María Carmen Terencio

Role of nuclear factor-κB and heme oxygenase-1 in the mechanism of action of an anti-inflammatory chalcone derivative in RAW 264.7 cells

The synthetic chalcone 3′,4′,5′,3,4,5‐hexamethoxy‐chalcone (CH) is an anti‐inflammatory compound able to reduce nitric oxide (NO) production by inhibition of inducible NO synthase protein synthesis. In this work, we have studied the mechanisms of action of this compound. CH (10–30 μM) prevents the overproduction of NO in RAW 264.7 macrophages stimulated with lipopolysaccharide (1 μg ml−1) due to the inhibition of nuclear factor κB (NF‐κB) activation. We have shown that treatment of cells with CH results in diminished degradation of the NF‐κB–IκB complex leading to inhibition of NF‐κB translocation into the nucleus, DNA binding and transcriptional activity. We also demonstrate the ability of this compound to activate NfE2‐related factor (Nrf2) and induce heme oxygenase‐1 (HO‐1). Our results indicate that CH determines a rapid but nontoxic increase of intracellular oxidative species, which could be responsible for Nrf2 activation and HO‐1 induction by this chalcone derivative.  This novel anti‐inflammatory agent simultaneously induces a cytoprotective response (HO‐1) and downregulates an inflammatory pathway (NF‐κB) with a mechanism of action different from antioxidant chalcones.

Diclofenac sodium and cyclosporin A inhibit human lens epithelial cell proliferation in culture

Abstract• Purpose: To investigate the effect of diclofenac sodium salt and cyclosporin A (CsA) on human lens epithelial cell (HLEC) growth in culture. • Methods: Cultures of HLEC were obtained from anterior capsules from extracapsular cataract surgery. Third-passage cells were seeded in 96-well plates in 0.1 ml culture medium. Cytotoxicity was estimated by the tetrazolium test in confluent monolayers after 24 h exposure to a wide range of concentrations of diclofenac and CsA. The effect of subcytotoxic concentrations of diclofenac and CsA on HLEC proliferation in subconfluent cultures was evaluated after 24 and 72 h of exposure. To investigate the relationship between PGEZ synthesis and the inhibitory effect of these drugs, after 24 h of exposure to diclofenac and CsA the production of PGE2 was measured by radioimmunoassay. We also tested the effect of exogenous PGE2 addition to diclofenac 72-h-treated cultures. • Results: Diclofenac and CsA (at concentrations ≥65 μM and ≥2.5 μM, respectively) inhibited the proliferation of subconfluent cultures of HLEC in a dose-dependent fashion. Diclofenac inhibits PGE2 synthesis, while CsA at high doses stimulates PGE2 synthesis of cultured HLEC. Exogenous PGE2 addition reversed in part the inhibitory effect of diclofenac.• Conclusions: Diclofenac and CsA at appropriate doses are effective in inhibiting cultured HLEC proliferation. This could be of interest to prevent posterior capsule opacification. Further in vivo experimental studies seem worthwhile.

Involvement of secretory phospholipase A2 activity in the zymosan rat air pouch model of inflammation.

1 In the zymosan rat air pouch model of inflammation we have assessed the time dependence of phospholipase A2 (PLA2) accumulation in the inflammatory exudates as well as cell migration, myeloperoxidase activity, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels. 2 A significant increase in PLA2 activity was detected in 1,200 g supernatants of exudates 8 h after injection of zymosan into rat air pouch. This event coincided with peaks in cell accumulation (mainly neutrophils) and myeloperoxidase activity in exudates and was preceded by a rise in eicosanoid levels. 3 This enzyme (without further purification) behaved as a secretory type II PLA2 with an optimum pH at 7 − 8 units, lack of selectivity for arachidonate release and dependence on mM calcium concentrations for maximal activity. 4 The PLA2 inhibitors manoalide and scalaradial inhibited this enzyme activity in vitro in a concentration‐dependent manner. Scalaradial also inhibited zymosan stimulated myeloperoxidase release in vitro. 5 Injection of the marine PLA2 inhibitor scalaradial together with zymosan into the pouch at doses of 0.5, 1 and 5 μmol per pouch resulted in a dose‐dependent inhibition of PLA2 activity in exudates collected 8 h later. Myeloperoxidase levels and cell migration were also decreased, while eicosanoid levels were not modified. 6 Colchicine administration to rats prevented infiltration and decreased PLA2 levels in the 8 h zymosan‐injected air pouch. 7 These results indicate that during inflammatory response to zymosan in the rat air pouch a secretory PLA2 activity is released into the exudates. The source of this activity is mainly the neutrophil which migrates into the pouch. 8 Scalaradial exerts anti‐inflammatory effects in the zymosan air pouch.

Avarol inhibits TNF-α generation and NF-κB activation in human cells and in animal models

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.

