Effects of Caffeoylquinic Acid Derivatives and C-Flavonoid from Lychnophora ericoides on in vitro Inflammatory Mediator Production

In this study we aimed at evaluating the effect of the major polar constituents of the medicinal plant Lychnophora ericoides on the production of inflammatory mediators produced by LPS-stimulated U-937 cells. The 6,8-di-C-beta-glucosylapigenin (vicenin-2) presented no effect on tumor necrosis factor (TNF)-alpha production, but inhibited, in a dose-dependent manner, the production of prostaglandin (PG) E(2) without altering the expression of cyclooxygenase (COX) -2 protein. 3,5-Dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, at lower concentrations, had small but significant effects on reducing PG E, levels; at higher doses these compounds stimulated PGE(2) and also TNF-alpha production by the cells. All the caffeoylquinic acid derivatives, in a dose-dependent fashion, were able to inhibit monocyte chemoattractant protein-3 synthesis/release, with 4,5-DCQ being the most potent at the highest tested concentration. These results add important information on the effects of plant natural polyphenols, namely vicenin-2 and caffeoylquinic acid derivatives, on the production of inflammatory mediators by cultured cells.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies. Published by Elsevier Ltd.

Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.

PURPOSE: This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo. METHODS: Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA. RESULTS: The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction. CONCLUSIONS: This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: Role of NO and prostanoids

Background and aim: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. Methods and results: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. Conclusion: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations (C) 2010 Elsevier B.V. All rights reserved.

Chronic administration of Abarema cochliacarpos attenuates colonic inflammation in rats

Inflammatory bowel diseases are characterized by a chronic clinical course of relapse and remission associated with self-destructive inflammation of the gastrointestinal tract. Active extracts from plants have emerged as natural potential candidates for its treatment. Abarema cochliacarpos (Gomes) Barneby & Grimes, Fabaceae (Barbatimão), is a native medicinal plant in to Brazil. Previously we have demonstrated in an acute colitis model a marked protective effect of a butanolic extract, so we decided to assess its anti-inflammatory effect in a chronic ulcerative colitis model induced by trinitrobenzensulfonic acid (TNBS). Abarema cochliacarpos (150 mg/day, v.o.) was administered for fourteen consecutive days. This treatment decreased significantly macroscopic damage as compared with TNBS. Histological analysis showed that the extract improved the microscopic structure. Myeloperoxidase activity (MPO) was significantly decreased. Study of cytokines showed that TNF-α was diminished and IL-10 level was increased after Abarema cochliacarpos treatment. In order to elucidate inflammatory mechanisms, expression of cyclooxygenase (COX)-2 and nitric oxide synthase (iNOS) were studied showing a significant downregulation. In addition, there was reduction in the JNK and p-38 activation. Finally, IκB degradation was blocked by Abarema cochliacarpos treatment being consistent with an up-regulation of the NF-kappaB-binding activity. These results reinforce the anti-inflammatory effects described previously suggesting that Abarema cochliacarpos could provide a source for the search for new anti-inflammatory compounds useful in ulcerative colitis treatment.

Dopplervelocimetria da artéria umbilical e controle glicêmico materno como marcadores de alterações vasculares e apoptóticas placentárias

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Effects of electroacupuncture on experimental periodontitis in rats

Background: Acupuncture has shown the capability of modulating the immuno-inflammatory response of the host. This study aims to evaluate the effects of electroacupuncture (EA) on ligature-induced periodontitis in rats. Methods: Thirty-two animals were divided into four groups: 1) control; 2) experimental periodontitis (EP); 3) sham-treated (EP/EA-sham); and 4) treated with EA (EP/EA). For the EP groups, a ligature was placed around the right mandibular first molars at day 1. Sessions of EA or EA-sham were assigned every other day. For EA treatment, large intestine meridian points LI4 and LI11 and stomach meridian points ST36 and ST44 were used. EA-sham was performed in off-meridian points. Animals were euthanized at day 11. Histomorphometric and microtomographic analyses were performed. Immunolabeling patterns for the receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) were assessed. Expressions of interleukin (IL)-1 beta, matrix metalloproteinase (MMP)-8, IL-6, and cyclooxygenase (COX)-2 messenger RNAs (mRNAs) were evaluated by quantitative reverse transcription-polymerase chain reaction. Data were analyzed statistically (P < 0.05, analysis of variance). Results: Histomorphometric and microtomographic analyses demonstrated that group EP/EA presented reduced alveolar bone loss when compared to group EP (P < 0.05). Reduced RANKL immunolabeling and fewer TRAP-positive multinucleated cells were observed in the EA-treated group in relation to group EP. No differences were observed in OPG expression among groups. EA treatment decreased the genic expression of IL-1 beta and MMP-8 (P < 0.05), increased the mRNA expression of IL-6 (P < 0.05), and did not modify the genic expression of COX-2 in animals with EP (P > 0.05). Conclusion: It can be concluded that EA reduced periodontal tissue breakdown and the expression of some proinflammatory mediators and a proresorptive factor in EP in rats.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Analgesic and anti-inflammatory actions of robenacoxib in acute joint inflammation in dog

