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Adenosine is a potent regulator of inflammation and immunity, but the role of adenosine receptors in keratinocytes remains controversial. We determined that in addition to A 2B receptors, human epidermal keratinocytes also express A 2A receptors, although to a lower extent. Through the use of selective adenosine receptor agonists and antagonists, we showed that physiological concentrations of adenosine activate A 2B receptors in normal human keratinocytes, inducing cell cycle arrest through the increase of intracellular calcium but not through cAMP signaling. In contrast, the selective activation of A 2A receptors by CGS-21680 induces keratinocyte proliferation via p38-mitogen-activated protein kinase activation. Adenosine and selective A 2A and A 2B agonists presented anti-inflammatory profiles independent of adenosine receptors but mediated by membrane phosphatase activation. Finally, keratinocyte exposure to diverse inflammatory cytokines altered adenosine receptor expression by reducing A 2B and increasing A 2A , a pattern also observed in psoriatic epidermis. Because increased epidermal turnover and inflammatory response are characteristics of psoriatic disease, further studies are needed to assess the role and consequences of the altered adenosine receptor expression in lesional and nonlesional psoriatic keratinocytes., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Benzo[b]thiophen-2-yl-3-bromo-5-hydroxy-5H-furan-2-one (BTH) is a simple and interesting synthetic derivative of petrosaspongiolide M, a natural compound isolated from a sea sponge with demonstrated potent anti-inflammatory activity through inhibition of the NF-κB signaling pathway. In the present study, we report the in vitro and in vivo pharmacological effect of BTH on some parameters related to the innate and adaptive response in the pathogenesis of psoriasis. BTH inhibited the release of some of the key psoriatic cytokines such as tumor necrosis factor α, IL-8, IL-6, and CCL27 through the downregulation of NF-κB in normal human keratinocytes. Moreover, it impaired signal transducers and activators of transcription 3 (STAT3) phosphorylation and translocation to the nucleus, which resulted in decreased keratinocyte proliferation. These results were confirmed in vivo in two murine models of psoriasis: the epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate and the imiquimod-induced skin inflammation model. In both cases, topical administration of BTH prevented skin infiltration and hyperplasia through suppression of NF-κB and STAT3 phosphorylation. Our results confirm the pivotal role of both transcriptional factors in skin inflammation, as occurs in psoriasis, and highlight the potential of small molecules as therapeutic agents for the treatment of this skin disease, with BTH being a potential candidate for future drug research.

Chondroitin sulfate (CS) is a natural glycosaminoglycan, formed by the 1-3 linkage of d-glucuronic acid to N-acetylgalactosamine, present in the extracellular matrix. It is used as a slow acting disease modifying agent in the treatment of osteoarthritis, and part of its beneficial effects are due to its antiinflammatory properties that result from an inhibitory effect on NF-κB signaling pathway. This ability raises the hypothesis that CS might be effective in other chronic inflammatory processes such as psoriasis, in which a deregulation of NF-κB is a key feature. In addition, psoriasis is characterized by an upregulation of STAT3 signaling pathway that is related to the epidermal hyperplasia. In the present study we report the pharmacological modulation of the NF-κB and STAT3 signaling pathways by CS in normal human keratinocytes. CS inhibited NF-κB activation and the release of some of the key psoriatic cytokines such as TNFα, IL-8, IL-6 and CCL27. Moreover, it impaired STAT3 translocation to the nucleus and significantly reduced STAT3 transcriptional activity by a mechanism that was independent from STAT3 phosphorylation. Our results confirm the interest of CS as a candidate for future drug research in the therapeutics of psoriasis given the need of more effective and safer oral medications for these patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Avarol is a marine sesquiterpenoid hydroquinone with interesting pharmacological properties including anti-inflammatory and antipsoriatic effects. In the present study we evaluated the pharmacological effect of avarol on some inflammatory parameters related to the pathogenesis of psoriasis. Avarol inhibited tumor necrosis factor-alpha (TNF-alpha) generation in stimulated human monocytes (IC(50) 1 microM) and TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB)-DNA binding in keratinocytes. In the mouse air pouch model, administration of avarol produced a dose-dependent reduction of TNF-alpha generation (ED(50) 9.2 nmol/pouch) as well as of interleukin (IL)-1beta, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) levels in pouch exudates. In the psoriasis-like model of 12-O-tetradecanoylphorbol-acetate-induced mouse epidermal hyperplasia, topical administration of avarol (0.6-1.2 micromol/site) reduced edema, myeloperoxidase activity, IL-1beta, IL-2 and eicosanoid levels in skin. Histopathological study confirmed the inhibition of epidermal hyperplasia as well as leukocyte infiltration. The reduction of cutaneous TNF-alpha by avarol was also detected by immunohistochemical analysis. Avarol was also capable of suppressing in vivo NF-kappaB nuclear translocation, determined in mouse skin. Our results suggested that antipsoriatic properties of avarol previously described could be mediated in part by the downregulation of several inflammatory biomarkers, such as TNF-alpha and NF-kappaB in psoriatic skin.

Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression.

