibited anti-high mobility group box 1 (HMGB1)/RAGE/ anti-nuclear factor-kappa B (NF-&kappa, B)/transforming growth factor-&beta, 1 (TGF-&beta, 1)/TGF-&beta, 1R/Smad 2/3/fibronectin signaling pathway and HIF-1&alpha, activation in AGE-stimulated NRK-52E cells. Conversely, the expression and activity of Sirt-1, the levels of ubiquitin-specific peptidase 22 (USP22), p-AMPK, glucagon-like peptide-1 receptor (GLP-1R), and heme oxygenase-1 (HO-1), and Nrf2 activation were remarkably increased by oligo-FO in AGE-stimulated cells. However, the above effects of oligo-FO were greatly diminished by inhibiting Sirt-1, HO-1, or GLP-1R activity. Similar changes of these pro-fibrotic genes in the kidney and a marked attenuation of renal injury and dysfunction were observed in oligo-FO-treated diabetic mice. These findings indicated that the inhibitory effects of the oligo-FO on diabetes-evoked renal fibrosis are mediated by suppressing TGF-&beta, 1-activated pro-fibrogenic processes via Sirt-1, HO-1, and GLP-1R dependence. Collectively, fucoidan-containing foods or supplements may be potential agents for ameliorating renal diseases due to excessive fibrosis.

Acute lung injury (ALI) has a high morbidity and mortality rate due to the serious inflammation and edema occurred in lung. Magnolol extracted from Magnolia officinalis, has been reported to exhibit anti-inflammatory, and antioxidant activities. Peroxisome proliferator-activated receptors (PPARs) are known to exert a cytoprotective effect against cellular inflammatory stress and oxidative injury. The aim of this study was to explore the involvement of PPAR-γ in the beneficial effect of magnolol in lipopolysaccharide (LPS)-induced ALI. We found that treatment with magnolol greatly improved the pathological features of ALI evidenced by reduction of lung edema, polymorphonuclear neutrophil infiltration, ROS production, the levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid (BALF), the expression of iNOS and COX-2, and NF-κB activation in lungs exposed to LPS. Importantly, magnolol is capable of increasing the PPAR-γ expression and activity in lungs of ALI. However, blocking PPAR-γ activity with GW9662 markedly abolished the protective and anti-inflammatory effects of magnolol. Taken together, the present study provides a novel mechanism accounting for the protective effect of magnolol in LPS-induced ALI is at least partly attributed to induction of PPAR-γ in lungs, and in turn suppressing NF-κB-related inflammatory responses.

Melaleuca alternifolia concentrate (MAC) is a mixture predominantly composed of monoterpenoids and sesquiterpenes, refined from the essential oil of the tea tree by removing up to 99% of the more toxic, hydrophobic monoterpenes. MAC was examined here for its immunomodulatory effects on the human THP1 and murine RAW264.7 myeloid leukemic cell lines as models for macrophage-like cells. Firstly, MAC levels were determined that did not affect either the survival or proliferation of these cell lines in vitro. Next, the levels of lipopolysaccharide (LPS)-induced production of cytokines (IL-6, TNFα, IL-10, GM-CSF, IFNγ and IL-3) were examined from the myeloid cell lines using multiplex assays. Many of the LPS-inducible cytokines produced by either cell lines could be significantly inhibited by MAC. Closer examination of the mechanism of action of MAC showed that it inhibited the LPS-induced activation of IκB phosphorylation and nuclear factor (NF)-κB signalling and translocation, inhibiting iNOS protein expression and NO production. These results demonstrate that MAC exerts its immunomodulatory effects by inhibiting NF-κB signalling activation and levels of cytokine production by macrophage-like cell lines.

Magnolol (1) isolated from Magnolia officinalis exhibits many beneficial effects such as anti-inflammatory and antioxidant activity. The aim of this study was to evaluate the effects of magnolol (1) on RANKL-induced osteoclast differentiation and investigate the underlying molecular mechanisms. Treatment with magnolol (1) significantly inhibited osteoclast differentiation of RAW 264.7 macrophages and bone-resorbing activity of osteoclasts in the RANKL-induced system. Moreover, RANKL-activated JNK/ERK/AP-1 and NF-κB signaling, ROS formation, and NFATc1 activation were attenuated by magnolol (1). A novel finding of this study is that magnolol (1) can increase heme oxygenase-1 (HO-1) expression and Nrf2 activation in RANKL-stimulated cells. Blocking HO-1 activity with tin protoporphyrin IX markedly reversed magnolol (1)-mediated inhibition of osteoclast differentiation, NFATc1 nuclear translocation, and MMP-9 activity, suggesting that HO-1 contributes to the attenuation of NFATc1-mediated osteoclastogenesis by magnolol (1). Therefore, the inhibitory effect of magnolol (1) on osteoclast differentiation is due to inhibition of MAPK/c-fos/AP-1 and NF-κB signaling as well as ROS production and up-regulation of HO-1 expression, which ultimately suppresses NFATc1 induction. These findings indicate that magnolol (1) may have potential to treat bone diseases associated with excessive osteoclastogenesis.

