Oxygen free radicals and the systemic inflammatory response

The generation of oxygen free radicals is known to be involved in the development of the systemic inflammatory response syndrome. In addition to their actions as noxious mediators generated by inflammatory cells, these molecules play also a crucial role contributing to the onset and progression of inflammation in distant organs. In the early stages of the process, free radicals exert their actions via activation of nuclear factors, as NFkappaB or AP-1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion molecules. Finally, free radicals exert their toxic effects at the site of inflammation by reacting with different cell components, inducing loss of function and cell death. This review focuses on progress in the understanding the different actions of free radicals at the sequential stages of the development of the systemic inflammatory response.     

Screening and evaluation of antioxidant activity of some pyridazine derivatives

Antioxidants are compounds that can delay, inhibit, or prevent the oxidation of materials that can be oxidized by scavenging free radicals and help in diminishing oxidative stress. They belong to different chemical classes. Recently there are studies related to pyridazinone derivatives for their antioxidant activities. Since there are evidences implicates reactive oxygen species and nitric oxide as mediators of inflammation and/or tissue damage in inflammatory and arthritic disorders it was though that compounds that have both antioxidant and anti-inflammatory activities would have been essential for the inflammatory diseases. Based on these findings a series of 2H-pyridazine-3-one and 6-chloropyridazine analogues that have anti-inflammatory activity was tested in vitro on superoxide formation and effects on lipid peroxidation were determined against alpha-tocopherol. Most of the compounds have strong inhibitory effect on superoxide anion (between 84% - 99%) at 10(- 3) M concentration. In addition, these compounds showed similar activity to alpha-tocopherol at 10(- 3) M concentrations.     

Oxygen radicals in the adult respiratory distress syndrome, in myocardial ischemia and reperfusion injury, and in cerebral vascular damage

Recent work suggests that oxygen radicals may be important mediators of damage in a wide variety of pathologic conditions. In this review we consider the evidence supporting the participation of oxygen radicals in the adult respiratory distress syndrome, in ischemia reperfusion injury in the myocardium, and in cerebral vascular injury in acute hypertension and traumatic brain injury. In the adult respiratory distress syndrome there is active sequestration of polymorphonuclear neutrophils in the pulmonary vascular system. There is evidence that activation of these neutrophils results in the production of oxygen radicals which injure the capillary membrane and increase permeability, leading to progressive hypoxia and decreased lung compliance which are hallmarks of the syndrome. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. The metabolism of arachidonic acid is the source of oxygen free radical production in these conditions. In myocardial ischemia and reperfusion injury, the ischemic myocyte is "primed" for free radical production. With reperfusion and reintroduction of molecular oxygen there is a burst of oxygen radical production resulting in extensive tissue destruction. Myocardial ischemia--reperfusion injury shares in common with the other two syndromes activation of the arachidonic acid cascade and acute inflammation. Thus it would appear that the generation of toxic oxygen species may represent a final common pathway of tissue destruction in several pathophysiologic states.     

Regulation of inflammation in cancer by eicosanoids

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.     

Prostanoids and their receptors that modulate dendritic cell-mediated immunity

Dendritic cells (DC) are essential for the initiation of immune responses by capturing, processing and presenting antigens to T cells. In addition to their important role as professional APC, they are able to produce immunosuppressive and pro-inflammatory prostanoids from arachidonic acid (AA) by the action of cyclooxygenase (COX) enzymes. In an autocrine and paracrine fashion, the secreted lipid mediators subsequently modulate the maturation, cytokine production, Th-cell polarizing ability, chemokine receptor expression, migration, and apoptosis of these extremely versatile APC. The biological actions of prostanoids, including their effects on APC-mediated immunity and acute inflammatory responses, are exerted by G protein-coupled receptors on plasma membrane. Some COX metabolites act as anti-inflammatory lipid mediators by binding to nuclear receptors and modulating DC functions. Although the role of cytokines in DC function has been studied extensively, the effects of prostanoids on DC biology have only recently become the focus of investigation. This review summarizes the current knowledge about the role of prostanoids and their receptors in modulating DC function and the subsequent immune responses.     

