Effect of quercetin and role of nitric oxide pathway in chloroquine-induced scratching

Abstract Nitric oxide (NO) is an abundant mediator which is demonstrated to be involved in pruritus. Assuming that the increased NO also mediates chloroquine-induced pruritus, which is a frequent complication seen in the chronic chloroquine treatment, the current study aimed to investigate the effect of quercetin and the role of NO in chloroquine-induced pruritus in C57BL/6 mice. Model was created with subcutaneous chloroquine (400µg/site) injection to the nape of the mice. Effect of quercetin and role of NO were investigated with administration of quercetin, and co-administration with L-NAME, 7-NI and L-arginine before chloroquine injection. Locomotor activity was assessed by activity cage and number of the scratching bouts after chloroquine injection was recorded for 30 minutes. Our results show that quercetin significantly reduced scratching bouts at the doses of 10, 20, 40 and 80 mg/kg. Locomotor activity was decreased at the 40 and 80 mg/kg doses of quercetin. Additionally, decrease of the number of scratching bouts by quercetin prevented by L-arginine treatment, while L-NAME and 7-NI enhanced the anti-pruritic effect of sub-effective doses of quercetin. Therefore, our study demonstrated that acute injection of quercetin significantly diminished chloroquine-induced scratching behavior, and this effect is partly mediated by inhibition of neuronal nitric oxide synthase enzyme.

Isolation of an acidic polysaccharide from the flowers of Leucosceptrum canum Smith and its immunomodulatory activity evaluation.

A water-soluble polysaccharide (LCP-05) was isolated from the flowers of Leucosceptrum canum Smith. LCP-05 was an acidic polysaccharide with a molecular weight of approximately 8.9 kDa. Monosaccharide composition analysis indicated that LCP-05 was composed of Man, Rha, GlcA, GalA, Glc, Gal and Ara in a molar ratio of 0.83:1.68:0.33:2.15:1.00:1.45:1.22. The framework of LCP-05 was speculated to be a branched rhamnogalacturonan with the backbone consisting of α-1,2,4-linked Rhap and α-1,4-linked GalAp, and bearing branches at the O-4 position of the Rha residues. The side chains are terminated primarily with the Araf and Glcp residues. LCP-05 was found to be able to significantly induce the production of NO, IL-6, and TNF-α in RAW 264.7 cells, and to induce RAW 264.7 cell's suppressive effect on both cell growth and cell migration of 4 T1 mammary breast cancer cells.

Structural and immunological studies on the polysaccharide from spores of a medicinal entomogenous fungus Paecilomyces cicadae.

A neutral branched heteropolysaccharide (Pc0-1) was purified from the spores of Paecilomyces cicadae, which parasitized in the bamboo cicada (Platylomia pieli Kato). The structure of Pc0-1 was analyzed by HPLC, IR, methylation and NMR spectroscopy. The results reveal that Pc0-1, with an average molecular weight of 18 × 103 kDa, consists of glucose, galactose, mannose and arabinose in the molar ratio of 8:5:4:1. Some of the glucose residues have methyl modification at O-6 position. The Pc0-1 polysaccharide has a core structure containing 1,2-linked α-d-Manp residues as the backbone and branches at the O-3 and O-6 of the α-d-Manp residues. The inner part of the side-chains is comprised of 1,4-linked α-d-Glcp and 1,4-linked 6-O-Me-α-d-Glcp residues. 1,2-linked ß-Galf and minor 1,4-linked Arap and 1,3 or 4-linked Arap residues were occasionally linked at the outside of the side-chains. The side-chains have a single terminal residue of α-d-Glcp, α-Manp, ß-Galf or minor Arap (minor). Studies on the bioactivity of Pc0-1 on the macrophages show it exhibit moderate immunostimulating activity through increasing the production of nitric oxide (NO) and enhancing the secretion of major inflammatory cytokines by macrophages, such as TNF-α, IL-1ß, IL-6, in RAW 264.7 cells. We examined the effect of Pc0-1 on induced NO and cytokine production in macrophages using anti-PRR antibodies to investigate the membrane receptor for the polysaccharide. The results show that Pc0-1 mainly activates macrophages through their mannose receptor (MR). TLR4 and TLR2 also participated in the recognition of Pc0-1.

Modulating the Bioactivity of Nitric Oxide as a Therapeutic Strategy in Cardiac Surgery.