Background: Magnolol, a major bioactive component extracted from Magnolia officinalis, exerts several beneficial effects, such as anti-inflammatory and anti-hypertensive activities. Purpose: In this study, we investigated whether magnolol has a protective effect on pneumonectomy and monocrotaline-induced pulmonary arterial hypertension (PAH) in rats. Design/Methods: The alterations of right ventricular (RV) hypertrophy, pulmonary vascular remodeling, histopathological parameters, and related gene expression and signaling pathways in lungs by magnolol treatment were studied in the PAH rats. Results: Administration of magnolol greatly ameliorated the characteristic features of PAH, including increased pulmonary arterial pressure, RV hypertrophy, and pulmonary vascular remodeling. Moreover, magnolol inhibited angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 receptor (AT-1R) cascade, whereas upregulates ACE2 in the lungs of PAH rats. The overexpression of endothelin-1 (ET-1) and ETA receptor occurred in the PAH rats was significantly attenuated by magnolol through inhibition of Akt/ERK1/2/GSK3β/β-catenin pathway. Compared with that of untreated PAH rats, higher expression of endothelial nitric oxide synthase, and lower expression of inducible nitric oxide synthase and O2− production in lungs were observed in magnolol-treated PAH rats. Conclusion: We demonstrated that treatment with magnolol reduces the development of PAH induced by pneumonectomy and monocrotaline in rats, and suppressing Ang II and ET-1-mediated processes may contribute to its protective effects. These findings suggest that magnolol may be a potential agent for PAH therapy.

Platelet hyperactivity plays a critical role for initiating several vascular diseases such as atherothrombosis. Therefore, development of effective antiplatelet agents is necessary for ameliorating platelet-related diseases. In this study, we investigated the effects of the new synthesized compound, MP407 on platelet aggregation and further elucidated the underlying mechanisms. Our results demonstrated that MP407 dose-dependently inhibited collagen-induced platelet aggregation, thromboxane B2 (TXB2) production, intracellular Ca2+ mobilization, platelet membrane GPIIb/IIIa expression, and the phosphorylation of Akt, GSK3β, p38MAPK, and phospho (Ser) PKC substrate (p47). Moreover, MP407 is able to increase the cyclic AMP formation both in resting and activated platelets. However, blocking cyclic AMP formation with 2'5'-ddAdo, an inhibitor of adenylate cyclase, greatly reversed the antiplatelet activity of MP407 and related platelet-activating pathways. MP407 also enhanced VASP phosphorylation at Ser157 in collagen-stimulated platelets, which was attenuated by addition of 2'5'-ddAdo. Therefore, the antiplatelet activity of MP407 may be modulated by cyclic AMP-dependent regulation of Akt, GSK3β, p38MAPK and VASP phosphorylation. Notably, treatment with MP407 markedly reduced the pulmonary thrombosis and the numbers of paralysis and death in mice induced by ADP injection, but did not affect the bleeding time. Taken together, MP407 may be a potential candidate or lead compound for developing novel antiplatelet or antithrombotic agents for platelet hyperactivity-triggered disease therapy.

Baicalein (BE), a phenolic flavonoid extracted mainly from the root of Scutellaria baicalensis Georgi, a Chinese herb, is traditionally used in oriental medicine. Several studies have demonstrated that BE exerts many beneficial effects including anti-inflammatory and antioxidant activities. However, its effect on acute lung injury (ALI) and the molecular mechanisms involved remain unclear and warrant further investigation. The aim of the study is to investigate whether BE improves lipopolysaccharide (LPS, intratracheally, i.t.)-induced ALI in rats, and further study the underlying mechanisms of its activity.Rats were administrated with LPS (5mg/kg/body weight, i.t.) through a 24-gauge catheter to establish the ALI model. The effects of BE on the levels of pro-inflammatory cytokines, nitrite/nitrate in bronchoalveolar lavage fluid, and the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nuclear factor-kappa B (NF-κB) activation as well as the histopathological changes were evaluated.Results showed that BE (20mg/kg, i.p.) treatment markedly attenuated LPS-induced lung edema, elevation of the levels of IL-1β, TNF-α, IL-6, CINC-3, and nitrite/nitrate in bronchoalveolar lavage fluid accompanied by a remarkable improvement of lung histopathological symptoms. The LPS-enhanced inflammatory cell infiltration and myeloperoxidase activity, O2(-) formation and the expression of inducible nitric oxide synthase and nitrotyrosin in lungs were all attenuated by BE. Notably, BE could augment Nrf2/HO-1 cascade, but inhibited NF-κB activation in LPS-instilled lungs that was strongly reversed by blocking HO-1 activity.This study is the first to demonstrate that BE protects against LPS-induced ALI in rats. The underlying mechanisms may include inhibition of NF-κB-mediated inflammatory responses and upregulation of Nrf2/HO-1 pathway, which ultimately alleviates the pathological symptoms of ALI.

Background and PurposeThe transcription factor NF-κB, stimulates platelet aggregation through a non-genomic mechanism. Nifedipine, a voltage-gated L-type calcium channel blocker, is widely used to treat hypertension. Nifedipine also displays antiplatelet activity, but the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiplatelet effects of nifedipine are mediated by regulating NF-κB-dependent responses. Experimental ApproachPlatelet aggregation was measured turbidimetrically using an aggregometer. NF-κB and PPAR activation, intracellular Ca2+ mobilization, PKCα activity, surface glycoprotein IIb/IIIa (GPIIb/IIIa) expression and platelet activation-related signalling pathways were determined in control and nifedipine-treated platelets in the presence or absence of PPAR antagonists or betulinic acid, a NF-κB activator. Key ResultsExposure of platelets to nifedipine significantly increased the PPAR-β/-γ activity in activated human platelets. Treatment with nifedipine reduced collagen-induced NF-κB events, including the phosphorylation of IκB kinase-β, IκBα and p65NF-κB, which were markedly attenuated by GSK0660, a PPAR-β antagonist, or GW9662, a PPAR-γ antagonist. Furthermore, the interaction of PPAR-β/-γ with NF-κB and the PPAR-β/-γ-up-regulated NO/cGMP/PKG1 cascade may contribute to inhibition of NF-κB activation by nifedipine. Suppressing PPAR-β/-γ activity or increasing NF-κB activation greatly reversed the inhibitory effect of nifedipine on collagen-induced platelet aggregation, intracellular Ca2+ mobilization, PKCα activity and surface GPIIb/IIIa expression. Conclusions and ImplicationsPPAR-β/-γ-dependent inhibition of NF-κB activation contributes to the antiplatelet activity of nifedipine. These findings provide a novel mechanism underlying the beneficial effects of nifedipine on platelet hyperactivity-related vascular and inflammatory diseases.