The inhibitory effect of a series of 6 cycloamphilectenes, novel marine diterpenes based on amphilectene skeletons and isolated from the Vanuatu sponge Axinella sp., on NO, PGE(2) and TNFalpha production in murine peritoneal macrophages was studied. These compounds reduced potently nitric oxide production in a concentration-dependent manner with IC(50) values in the submicromolar range (0.1-4.3 microM). Studies on intact cells and Western blot analysis showed that the more potent cycloamphilectenes reduced the expression of inducible nitric oxide synthase without affecting cyclo-oxygenase-2 expression. Among them cycloamphilectene 2, the unique compound bearing an exocyclic methylene group, was able to reduce NO production without affecting TNFalpha release. Cycloamphilectene 2, which is an inhibitor of the nuclear factor-kB pathway, exhibited topical anti-inflammatory activity.

The inhibitory effect of cavernolide, a novel C2, terpene lactone isolated from the sponge Fasciospongia cavernosa, on PLA2 and other enzyme activities involved in the inflammatory process was studied. Cavernolide inhibited human synovial sPLA2 in a concentration-dependent manner with an IC50 value of 8.8 microM. Besides, this compound decreased in the nanomolar range the myeloperoxidase degranulation process using different stimuli. Cavernolide also inhibited TNFalpha, NO and PGE2 production in intact cell experiments. NO and PGE2 reduction was the consequence of the inhibition on iNOS and COX-2 expression because it did not affect inducible nitric oxide synthase and cyclooxygenase-2 activities in intact cells.

The effects of dichloromethane and methanol extracts from the marine invertebrates Leptogorgia ceratophyta, Holothuria tubulosa, Coscinasterias tenuispina and Phallusia fumigata on carrageenan-induced paw oedema in mice were investigated. The dichloromethane extract of Coscinasterias tenuispina and the methanol extract of Holothuria tubulosa administered p.o. at 50, 100 and 150 mg/kg, inhibited oedema in a dose-dependent manner 3 h after administration of carrageenan. Both extracts partially decreased elastase activity and PGE2 levels measured in homogenates from inflamed paws, without affecting the levels of this prostanoid present in stomach homogenates. As observed with the selective inhibitor NS398, both extracts can decrease cyclo-oxygenase activity in inflamed tissues but do not modify the constitutive cyclo-oxygenase enzyme. Therefore, these extracts represent new marine resources for the isolation of novel agents active on inflammatory conditions.

The novel analgesic filenadol (d, 1-erythro-1-(3',4'-methylenedioxyphenyl)-1- morpholinopropan-2-ol) inhibited phenyl-p-benzoquinone-induced writhing in mice with ID50 values of 68.8 (p.o.), 1.67 (i.v.) and 0.48 (i.c.v.) mg/kg. Hyperalgesia induced by arachidonic acid, PGE2 or LTB4 in this test was also decreased by filenadol (ID50 = 24.4, 3.7 and 50.1 mg/kg p.o., respectively). This compound was effective on PGE2, LTB4, bradykinin, PAF or IL-1 beta-induced decrease in pain threshold in the rat paw pressure model and almost totally suppressed the writhing induced by zymosan in mice, while peritoneal production of 6-ketoPGF1 alpha was inhibited by 48.5-62% and only at 100 mg/kg significant inhibition of LTC4 was achieved. The late phase of formalin-induced pain response in mice was prevented by filenadol, without affecting the oedema. Filenadol is an antinociceptive agent that reduces the hyperalgesic effects of inflammatory mediators besides inhibiting partially the synthesis of eicosanoids.

The marine metabolites pacifenol, stypotriol triacetate and epitaondiol were tested for their effects on a number of inflammatory responses. Epitaondiol exhibited a potent topical anti-inflammatory activity related to inhibition of leukocyte accumulation. The other compounds showed a lower potency, similar to that of indomethacin. None of the compounds affected superoxide generation by human neutrophils but pacifenol effectively inhibited the degranulation response. This compound and epitaondiol decreased the release of eicosanoids with a higher potency on the cyclo-oxygenase pathway. Only epitaondiol inhibited human recombinant synovial phospholipase A2 activity in a concentration-dependent manner.

The effects of the natural flavonoid hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) on bromobenzene-induced hepatotoxicity in mice were investigated. We found a correlation between liver injury and hepatic lipid peroxidation besides a strong liver glutathione depletion due to the toxicant. Hispidulin at doses between 50 and 150 mg/kg i.p. compared favourably with the reference compound N-acetyl-L-cysteine for inhibition of liver injury and lipid peroxidation. The flavonoid at the highest dose tested was also able to counteract reduced glutathione depletion induced by bromobenzene in starved mice. These hepatoprotective effects can be related to the antioxidant properties of hispidulin.

Six flavonoid derivatives were tested for their influence on Naja naja and human recombinant synovial phospholipase A2. They showed a selectivity for the last enzyme with IC50 = 14.3, 17.6, 12.2 and 28.2 microM for quercetagetin, kaempferol-3-O-galactoside, scutellarein and scutellarein-7-O-glucuronide, respectively, while reduced effects were observed for hispidulin and hibifolin. After topical application all the flavonoids inhibited 12-O-tetradecanoylphorbol-13-acetate-induced ear oedema in mice with a potency comparable to that of indomethacin and they were also able to inhibit carrageenan-induced mouse paw oedema at a dose of 150 mg/kg p.o. The blockade of the free hydroxyl at C-7 or C-6 reduced the anti-inflammatory activity and also the inhibitory effect on human recombinant synovial phospholipase A2. These results are in accordance with the notion that group II phospholipases A2 may play a role in experimental inflammation, although several mechanisms seems to be involved in the anti-inflammatory effect of this group of flavonoids.