Cardiac surgery, including cardioplegic arrest and extracorporeal circulation, causes endothelial dysfunction, which can lead to no-reflow phenomenon and reduction of myocardial pump function. Nitric oxide (NO) deficiency is involved in this pathologic process, thereby providing a fundamental basis for the use of NO replacement therapy. Presently used drugs and additives to cardioplegic and heart preservation solutions are not able to reliably protect endothelial cells and cardiomyocytes from ischemia-reperfusion injury. This review discusses promising NO-releasing compounds of various chemical classes for cardioplegia and reperfusion, which effectively maintain NO homeostasis under experimental conditions, and presents the mechanisms of their action on the cardiovascular system. Incomplete preclinical studies and a lack of toxicity assessment, however, hinder translation of these drug candidates into the clinic. Perspectives for modulation of endothelial function using NO-mediated mechanisms are discussed. They are based on the cardioprotective potential of targeting vascular gap junctions and endothelial ion channels, intracoronary administration of progenitor cells, and endothelial-specific microRNAs. Some of these strategies may provide important therapeutic benefits for human cardiovascular interventions.

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial.

BACKGROUND: Measurement of the reactive hyperemia index (RHI) using peripheral arterial tonometry (PAT) has shown benefits in the evaluation of vascular endothelial function and prediction of cardiovascular disease prognosis. Thus, it is important to examine the factors that promote the RHI. In this study, we aimed to investigate the effect of molecular hydrogen (H2) on reactive hyperemia-PAT of the small arteries of fingers in healthy people. METHODS: To determine the efficacy of H2 for improving peripheral vascular endothelial function, water containing high H2 concentrations was administered to participants, and the Ln_RHI was measured in the finger vasculature. Sixty-eight volunteers were randomly divided into two groups: a placebo group (n = 34) that drank molecular nitrogen (N2)-containing water and a high H2 group (n = 34) that drank high H2 water (containing 7 ppm of H2: 3.5 mg H2 in 500-mL water). The Ln_RHI was measured before ingesting the placebo or high H2 water, 1 h and 24 h after the first ingestion, and 14 days after daily ingestion of high H2 water or the placebo. The mixed effects model for repeated measures was used in data analysis. RESULTS: The high H2 group had a significantly greater improvement in Ln_RHI than the placebo group. Ln_RHI improved by 22.2% (p<0.05) at 24 h after the first ingestion of high H2 water and by 25.4% (p<0.05) after the daily consumption of high H2 water for 2 weeks. CONCLUSIONS: Daily consumption of high H2 water improved the endothelial function of the arteries or arterioles assessed by the PAT test. The results suggest that the continuous consumption of high H2 water contributes to improved cardiovascular health.

Resveratrol and its dimers ε-viniferin and δ-viniferin in red wine protect vascular endothelial cells by a similar mechanism with different potency and efficacy.

Red wine compounds have been reported to reduce the rate of atherosclerosis by inducing nitric oxide (NO) production and antioxidant enzyme expression in vascular endothelial cells (VECs). The present study compared the effects of the three red wine compounds resveratrol and its dimers, ε-viniferin and δ-viniferin, on VECs function for the first time. Both 5 µM ε-viniferin and δ-viniferin, but not 5 µM resveratrol, significantly stimulated wound repair of VECs. Increased levels of wound repair induced by 10 and 20 µM ε-viniferin were significantly higher than those stimulated by 10 and 20 µM resveratrol, respectively. These stimulatory effects of the three compounds were suppressed by the NO synthase inhibitor L-NAME. When VECs were exposed to each compound, endothelial NO synthase was activated and the expression of sirtuin 1 (SIRT1) and HO-1 was induced. Addition of the SIRT1 and HO-1 inhibitors EX527 and ZnPPiX, respectively, suppressed wound repair stimulated by the three compounds, demonstrating that SIRT1 and HO-1 are involved in these wound repair processes. Furthermore, each compound induced the suppression of H2 O2 -dependent reduction of cell viability as well as the expression of the antioxidant enzyme catalase. These data suggest that not only resveratrol, but also its dimers, ε-viniferin and δ-viniferin, may be effective in preventing atherosclerosis by a similar molecular mechanism with different potency and efficacy.

Endothelial Sphingolipid De Novo Synthesis Controls Blood Pressure by Regulating Signal Transduction and NO via Ceramide.

Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus-related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)-the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.

VEGF and bFGF induction by nitric oxide is associated with hyperbaric oxygen-induced angiogenesis and muscle regeneration.

Hyperbaric oxygen (HBO) treatment promotes early recovery from muscle injury. Reactive oxygen species (ROS) upregulation is a key mechanism of HBO, which produces high O2 content in tissues through increased dissolution of oxygen at high pressure. Nitric oxide (NO), a type of ROS, generally stabilizes hypoxia-inducible factor (HIF) 1α and stimulates secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from endothelial cells and macrophages, which then induces angiogenesis. The purpose of the present study was to investigate whether HBO could promote angiogenesis via induction of NO and induce muscle regeneration in contused rat skeletal muscles. The HBO protocol consisted of 2.5 atmospheres absolute (ATA) 100% oxygen for 120 minutes, once a day for 5 consecutive days. We also evaluated the effects of a ROS inhibitor (NAC) or NOS-specific inhibitor (L-NAME) on HBO. HBO significantly increased NO3-, VEGF, and bFGF levels and stabilized HIF1α within 1 day. HBO promoted blood vessel formation at 3-7 days and muscle healing at 5-7 days after contusion. Administration of both NAC and L-NAME before HBO suppressed angiogenesis and muscle regeneration even after HBO. HBO thus promoted angiogenesis and muscle regeneration mainly through generation of NO in the early phase after muscle contusion injury.

Effect of tadalafil and nitric oxide agonist sodium nitroprusside on penicillin-induced epileptiform activity.

Objective: Comorbidity of erectile dysfunction (ED) and epilepsy is not rare. Tadalafil is widely used in the treatment of ED and shows its effect by increasing nitric oxide (NO) level. Previous studies demonstrated that ED treatment drugs increased epileptiform activity in clinical studies and various experimental epilepsy models. Therefore, it is important to know whether an ED treatment drug has proconvulsion or anticonvulsant properties. This study was designed to demonstrate the effect of tadalafil and NO agonist sodium nitroprusside during penicillin-induced seizures in rats. The experimental penicillin epilepsy model is preferred in clinical studies of partial epilepsy.Methods: A single dose of penicillin (500 units) intracortical (i.c.) injection into the left sensorimotor cortex induced epileptiform activity. In the first set of experiments, tadalafil (20 mg/kg/intraperitoneal [i.p.]) and sodium nitroprusside (50 µg/intracerebroventricular [µg/i.c.v]) were administered 30 min after penicillin injection.In the second set of experiments, tadalafil (i.p) was administered 30 min after penicillin injection and sodium nitroprusside was administered simultaneously with the tadalafil injection.Results: Tadalafil, sodium nitroprusside and tadalafil+sodium nitroprusside groups decreased the frequency and amplitude of epileptiform activity in rats. Spike frequency of all groups decreased significantly 10 min after the administration and this decrease continued for 180 min. The mean amplitude of epileptiform activity significantly decreased 120 min after penicillin application in tadalafil or sodium nitroprusside applications. But this decrease was observed 110 min after tadalafil+sodium nitroprusside combined application.Conclusion: Data from the present study indicate that tadalafil has an anticonvulsion effect against penicillin-induced epileptiform activity in rats.

Nitric oxide-enhancing or -releasing agents as antithrombotic drugs.

Nitric oxide (NO) is a powerful biological mediator provided with a number of activities of relevance for the prevention of thrombosis, like vasodilation, inhibition of platelet adhesion and aggregation, prevention of smooth muscle cell proliferation. Several cells in the circulation release NO, like endothelial cells which are the largest source, red blood cells, platelets and white blood cells, and conditions associated with an impaired production or bioavailability of NO predispose to arterial and venous thrombosis. It seems thus logical to use NO as an antithrombotic agent. However, given the extremely short half-life, limited water solubility and radical nature of this mediator, several chemical strategies to generate drugs releasing NO and/or favouring its endogenous production/bioavailability have been developed. Here we review the pharmacologic approaches to enhance endogenous NO or to induce NO-release developed over the last decades for their effects on platelet activation in vitro and in vivo and on thrombosis, in animal models and in humans. One limitation to the development of NO-releasing agents as antithrombotic drugs is represented by their concomitant vasodilatory action which, by inducing hypotension, limits their applicability. Further pharmacologic and clinical research of novel NO-enhancing and/or -releasing molecules is highly warranted in order to fully exploit the great antithrombotic potential of NO.

Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide.

We previously demonstrated that co-exposing pre-steatotic hepatocytes to benzo[a]pyrene (B[a]P), a carcinogenic environmental pollutant, and ethanol, favored cell death. Here, the intracellular mechanisms underlying this toxicity were studied. Steatotic WIF-B9 hepatocytes, obtained by a 48h-supplementation with fatty acids, were then exposed to B[a]P/ethanol (10 nM/5 mM, respectively) for 5 days. Nitric oxide (NO) was demonstrated to be a pivotal player in the cell death caused by the co-exposure in steatotic hepatocytes. Indeed, by scavenging NO, CPTIO treatment of co-exposed steatotic cells prevented not only the increase in DNA damage and cell death, but also the decrease in the activity of CYP1, major cytochrome P450s of B[a]P metabolism. This would then lead to an elevation of B[a]P levels, thus possibly suggesting a long-lasting stimulation of the transcription factor AhR. Besides, as NO can react with superoxide anion to produce peroxynitrite, a highly oxidative compound, the use of FeTPPS to inhibit its formation indicated its participation in DNA damage and cell death, further highlighting the important role of NO. Finally, a possible key role for AhR was pointed out by using its antagonist, CH-223191. Indeed it prevented the elevation of ADH activity, known to participate to the ethanol production of ROS, notably superoxide anion. The transcription factor, NFκB, known to be activated by ROS, was shown to be involved in the increase in iNOS expression. Altogether, these data strongly suggested cooperative mechanistic interactions between B[a]P via AhR and ethanol via ROS production, to favor cell death in the context of prior steatosis.

Effect of gamma irradiation on structure, physicochemical and immunomodulatory properties of Astragalus polysaccharides.

Astragalus polysaccharides (APS) were treated with different gamma irradiation doses (10, 25, 50, 100 and 150 kGy) to investigate the effects of gamma radiation processing on structure, physicochemical and immunomodulatory properties. The results revealed both the number-average and weight-average molecular weight of APS significantly decreased with increasing irradiation dose, whereas the solubility was increased after irradiation. A decrease in the apparent viscosity, as well as an increase in amount of small fragments of APS granules was also observed with increasing irradiation dose. FT-IR spectra indicated that gamma irradiation introduced no significant changes into the functional group status of APS. High irradiation dose (>50 kGy) caused a significant increase of yellowness and a slightly decrease of thermal stability of APS. Further, the immunomodulatory activity of irradiated APS was evaluated on Caco2 cells. APS irradiated at dose of 25 kGy exhibited the highest ability to induce nitric oxide production and up-regulate the mRNA expression of inflammatory cytokines, occludin, zonula occludens protein-1 (ZO-1) and toll-like receptor 4 (TLR4), as well as the protein expression of ZO-1 and TLR4. These findings indicate that gamma irradiation modification with a proper dose enhance immunomodulatory activity of APS by improving physicochemical properties without changing the functional groups.

Schiff bases containing a furoxan moiety as potential nitric oxide donors in plant tissues.

Stable Schiff bases containing a furoxan moiety are synthesized as single regioisomers by the reaction of 3-methyl-2-oxy-furazan-4-carbaldehydewith various amino compounds at room temperature. The structures of synthesized compounds were fully characterized by multinuclear NMR spectroscopy and X-ray crystallography. The effect of synthesized Schiff bases containing a furoxan moiety on biological generation of reactive oxygen species and nitric oxide in plant tissues was investigated for the first time by fluorescence microscopy and the released NO identified as nitrite with Griess reagent. There is a good correlation between the biological generation of NO determined by fluorescence microscopy and with Griess reagent. Some of the synthesized compounds exhibited both nitric oxide and reactive oxygen species generation abilities and represent potential NO donors in plant tissues.

Homocysteine sensitizes the mouse neuromuscular junction to oxidative stress by nitric oxide.