Gametogenesis is a complex process wherein germ cells develop from primordial diploid cells into haploid gametes. To understand the mechanisms controlling gametogenesis, we identified a novel germ-cell-specific gene, Gcse. Gcse produces two major transcripts that are 1589 bp (Gcse-l) and 906 bp (Gcse-s) in length. Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) analyses of multiple tissues reveal that Gcse-l is expressed in both adult testes and ovaries, but Gcse-s is expressed only in adult testes. During female gonad development, Gcse-l is expressed from embryonic day 13.5 to adulthood, specifically in oocytes, and maintained in ovulated and fertilized eggs. However, Gcse-s signals were detected only in ovulated oocytes and fertilized eggs but not in adult ovary. During male gonad development, strong Gcse-l signals were detected in late pachytene spermatocytes and round spermatids. However, Gcse-s transcripts exist only in round spermatids. Furthermore, the expression of GCSE-L proteins and their subcellular localizations within cells are stage specific. GCSE-L is detected in the nucleus of late pachytene spermatocytes. During meiosis, GCSE-L is translocated to acrosome regions in spermatids and maintained in the acrosome of spermatozoa. GCSE-L colocalizes with acrosin and lectin peanut agglutinin in the Golgi apparatus. However, GCSE-S proteins are expressed only in the nucleus of spermatids. From these results, we suggest that GCSE proteins play roles in meiosis and may be involved in acrosome biogenesis during spermiogenesis.

The use of an electrospun polycaprolactone (PCL) nonwoven mat that is coated with a layer of chitosan (CS) containing active ingredient [tea tree oil (TTO)] represents an effective strategy for producing functional dressings. CS-coated porous PCL nonwoven mat (CS3/PCLNM) with various concentrations of active ingredients were produced and investigated. In vitro, active ingredient-containing CS3/PCLNM is effective in inhibiting the formation of nitrite and the growth of Staphylococcus aureus. Both active ingredient TTO and CS have been proven to reach their maximum amount of releases within 24 h of contact with water-based environment. In vivo, full-thickness skin removal (1.2 cm × 1.2 cm) was performed on the back of the C57BL6/J mice in noninfected and infected animal models. Four groups of functional dressings were tested in this work including Tegderm™, PCLNM, CS3/PCLNM, and 100 μL TTO-CS3/PCLNM. After 7 days post-treatment, the bacterial levels were found to be significantly lower in both CS3/PCLNM and 100 μL TTO-CS3/PCLNM-treated groups than in the control group (81.6 ± 18.1% and 93.7 ± 9.57% of reductions in the bacterial load in the pus relative to the control group, respectively). Additionally, based on the histological analyses, the 100 μL TTO-CS3/PCLNM-treated group outperformed all other groups in wound healing.

The accumulation of advanced glycation end products (AGEs) in diabetic patients induces vascular endothelial injury. Promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor that can be activated by low-temperature far-infrared (FIR) irradiation to exert beneficial effects on the vascular endothelium. In the present study, we investigated the influence of FIR-induced PLZF activation on AGE-induced endothelial injury both in vitro and in vivo. FIR irradiation inhibited AGE-induced apoptosis in human umbilical vein endothelial cells (HUVECs). PLZF activation increased the expression of phosphatidylinositol-3 kinases (PI3K), which are important kinases in the autophagic signaling pathway. FIR-induced PLZF activation led to autophagy in HUVEC, which was mediated through the upregulation of PI3K. Immunofluorescence staining showed that AGEs were engulfed by HUVECs and localized to lysosomes. FIR-induced autophagy promoted AGEs degradation in HUVECs. In nicotinamide/streptozotocin-induced diabetic mice, FIR therapy reduced serum AGEs and AGEs deposition at the vascular endothelium. FIR therapy also reduced diabetes-induced inflammatory markers in the vascular endothelium and improved vascular endothelial function. These protective effects of FIR therapy were not found in PLZF-knockout mice. Our data suggest that FIR-induced PLZF activation in vascular endothelial cells protects the vascular endothelium in diabetic mice from AGE-induced injury.

This study demonstrates a facile method developed to generate a chitosan/polycaprolactone (CS/PCL) nonwoven mat. All nonwoven mats are composed of microfibers with an average diameter of 2.51 ± 0.69 μm. The X-ray photoelectron spectroscopy data indicate that positively charged nitrogen was generated on the surface of the mats after undergoing CS coating. By using a non-contacting electrostatic voltmeter, we determined that the nonwoven mats exhibited a positive potential and the charge density of the CS/PCL nonwoven mat was in proportion to the thickness of the CS overlayer. Moreover, platelet aggregation and anti-bacterial ability were enhanced by the CS/PCL nonwoven mat as compared to that of PCL nonwoven mat alone. The enhancements of the CS/PCL nonwoven mat on platelet aggregation are further promoted by incorporating a 1 mM calcium ion in its CS overlayer. We also find that the addition of tea tree oil in the CS overlayer significantly inhibited LPS-induced nitrite formation in Raw 264.7 macrophages. In conclusion, our CS/PCL nonwoven mat possesses pharmacological effects including an increase of platelet aggregation, anti-bacterial, anti-adhesive, and anti-inflammatory activities. The performance of this CS/PCL nonwoven mat can be further promoted by incorporating active compounds to exert therapeutic effects in wound healing.