Inhibition of the NF-κB signaling pathway mediates the anti-inflammatory effects of petrosaspongiolide M

Petrosaspongiolide M (PT) is a potent secretory phospholipase A(2) inhibitor and anti-inflammatory agent. This marine metabolite reduced the production of nitrite, prostaglandin E(2), and tumor necrosis factor-alpha in the mouse air pouch injected with zymosan. These effects were also observed in mouse peritoneal macrophages stimulated with zymosan. Inhibition of these inflammatory mediators was related to reductions in inducible nitric oxide synthase, cyclo-oxygenase-2, and tumor necrosis factor-alpha expression. Since nuclear factor-kappaB (NF-kappaB) appears to play a central role in the transcriptional regulation of these proteins by macrophages, we investigated the effects of PT on this transcription factor. We found that PT was a potent inhibitor of the NF-kappaB pathway since at 1 microM it strongly decreased NF-kappaB-DNA binding in response to zymosan, in mouse peritoneal macrophages. Our study also indicated that PT could interfere with a key step in NF-kappaB activation, the phosphorylation of IkappaBalpha, resulting in inhibition of IkappaBalpha degradation. The control of a wide range of mediators by PT suggests a potentially wide therapeutic spectrum for this marine metabolite in inflammatory conditions.

Antipsoriatic effects of avarol-3′-thiosalicylate are mediated by inhibition of TNF-α generation and NF-κB activation in mouse skin

Avarol is a marine sesquiterpenoid hydroquinone with anti‐inflammatory and antipsoriatic properties. The aim of this study was to evaluate the in vitro and in vivo pharmacological behaviour of the derivative avarol‐3′‐thiosalicylate (TA) on some inflammatory parameters related to the pathogenesis of psoriasis.

1-(2,3,4-trimethoxyphenyl)-3-(3-(2-chloroquinolinyl))-2-propen-1-one, a chalcone derivative with analgesic, anti-inflammatory and immunomodulatory properties

Abstract:Objetive and Design: The synthetic chalcone derivative 1-(2,3,4-trimethoxyphenyl)-3-(3-(2-chloroquinolinyl))-2-propen-1-one (TQ) was evaluated for its immunomodulatory and anti-inflammatory efficacy in vitro and in vivo. Material and Subjets: Human neutrophils and lymphocytes from healthy volunteers and RAW 264.7 murine macrophages. Swiss mice and Lewis rats were randomly divided into groups of six animals. Treatment: TQ was orally administered in all in vivo assays (10–30 mg/kg). Methods: Elastase, superoxide and LTB4 release were assayed in human neutrophils, NO/PGE2 production and NF-κB activation in RAW 264.7, and 3H thymidine incorporation in human lymphocytes. Zymosan-stimulated air pouches, DNFB-DTH, PBQ-induced writhings and formalin-induced pain were assayed in mice. Adjuvant-induced arthritis was tested in rats. Dunnetts t-test was employed for statistical analysis. Results: Human T-cell proliferation, neutrophil functions and NO/PGE2 production in murine macrophages were inhibited by TQ (IC50 in the μM range), which showed anti-inflammatory, immunomodulatory and analgesic effects. Conclusions: Our findings indicate the potential interest of TQ in the modulation of some immune and inflammatory responses probably by NF-κB inhibition.

INHIBITION OF INFLAMMATORY RESPONSES BY EPITAONDIOL AND OTHER MARINE NATURAL PRODUCTS

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.

ANTIOXIDANT PROFILE OF MONO- AND DIHYDROXYLATED FLAVONE DERIVATIVES IN FREE RADICAL GENERATING SYSTEMS

Abstract A number of free radical generating systems were used to investigate the antioxidant properties and structure-activity relationships of a series of monohydroxylated and dihydroxylated flavones. Ortho-dihydroxylated flavones showed the highest inhibitory activity on en zymic and non-enzymic microsomal lipid peroxidation as well as on peroxyl radical scavenging. Most flavones were weak scavengers of hydroxyl radical, while ortho-dihydroxylated flavones interacted with superoxide anion generated by an enzymic system or by human neutrophils. This series of compounds did not exert cytotoxic effects on these cells. Scavenging of superoxide and peroxyl radicals may determ ine the antioxidant properties of these active flavones.

Inhibition of 5-lipoxygenase activity by the natural anti-inflammatory compound aethiopinone.

Abstract:Objetive and Design: We have investigated the mechanisms of action of aethiopinone, an anti-inflammatory compound from Salvia aethiopis L. roots.¶Material and Subjects: Human neutrophils from healthy volunteers and murine peritoneal macrophages. Swiss mice were randomly divided into groups of six animals.¶Treatment: Test compounds were applied topically in the mouse ear oedema test. In the air pouch, mice received aethiopinone (0.001-0.5 μmol/pouch or 12.5-50 mg/kg p.o.).¶Methods: LTB4 production was assayed in human neutrophils and COX-2 and iNOS activities in murine macrophages. Air pouches were induced subcutaneously in mice and injected with zymosan on the day six. Mouse ear oedema was induced by arachidonic acid. Dunnetts t-test was employed for statistical analysis.¶Results: We have observed potent inhibitory effects on human neutrophil LTB4 production without effects on COX or NOS activities. Aethiopinone is an in vitro inhibitor of 5-LO from human neutrophils (IC50 = 0.11 μM). In addition, aethiopinone reduced leukocyte accumulation and showed in vivo inhibitory activity on this enzyme.¶Conclusions: Our results indicate that inhibition of 5-LO could participate in the anti-inflammatory properties of this natural product.¶