The objectives of this study were to establish dose-response and blood concentration-response relationships for robenacoxib, a novel nonsteroidal anti-inflammatory drug with selectivity for inhibition of the cyclooxygenase (COX)-2 isoenzyme, in a canine model of synovitis. Acute synovitis of the stifle joint was induced by intra-articular injection of sodium urate crystals. Robenacoxib (0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg), placebo and meloxicam (0.2 mg/kg) were administered subcutaneously (s.c.) 3 h after the urate crystals. Pharmacodynamic endpoints included data from forceplate analyses, clinical orthopaedic examinations and time course of inhibition of COX-1 and COX-2 in ex vivo whole blood assays. Blood was collected for pharmacokinetics. Robenacoxib produced dose-related improvement in weight-bearing, pain and swelling as assessed objectively by forceplate analysis (estimated ED(50) was 1.23 mg/kg for z peak force) and subjectively by clinical orthopaedic assessments. The analgesic and anti-inflammatory effects of robenacoxib were significantly superior to placebo (0.25-4 mg/kg robenacoxib) and were non-inferior to meloxicam (0.5-4 mg/kg robenacoxib). All dosages of robenacoxib produced significant dose-related inhibition of COX-2 (estimated ED(50) was 0.52 mg/kg) but no inhibition of COX-1. At a dosage of 1-2 mg/kg administered s.c., robenacoxib should be at least as effective as 0.2 mg/kg of meloxicam in suppressing acute joint pain and inflammation in dogs.

Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

Ultraviolet radiation modulates the immune system through the platelet -activating factor receptor

Ultraviolet radiation plays a critical role in the induction of non-melanoma skin cancer. UV radiation is also immune suppressive. Moreover, UV-induced systemic immune suppression is a major risk factor for skin cancer induction. Previous work had shown that UV exposure in vivo activates a cytokine cascade involving PGE2, IL-4, and IL-10 that induces immune suppression. However, the earliest molecular events that occur immediately after UV-exposure, especially those upstream of PGE2, were not well defined. To determine the initial events and mediators that lead to immune suppression after a pathological dose of UV, mouse keratinocytes were analyzed after sunlamp irradiation. It is known that UV-irradiated keratinocytes secrete the phospholipid mediator of inflammation, platelet-activating factor (PAF). Since PAF stimulates the production of immunomodulatory compounds, including PGE2, the hypothesis that UV-induced PAF activates cytokine production and initiates UV-induced immune suppression was tested. Both UV and PAF activated the transcription of cyclooxygenase (COX)-2 and IL-10 reporter gene constructs. A PAF receptor antagonist blocked UV-induced IL, 10 and COX-2 transcription. PAF mimicked the effects of UV in vivo and suppressed delayed-type hypersensitivity (DTH), and immune suppression was blocked when UV-irradiated mice were injected with a PAF receptor antagonist. This work shows that UV generates PAF-like oxidized lipids, that signal through the PAF receptor, activate cytokine transcription, and induce systemic immune suppression. ^

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFκB

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin’s inhibition of NFκB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFκB, or via the “inflammatory” cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFκB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFκB.

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.

Rheumatoid arthritis: links with cardiovascular disease and the receptor for advanced glycation end products

Cardiovascular (CV) disease is increased in patients with chronic inflammatory disease, including rheumatoid arthritis (RA). Furthermore it has become clear at a pathophysiological level, that atherosclerosis has striking similarities with autoimmune disease. This realization has come at a time of paradigm shift in how rheumatologists manage RA, with the availability of biological agents targeting key inflammatory cytokines. This review will focus on the possible causes of increased vascular disease in RA, including the role of traditional CV risk factors. Mechanisms potentially at play, such as C-reactive protein (CRP), altered coagulation, and cyclooxygenase (COX) -2 inhibitors will be covered in brief. The Receptor for Advanced Glycation End Products (RAGE) has been identified as a candidate molecule influencing response to ongoing inflammation and autoimmunity. There will be a focus on the role of RAGE in CV disease and RA. As has been the case with many novel molecules, functional polymorphisms are thought to alter disease expression and assist us in coming to terms with the biological activities of the parent molecule. The review will conclude with a discussion of the potential role of the RAGE Glycine 82 Serine polymorphism

The Nrf2-ARE activation protect neurones against oxidative stress

Oxidative stress has been implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease. The transcription factor, Nrf2 (nuclear factor E2-related factor 2) that binds to the antioxidant responsive element (ARE) activates a battery of genes encoding enzymes and factors essential for neuronal survival. We have investigated the hypothesis that a downstream product of cyclooxygenase(COX-2), 15-deoxy-delta (12, 14)-prostagland in J2 (15d-PGJ2) has protective effects by activating the Nrf2 pathway during oxidative stress.Human neuroblastoma cells (SHSY5Y) were differentiated intoneuronal-like cells as described previously (Gimenez-Cassina et al.,2006). SHSY5Y cells were co-treated with 10 mM buthionine sulfoximine (BSO) 7 10 mM 15d-PGJ2. Cell viability was measured by MTT assay and cellular glutathione (GSH) levels were measured after treating cells for0.5-24 hours by GSH recycling assay. Cellular Nrf2 levels were determined by immunoblotting. IL-6 levels were measured by ELISA.15d-PGJ2 alone lowered GSH levels 30min after the treatment(12.870.64 nmol/mg protein) and returned to untreated control levels at 16hours (28.173.6 nmol/mg protein; Po0.01). Compared to intracellular GSH levels in untreated cells (27.871.8 nmol/mg protein) BSO treatment alone significantly decreased GSH (9.672.1 nmol/mg protein;Po0.001) but co-incubation with 15d-PGJ2 for 24 hours prevented the depletion elicited by BSO(21.372.7 nmol/mg protein). Compared to untreated cells BSO treatment decrease dIL-6 secretion (from 0.941.6ng/ml to 0.6971.3ng/ml) and total Nrf2 protein levels (by21%). Co-incubation with15d-PGJ2 for 24 hours with BSO did not change IL-6(0.6771.4ng/ml) or total Nrf2 level at any time point. This study suggests that neuronal toxicity resulting from glutathione depletion canbere stored by the induction of Nrf2-ARE pathway and the role of the Nrf2 signalling merits further investigation in neurodegenerative diseases.