Magnolol has shown inhibitory effects on NO production and TNF-alpha production in lipopolysaccharide (LPS)-activated macrophages and LPS-induced acute lung injury; however, the poor solubility of magnolol has hindered its clinical success. In this study, magnolol-loaded microparticles were prepared via single emulsion method from a polyketal polymer, termed PK3. The particle sizes of magnolol-loaded PK3 microparticle is 3.73 ± 0.41 μm, and was suitable for phagocytosis by macrophages and pulmonary drug delivery. PK3 microparticles exhibited excellent biocompatibility both in vitro and in vivo. More importantly, intratracheal delivery of these magnolol-loaded microparticles significantly reduced the lung inflammatory responses at low dosage of magnolol (0.5 mg/kg), and have great clinical potential in treating acute lung injury.

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, β/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.

Alpha-lipoic acid (ALA) is often used as a dietary supplement to prevent and treat chronic diseases associated with excessive oxidative stress. The aim of this study was to investigate the mechanisms of the antiplatelet activity of ALA. ALA significantly inhibited collagen-induced platelet aggregation, thromboxane B(2) (TXB(2)) formation, Ca(2+) mobilization, and protein kinase Calpha (PKCalpha) activation, but ALA itself increased cyclic AMP formation in rabbit washed platelets. However, the effects of ALA on the above platelet responses were markedly reversed by the addition of 2'5'-ddAdo, an adenylate cyclase inhibitor. Additionally, increased reactive oxygen species (ROS) formation and cyclooxygenase-1 activity stimulated by arachidonic acid were inhibited by ALA. In conclusion, we demonstrated that ALA possesses an antiplatelet activity, which may be associated with an elevation of cyclic AMP formation, involving subsequent inhibition of TXA(2), Ca(2+) mobilization, and PKCalpha-mediated pathways. Moreover, inhibition of ROS formation and increase of platelet membrane fluidity may also involve its actions.

Partial liquid ventilation with various types of perfluorocarbon (PFC) has been shown to be beneficial in treating acute lung injury, a clinical outcome that may involve the anti-inflammatory activity of PFC. FC-77 is a type of PFC with relatively higher vapor pressure and evaporative loss than other PFCs during partial liquid ventilation. Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been proposed to play a crucial role in the pathogenesis of inflammatory diseases. However, whether the iNOS/NO pathway is affected by FC-77 is unknown. Thus, the aim of this study was to investigate whether FC-77 inhibits iNOS expression and NO production in lipopolysaccharide (LPS)–treated RAW 264.7 macrophages. We found that treatment with FC-77 significantly attenuated LPS-induced iNOS expression/activity and production of NO and reactive oxygen species (ROS). FC-77 also attenuated LPS-induced pro-inflammatory cytokine formation, but enhanced interleukin-10 production. Furthermore, the LPS-induced degradation of cytosolic IκB-α and activation of nuclear transcription factor-κB (NF-κB) were also inhibited by FC-77. In conclusion, the present study is the first to demonstrate that FC-77 decreases LPS-induced NO production in macrophages, which may be associated with the suppression of pro-inflammatory cytokines, and ROS production, as well as NF-κB activation. These results also provide a novel explanation for its anti-inflammatory activity. Keywords:: perfluorocarbon, nitric oxide, lipopolysaccharide, cytokine, macrophage

Background and purpose: It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin. Experimental approach: After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately. Key results: Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC50 range of 52–158 μM. We also demonstrated that simvastatin (20–80 μM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64±0.12 to 3.52±0.21–5.10±0.14 μmol min−1 mg protein−1) and guanylyl cyclase activity (from 142.9±7.2 to 163.5±17.5–283.8±19.5 pmol min−1 mg protein−1) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase. Conclusions and implications: Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin. British Journal of Pharmacology (2008) 153, 1281–1287; doi:10.1038/bjp.2008.19; published online 11 February 2008

C-phycocyanin (C-PC), found in blue green algae, is often used as a dietary nutritional supplement. C-PC has been found to have an anti-inflammatory activity and exert beneficial effect in various diseases. However, little is known about its mechanism of action. Overproduction of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of inflammation. The aim of this study was to determine whether C-PC inhibits production of nitrite, an index of NO, and iNOS expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results indicated that C-PC significantly inhibited the LPS-induced nitrite production and iNOS protein expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-alpha) formation but had no effect on interleukin-10 production in macrophages. Furthermore, C-PC also suppressed the activation of nuclear factor-kappaB (NF-kappaB) through preventing degradation of cytosolic IkappaB-alpha in LPS-stimulated RAW 264.7 macrophages. Thus, the inhibitory activity of C-PC on LPS-induced NO release and iNOS expression is probably associated with suppressing TNF-alpha formation and nuclear NF-kappaB activation, which may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.

Skeletal muscle atrophy, the most prominent phenotypic feature of cancer cachexia, is often observed in cancer patients undergoing chemotherapy. Magnolol (M) extracted from Magnolia officinalis exhibits several pharmacological effects including anti-inflammatory and anticancer activities. In this study, we investigated whether magnolol supplementation protects against the development of cachexia symptoms in bladder cancer-bearing mice undergoing chemotherapy. Combined treatment of magnolol with chemotherapeutic drugs, such as gemcitabine and cisplatin (TGCM) or gemcitabine (TGM), markedly attenuates the body weight loss and skeletal muscle atrophy compared with conventional chemotherapy (TGC). The antiatrophic effect of magnolol may be associated with inhibition of myostatin and activin A formation, as well as FoxO3 transcriptional activity resulting from Akt activation, thereby suppressing ubiquitin ligases MuRF-1 and MAFbx/atrogin-1 expression, as well as proteasomal enzyme activity. Notably, magnolol-induced insulin-like growth factor 1 (IGF-1) production and related protein synthesis may also contribute to its protective effects. The decreased food intake, and intestinal injury and dysfunction observed in the mice of TGC group were significantly improved in the TGCM and TGM groups. Moreover, the increased inflammatory responses evidenced by elevation of proinflammatory cytokine formation and NF-κB activation occurred in the atrophying muscle of TGC group were markedly inhibited in mice of combined treatment with magnolol. In summary, these findings support that magnolol is a promising chemopreventive supplement for preventing chemotherapy-induced skeletal muscle atrophy associated with cancer cachexia by suppressing muscle protein degradation, and inflammatory responses, as well as increasing IGF-1-mediated protein synthesis.