Homocysteine (HCY), a redox-active metabolite of the methionine cycle, is of particular clinical interest because of its association with various neurodegenerative diseases including amyotrophic lateral sclerosis. It has been previously established that HCY exacerbates damage to motor neurons from reactive oxygen species (ROS) such as hydrogen peroxide. To assess the role of HCY at the mammalian neuromuscular junction, neurotransmission was monitored by electrophysiology at the mouse epitrochleoanconeus muscle. Preparations were preincubated in HCY before inducing ROS and recordings were taken before and after ROS treatment. In this study, HCY was observed to sensitize the neuromuscular junction to ROS-induced depression of spontaneous transmission frequency, an effect we found to be mediated by a N-methyl-D-aspartate receptor (NMDAR) and nitric oxide (NO). The NMDAR antagonist D, L-2-amino-5-phosphonopentanoic acid prevented the HCY-induced sensitization to oxidative stress. Disrupting NO activity with either the nitric oxide synthase I antagonist Nω-nitro-L-arginine methyl ester hydrochloride or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium salt also prevented sensitization. Moreover, replacing HCY with the exogenous NO donor Diethylamine NONOate diethylammonium was sufficient to reconstitute the effects of HCY-induced sensitization to ROS. Interestingly, a novel secondary effect was observed where HCY itself depresses quantal content, an effect found to be mediated by NMDARs independently of nitric oxide and ROS. Collectively, these data present a novel model of two distinct pathways through which HCY alters neurotransmission at the neuromuscular junction. Characterizing HCY's mechanism of action is of particular clinical relevance as many treatments for amyotrophic lateral sclerosis are centered on mitigating HCY-induced pathologies.

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Monosodium glutamate impairs the contraction of uterine visceral smooth muscle ex vivo of rat through augmentation of acetylcholine and nitric oxide signaling pathways.

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways.

Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation.

Downy mildew of pearl millet caused by the biotrophic oomycete Sclerospora graminicola is the most devastating disease which impairs pearl millet production causing huge yield and monetary losses. Chitosan nanoparticles (CNP) were synthesized from low molecular weight chitosan having higher degree of acetylation was evaluated for their efficacy against downy mildew disease of pearl millet caused by Sclerospora graminicola. Laboratory studies showed that CNP seed treatment significantly enhanced pearl millet seed germination percentage and seedling vigor compared to the control. Seed treatment with CNP induced systemic and durable resistance and showed significant downy mildew protection under greenhouse conditions in comparison to the untreated control. Seed treatment with CNP showed changes in gene expression profiles wherein expression of genes of phenylalanine ammonia lyase, peroxidase, polyphenoloxidase, catalase and superoxide dismutase were highly upregulated. CNP treatment resulted in earlier and higher expression of the pathogenesis related proteins PR1 and PR5. Downy mildew protective effect offered by CNP was found to be modulated by nitric oxide and treatment with CNP along with NO inhibitors cPTIO completely abolished the gene expression of defense enzymes and PR proteins. Further, comparative analysis of CNP with Chitosan revealed that the very small dosage of CNP performed at par with recommended dose of Chitosan for downy mildew management.

Binding of cholera toxin B subunit to intestinal epithelial cells.

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (Kd 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α1 and 131-135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10µM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells.

Valproate Plays a Protective Role against Migraine by Inhibiting Protein Kinase C Signalling in Nitroglycerin-treated Mice.

Migraine is a common disease with a high morbidity. Valproate (VP) is used as an anti-epilepsy drug in clinic. This study aimed to investigate the role of VP in nitroglycerin (NTG)-induced migraine using a mouse model. NTG was employed by intraperitoneal injection to induce a migraine model in mice. The NTG administration caused mouse head discomforts, decreased tolerance to cold or hot stimulation and increased content of nitric oxide, calcitonin gene-related peptide and neuropeptide Y in serum, which were ameliorated by intraperitoneal injection of VP. The levels of two inflammatory factors, interleukin (IL)-1ß and inducible nitric oxide synthase, in dura mater were increased by NTG treatment, while the increase was attenuated by application of VP. In addition, the phosphorylation levels of protein kinase C (PKC) α, γ, δ and ε were increased by NTG and decreased by VP. However, their total expression at the transcriptional and translational levels did not change significantly. Two substrates of PKC, cAMP-response element binding protein 1 and signal transducer and activator of transcription 1 were also phosphorylated by NTG application, and the phosphorylation level was attenuated by VP, consistent with the change of PKC informs. Together, we demonstrated that VP prevented damage due to migraine by inhibiting PKC signalling in NTG-injected mice, which may provide a basis for investigating the clinical treatment of migraine.