Magnolol isolated from Magnolia officinalis, a Chinese medical herb, exhibits an anti-inflammatory activity and a protective effect against periodontitis. The inflammation caused by lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) has been considered a key inducer in the development of periodontitis. In this study, we investigated whether magnolol inhibits P. gingivalis LPS-evoked inflammatory responses in RAW 264.7 macrophages and the involvement of heme oxygenase-1 (HO-1). Magnolol significantly activated p38 MAPK, Nrf-2/HO-1 cascade and reactive oxygen species (ROS) formation. Notably, the Nrf-2 activation and HO-1 induction by magnolol were greatly diminished by blocking p38 MAPK activity and ROS production. Furthermore, in P. gingivalis LPS-stimulated macrophages, magnolol treatment remarkably inhibited the inflammatory responses evidenced by suppression of pro-inflammatory cytokine, prostaglandin E2, nitrite formation, and the expression of inducible nitric oxide synthase and cyclooxygenase-2, as well as NF-κB activation accompanied by a significant elevation of Nrf-2 nuclear translocation and HO-1 expression/activity. However, inhibiting HO-1 activity with tin protoporphyrin IX markedly reversed the anti-inflammatory effects of magnolol. Collectively, these findings provide a novel mechanism by which magnolol inhibits P. gingivalis LPS-induced inflammation in macrophages is at least partly mediated by HO-1 activation, and thereby promoting its clinical use in periodontitis.

C-phycocyanin (cpc), a biliprotein isolated fromSpirulina platensis, has been reported to exert many therapeutic and nutritional values. In the present study, we examined whether cpc has an antiplatelet activityin vitroand further investigated the possible anti-aggregatory mechanisms involved. Our results showed that preincubation of cpc (1–50μg/ml) with rabbit washed platelets dose-dependently inhibited the platelet aggregation induced by collagen (10μg/ml) or arachidonic acid (100μm), with an ic50of about 10μg/ml. Furthermore, the thromboxane B2formation caused by collagen or arachidonic acid was significantly inhibited by cpc due to suppression of cyclooxygenase and thromboxane synthase activity. Similarly, the rise of platelet intracellular calcium level stimulated by arachidonic acid and collagen-induced platelet membrane surface glycoprotein IIb/IIIa expression were also attenuated by cpc. In addition, cpc itself significantly increased the platelet membrane fluidity and the cyclic AMP level through inhibiting cyclic AMP phosphodiesterase activity. These findings strongly demonstrate that cpc is an inhibitor of platelet aggregation, which may be associated with mechanisms including inhibition of thromboxane A2formation, intracellular calcium mobilization and platelet surface glycoprotein IIb/IIIa expression accompanied by increasing cyclic AMP formation and platelet membrane fluidity.

Background: The role of nitric oxide (NO) in the pathogenesis of cyclosporin A (CsA)-induced gingival overgrowth is still unknown. The purpose of this study was to evaluate the effect of CsA on the expression of nitric oxide synthases (NOS) in the gingival tissue of rats. Methods: Thirty male Sprague-Dawley rats were randomly assigned to a control and two test groups. Rats in each group received CsA (0, 10, or 30 mg/kg) daily by gastric feeding for 4 weeks. The plasma NO and the NOS enzyme activities were assayed at week 4 in the blood samples and in the gingiva and lung tissue specimens, respectively. The distribution of inducible nitric oxide synthase (iNOS) was further evaluated in tissues obtained from the gingiva and lung at the end of weeks 1 and 4 by immunohistochemistry. Results: In the CsA-treated animals, increased levels of plasma nitrites/nitrates were measured in comparison to those in control rats. Significantly greater iNOS enzyme activities were detected in lung and gingival tissues obtained from CsA-treated animals than from control animals. In addition, cells positively staining for iNOS were clearly observed in both gingival and lung tissues obtained from the CsA-treated animals by immunohistochemistry, whereas a few stained cells were found in those from the control group. The quantity of cells positively stained for iNOS was greater in tissue from week 4 than week 1. Conclusions: The effect of CsA on gingival iNOS expression was evaluated in rats for 4 weeks. A greater iNOS expression in the gingiva was observed after CsA therapy by both enzyme activities and immunohistochemica staining. Therefore, we suggest that CsA can increase gingival iNOS expression, which may play an important role in cyclosporin-induced gingival overgrowth.

This study tested whether FC-77, a perfluorochemical, inhibits inflammatory responses in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. The possible anti-inflammatory mechanisms involved were also investigated.RAW 264.7 macrophages were simultaneously incubated with pure FC-77 at final volume of 10% or 30% (v/v) and LPS (1 microg/ml) for 12 or 24 h on a mechanical shaker. In some tests FC-77 was added after cells stimulated by LPS for 12 h. Then the levels of prostaglandin E2(PGE2), tumor necrosis factor alpha (TNF-alpha), and interleukins (IL)-1beta, -6), and -10 were measured in medium. Alterations in cyclooxygenase (COX) 2 expression and nuclear transcription factor (NF) kappaB activation in cells were also evaluated.Pre- or posttreatment with FC-77 dose-dependently reduced the LPS-induced TNF-alpha, IL-1beta, and IL-6 formation and enhanced IL-10 production compared to LPS-stimulated alone cells. FC-77 also attenuated the LPS-induced PGE2 formation accompanied by suppressing COX-2 expression, the degradation of cytosolic inhibitor of kappaB-alpha and NF-kappaB activation.FC-77 inhibits inflammatory responses in LPS-stimulated macrophages by a mechanism that involves the attenuation of NF-kappaB dependent induction of COX-2/PGE2 pathway and the pro- /anti-inflammatory cytokine ratio.

The direct acylation of trimethoxyphenol (1) with substituted cinnamoyl chlorides followed by Fries rearrangement and cyclization afforded a practical route for the synthesis of novel baicalein derivatives 4 functionalized on the B-ring in good overall yields. In the methylthiazoletetrazolium bromide (MTT) assay, none of the synthetic polyhydroxyflavonoids were cytotoxic at concentrations up to 200 μm on lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages over 24 h, while in the same cells they significantly inhibited NO production. Among the derivatives, 4d (IC50 = 46.1 ± 0.3 μm) was found to exhibit the most potent activity compared with N-nitro-L-arginine methyl ester (L-NAME, IC50 > 300 μm). Compounds 4b, 4e, 4f, 4h and 4i remarkably inhibited platelet aggregation induced by arachidonic acid and collagen in rabbit washed platelets compared with aspirin. Analysis of their structure-activity relationships indicated that, in the structural modification on the B-ring of baicalein (4a), introduction of appropriate electro-withdrawing substituents such as 2-CI (4b), 4-CI (4d), and 4-phenyl (4i) notably increased the potency on the inhibition of LPS-activated NO production and arachidonic acid- and collagen-induced aggregation. Baicalein itself was equally effective in the inhibition of LPS-activated NO production and collagen-induced aggregation but less active against arachidonic acid-induced aggregation. Our in-vitro results suggested that by appropriate structural modification of baicalein it may be possible to develop novel therapeutic agents against platelet-aggregation and inflammation.

UNLABELLED We tested baicalin for its antiinflammatory and analgesic effects (and the mechanisms) in a rat model of carrageenan-evoked thermal hyperalgesia. Pre- or posttreatment with baicalin (10, 30, or 100 mg/kg intraperitoneally) caused a significant analgesic effect with a similar effect of dose-matched ibuprofen. Furthermore, baicalin dose-dependently attenuated tumor necrosis factor-alpha (from 3510 +/- 150 pg/mL to 2860 +/- 148 pg/mL to 1480 +/- 210 pg/mL), interleukin (IL)-1beta (from 3210 +/- 210 pg/mL to 2200 +/- 140 pg/mL to 750 +/- 95 pg/mL), and IL-6 (from 58.5 +/- 9.8 pg/mL to 38.5 +/- 9.0 to 21.0 +/- 8.1 ng/mL) formation but enhanced IL-10 (from 18.1 +/- 2.5 pg/mL to 36.1 +/- 5.5 pg/mL to 71.2 +/- 9.5 pg/mL) production in paw exudates at 4 h after carrageenan injection. Prostaglandin E(2) (PGE(2)) and nitrate formation in the carrageenan-injected paws were dose-dependently inhibited by baicalin (10-100 mg/kg intraperitoneally) (PGE(2): from 15.9 +/- 2.1 ng/mL to 12.1 +/- 1.6 ng/mL to 6.2 +/- 1.8 ng/mL; nitrate: from 39.8 +/- 4.8 microM to 27.5 +/- 3.0 microM to 17.2 +/- 1.6 microM) at 4 h but not at 1.5 h after carrageenan injection. Increased myeloperoxidase activity in carrageenan-injected paws was also dose-dependently reduced by baicalin. These findings suggest that the antiinflammatory and analgesic mechanisms of baicalin may be associated with the inhibition of critical inflammatory mediators, including nitric oxide, PGE(2), and proinflammatory cytokines, accompanied by an increase in IL-10 production, as well as neutrophil infiltration at sites of inflammation. IMPLICATIONS Our results showed that baicalin possesses an analgesic effect in carrageenan-evoked thermal hyperalgesia. The possible mechanisms of action of baicalin may be associated with the inhibition of proinflammatory mediator overproduction, including cytokines, nitric oxide, and prostaglandin E(2), as well as neutrophil infiltration. This implies that baicalin may be a potential therapeutic analgesic for inflammatory pain.

1. Paeonol was tested for its anti-inflammatory and analgesic effects in a rat model of carrageenan-evoked thermal hyperalgesia. The possible mechanisms involved in these effects were also investigated. 2. Pre- and post-treatment with paeonol (30, 50 or 100 mg kg(-1), i.p.) dose-dependently inhibited the carrageenan-evoked thermal hyperalgesia. 3. Treatment with paeonol dose-dependently inhibited tumour necrosis factor-alpha (TNF-alpha) and interleukin-lbeta (IL-1beta) formation, but enhanced IL-10 production in the rat paw exudates both at the early (1.5 h) and late phase (4 h) after carrageenan injection. However, inhibition of IL-6 formation by paeonol was only observed at the late phase. 4. Paeonol dose-dependently decreased the formation of prostaglandin E(2) (PGE(2)) in rat paw exudates with a greater inhibition at the late phase. However, inhibition of nitrate generation was observed only during the late phase (at 4 h after carrageenan injection), accompanied by an attenuation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in paw tissue. 5. Elevated myeloperoxidase activity, an indicator of neutrophil infiltration, in carrageenan-injected paws was also dose-dependently reduced in paeonol-treated rats. 6. Our results suggest that the mechanisms by which paeonol exerts its anti-inflammatory and analgesic effects in this inflammatory model may be associated with decreased production of proinflammatory cytokines, NO and PGE(2) and increased production of IL-10, an anti-inflammatory cytokine, in carrageenan-injected rat paws. In addition, attenuation of the elevated iNOS and COX-2 protein expression as well as neutrophil infiltration in carrageenan-injected paws may also be involved in the beneficial effects of paeonol.

cope Several beneficial biological functions of fucoidan (FO) isolated from brown algae have been demonstrated. The purpose of this study was to investigate whether FO derived from Sargassum hemiphyllum ameliorates pancreatic β-cell damage and impaired insulin synthesis under diabetic condition. Methods and results The effects of FO were studied in streptozotocin (STZ)-treated pancreatic β-cell line, NIT-1cells, and mice. The cell apoptosis, protein analyses, histological examination, and pancreatic function assays were performed. The increased pancreatic β-cell apoptosis and decreased insulin secretion observed in STZ-treated NIT-1 cells and mice were greatly attenuated by FO. Moreover, FO has an ability to enhance glucagon-like peptide-1 receptor (GLP-1R) and sirtuin 1 (Sirt-1) activity through activation of AMPK/GAPDH/PDX-1 cascade in STZ-treated β cells. However, the effects of FO were significantly reversed by EX527, a specific Sirt-1 inhibitor. Similarly, the hyperglycemia, lower expression of Sirt-1, PDX-1, and GLP-1R in the pancreas of diabetic mice were markedly improved after FO administration. Conclusion We demonstrated that FO exhibits an anti-diabetic effect mainly through attenuation of β-cell death, thereby elevating insulin synthesis by upregulating PDX-1 and GLP1-R via a Sirt-1-dependent manner. Therefore, FO-containing food or supplements may have a therapeutic effect for diabetes by preventing β-cell damage and dysfunction. This article is protected by copyright. All rights reserved

Platelet hyperactivity often occursd in hypertensive patients and is a key factor in the development of cardiovascular diseases including thrombosis and atherosclerosis. Nifedipine, an L-type calcium channel blocker, is widely used for hypertension and coronary heart disease therapy. In addition, nifedipine is known to exhibit an antiplatelet activity, but the underlying mechanisms involved remain unclear. Several transcription factors such as peroxisome proliferator-activated receptors (PPARs) and nuclear factor kappa B (NF-κB) exist in platelets and have an ability to regulate platelet aggregation through a non-genomic mechanism. The present article focuses on describing the mechanisms of the antiplatelet activity of nifedipine via PPAR activation. It has been demonstrated that nifedipine treatment increases the activity and intracellular amount of PPAR-β/-γ in activated platelets. Moreover, the antiplatelet activity of nifedipine is mediated by PPAR-β/-γ-dependent upon the up-regulation of the PI3K/AKT/NO/cyclic GMP/PKG pathway, and inhibition of protein kinase Cα (PKCα) activity via an interaction between PPAR-β/-γ and PKCα. Furthermore, suppressing NF-κB activation by nifedipine through enhanced association of PPAR-β/-γ with NF-κB has also been observed in collagen-stimulated platelets. Blocking PPAR-β/-γ activity or increasing NF-κB activation greatly reverses the antiplatelet activity and inhibition of intracellular Ca2+ mobilization, PKCα activity, and surface glycoprotein IIb/IIIa expression caused by nifedipine. Thus, PPAR-β/-γ- dependent suppression of NF-κB activation also contributes to the antiplatelet activity of nifedipine. Consistently, administration of nifedipine markedly reduces fluorescein sodium-induced vessel thrombus formation in mice, which is considerably inhibited when the PPAR-β/-γ antagonists are administrated simultaneously. Collectively, these results provide important information regarding the mechanism by which nifedipine inhibits platelet aggregation and thrombus formation through activation of PPAR-β/-γ- mediated signaling pathways. These findings highlight that PPARs are novel therapeutic targets for preventing and treating platelet-hyperactivity-related vascular diseases.

The transcription factor NF-κB, stimulates platelet aggregation through a non-genomic mechanism. Nifedipine, a voltage-gated L-type calcium channel blocker, is widely used to treat hypertension. Nifedipine also displays antiplatelet activity, but the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiplatelet effects of nifedipine are mediated by regulating NF-κB-dependent responses.Platelet aggregation was measured turbidimetrically using an aggregometer. NF-κB and PPAR activation, intracellular Ca2+ mobilization, PKCα activity, surface glycoprotein IIb/IIIa (GPIIb/IIIa) expression and platelet activation-related signalling pathways were determined in control and nifedipine-treated platelets in the presence or absence of PPAR antagonists or betulinic acid, a NF-κB activator.Exposure of platelets to nifedipine significantly increased the PPAR-β/-γ activity in activated human platelets. Treatment with nifedipine reduced collagen-induced NF-κB events, including the phosphorylation of IκB kinase-β, IκBα and p65NF-κB, which were markedly attenuated by GSK0660, a PPAR-β antagonist, or GW9662, a PPAR-γ antagonist. Furthermore, the interaction of PPAR-β/-γ with NF-κB and the PPAR-β/-γ-up-regulated NO/cGMP/PKG1 cascade may contribute to inhibition of NF-κB activation by nifedipine. Suppressing PPAR-β/-γ activity or increasing NF-κB activation greatly reversed the inhibitory effect of nifedipine on collagen-induced platelet aggregation, intracellular Ca2+ mobilization, PKCα activity and surface GPIIb/IIIa expression.CONCLUSIONS AND IMPLICATIONSPPAR-β/-γ-dependent inhibition of NF-κB activation contributes to the antiplatelet activity of nifedipine. These findings provide a novel mechanism underlying the beneficial effects of nifedipine on platelet hyperactivity-related vascular and inflammatory diseases.

UNLABELLED It is still unclear which neurotransmitters are involved in labor pain. We measured the concentrations of excitatory amino acids, nitric oxide, and prostaglandin E2 in the cerebrospinal fluid (CSF) of pregnant women, particularly in those with labor pain. The patients included in the study consisted of women who underwent cesarean delivery either with labor pain (Labor Pain group, n = 40) or without labor pain (Nonlabor Pain group, n = 58). All patients received spinal anesthesia (intrathecal injection of 10-12 mg of bupivacaine) for the operation, and 2 mL of CSF was collected before bupivacaine injection. Concentrations of aspartate and glutamate (0.50 +/- 0.06 microM and 0.79 +/- 0.10 microM, respectively) were significantly larger in the Labor Pain group than in the Nonlabor Pain group (0.35 +/- 0.03 microM and 0.54 +/- 0.04 microM, P < 0.05). There were no significant differences in the concentrations of nitric oxide and prostaglandin E2 between the groups. A positive correlation was found between CSF concentrations of excitatory amino acids and labor pain. IMPLICATIONS The excitatory amino acids, aspartate and glutamate, play a role in labor pain. N-methyl-D-aspartate receptor antagonists may be useful for labor pain and postlabor uterine contraction pain relief.

We evaluated the effect of HCL-31D, a novel cAMP-specific phosphodiesterase inhibitor, on the induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and on survival in a murine model of severe endotoxaemia. Treatment of cultured RASMC with LPS and IFN-gamma resulted in an increase of nitrite, tumour necrosis factor (TNF-alpha) production and induction of iNOS mRNA. However, incubation with HCL-31D (1 approximately 50 microM) for 24 h caused significant attenuation of nitrite and TNF-alpha formation as well as iNOS mRNA induction in a dose-dependent manner but no effect on iNOS activity in RASMC. In addition, administration of HCL-31D (5 mg/kg, i.p.) resulted in that the increase of both plasma nitrate and TNF-alpha levels induced by LPS in vivo was significantly reduced in LPS-treated rats. Treatment of conscious mice with a high dose of LPS (60 mg/kg, i.p.) to ICR mice resulted in a 24-h survival rate of only 10%. However, administration of HCL-31D (5 mg/kg, i.p. at 0 h and 6 h after LPS) improved the 24-h survival to 50%, indicating that HCL-31D has a beneficial effect in murine model endotoxaemia. These effects may be mainly due to inhibition of TNF-alpha formation and of the induction of iNOS. We proposed that the elevation of cAMP levels by HCL-31D may be involved in the prevention of TNF-alpha formation and iNOS induction.

Septic shock is characterized by a decrease in systemic vascular resistance. Nevertheless, regional increases in vascular resistance can occur that may predispose mammals to organ dysfunction, including the acute respiratory distress syndrome. In the host infected by endotoxin (lipopolysaccharide, LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNFalpha) rapidly increases, and this cytokine production is regulated by agents elevating cyclic AMP. In this report, we present evidence that terbutaline, a beta2-agonist, inhibits TNFalpha production and enhances interleukin-10 (IL-10) release in the anesthetized rat treated with LPS. In addition, an overproduction of nitric oxide (NO, examined by its metabolites nitrite/nitrate) by inducible NO synthase (iNOS, examined by western blot analysis) is attenuated by pretreatment of LPS rats with terbutaline. Overall, pretreatment of rats with terbutaline attenuates the delayed hypotension and prevents vascular hyporeactivity to norepinephrine. In addition, pretreatment of mice with terbutaline also improves the survival in a model of severe endotoxemia. The infiltration of polymorphonuclear neutrophils into organs (e.g., lung and liver) from the surviving LPS mice treated with terbutaline was reduced almost to that seen in the normal controls. These findings suggest that the inhibition of TNFalpha and NO (via iNOS) production as well as the increment of IL-10 production contribute to the beneficial effect of terbutaline in animals with endotoxic shock.

The B chain of β-bungarotoxin 1–6 sequence, RGRHGD, presents the highest local average hydrophilicity measured by Kyte and Doolittle modeling analysis. The RGRHGD holds parts of both RGD and KGD peptides, which have been reported as having high binding affinity to GPIIb-IIIa. The present study evaluates whether the synthesized hexapeptide, RGRHGD, has an antiplatelet effect and further elucidates the possible mechanisms of action. RGRHGD dose-dependently inhibited rabbit platelet aggregation and adenosine triphosphate release induced by arachidonic acid, collagen, platelet-activating factor, thrombin, or U46619 with the IC50 range of 82.7 to 510 μg/mL. The platelet thromboxane B2 formation induced by collagen or thrombin was also significantly decreased by RGRHGD, but there was no effect on arachidonic acid-induced thromboxane B2 formation. In addition, RGRHGD also inhibited the rise of intracellular calcium level stimulated by arachidonic acid, collagen, or thrombin in Fura 2-AM-loaded platelets. The adenosine 3′,5′-cyclic monophosphate level of washed platelets was not affected by RGRHGD. In conclusion, these data indicate that the inhibitory effect of RGRHGD on platelet aggregation may be due to the attenuation of thromboxane A2 formation and intracellular calcium mobilization. In addition, this study may provide a useful method of finding potential therapeutic agents by using molecular modeling analysis.

The antiplatelet effect of the pyridazinone analogue, 4, 5-dihydro-6-[4-[2-hydroxy-3-(3,4 dimethoxybenzylamino)propoxy]naphth-1-yl]-3(2H)-pyridazinone (HCL-31D), was investigated in vitro with rabbit platelets. HCL-31D dose-dependently inhibited the platelet aggregation and ATP release induced by collagen (10 microg/ml), arachidonic acid (100 microM) or thrombin (0.1 U/ml) with an IC(50) of about 0.95-5.41 microM. HCL-31D (0.5-5 microM) increased the platelet cyclic AMP level in a dose-dependent manner. Furthermore, HCL-31D potentiated cyclic AMP formation caused by prostaglandin E(1) but not that caused by 3-isobutyl-1-methylxanthine (IBMX). HCL-31D also attenuated phosphoinositide breakdown and intracellular Ca(2+) elevation induced by collagen, arachidonic acid or thrombin. HCL-31D inhibited the formation of thromboxane B(2) induced by collagen or thrombin but not by arachidonic acid. In addition, HCL-31D did not affect platelet cylooxygenase and thromboxane synthase activity. These data indicate that HCL-31D is an inhibitor of phosphodiesterase and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.