Genetic factors determine the contribution of leukotrienes to acute inflammatory responses

Leukotrienes (LT) are potent lipid mediators synthesized by the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LT have been implicated in a broad spectrum of inflammatory processes. To investigate the influence of genetic factors on the contribution of LT to acute inflammation, we generated congenic 5-lipoxygenase-deficient 129, C57BL/6 (B6), and DBA/1Lac (DBA) mouse lines. Topical application of AA evoked a vigorous inflammatory response in 129 and DBA mice, whereas only a modest response was seen in B6 animals. The response to AA in 129 and DBA strains is LT dependent. In contrast, LT make little contribution to this response in B6 mice. AA-induced inflammation in B6 mice is prostanoid dependent, since this response was substantially reduced by treating B6 mice with a cyclooxygenase inhibitor. These data suggest that prostanoids are essential for AA-induced cutaneous inflammation in B6 mice, whereas LT are the major mediators of this response in 129 and DBA strains. In contrast, the response to AA in the peritoneal cavity is robust in the 129 and B6 strains, but was significantly blunted in DBA mice, showing that strain differences in the response to AA are tissue specific. Variations in these and other experimental models of inflammation appear to correlate directly with the ability of a particular mouse strain and a specific tissue to respond to LT, specifically LTC4. Taken together, these findings indicate that the relative contribution of prostanoids and LT to inflammatory responses is variable not only between strains but also between different tissues within these inbred mouse lines.     

Proinflammatory actions of thromboxane receptors to enhance cellular immune responses

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE(2), in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A(2) synthesis. To study the immunoregulatory actions of thromboxane A(2), we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP(-/-) spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.     

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.     

New 3- and 4-hydroxyfuranones as anti-oxidants and anti-inflammatory agents

Two series of new furanones substituted by methylsulfonylphenyl or methylsulfamidophenyl moieties were found to protect against oxidation damage by inhibiting or quenching free radicals and reactive oxygen species in in vitro experiments. The effect on lipid peroxidation was also examined. In addition, we investigated the activity of products in two models of inflammation: phorbol ester-induced ear edema in mice and carrageenan-induced paw edema in rat. The most powerful compounds and with reducing activity against DPPH (IC50=1779 and 57 microM, respectively), superoxide anion quenching capacity (IC50=511 and 49 microM, respectively), lipid peroxidation inhibitory effect and anti-inflammatory properties (about 50-65% inhibition of edema at 200 mg/kg ip in both tests used) were selected for further pharmacological and toxicological tests because of their attractive profile for the treatment of inflammatory diseases.     

Adenosine up-regulates cyclooxygenase-2 in human granulocytes: impact on the balance of eicosanoid generation

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine's anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE(2) in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE(2) on leukotriene B(4) formation and involved the EP(2) receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE(2) generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.     

Bioactive products formed in humans from fish oils

Resolvins, maresins, and protectins can be formed from fish oils. These specialized pro-resolving mediators (SPMs) have been implicated in the resolution of inflammation. Synthetic versions of such SPMs exert anti-inflammatory effects in vitro and when administered to animal models. However, their importance as endogenous products formed in sufficient amounts to exert anti-inflammatory actions in vivo remains speculative. We biased our ability to detect SPMs formed in healthy volunteers by supplementing fish oil in doses shown previously to influence blood pressure and platelet aggregation under placebo-controlled conditions. Additionally, we sought to determine the relative formation of SPMs during an acute inflammatory response and its resolution, evoked in healthy volunteers by bacterial lipopolysaccharide (LPS). Bioactive lipids, enzymatic epoxyeicosatrienoic acids (EETs), and free radical-catalyzed prostanoids [isoprostanes (iPs)] formed from arachidonic acid and the fish oils, served as comparators. Despite the clear shift from ω-6 to ω-3 EETs and iPs, we failed to detect a consistent signal, in most cases, of SPM formation in urine or plasma in response to fish oil, and in all cases in response to LPS on a background of fish oil. Our results question the relevance of these SPMs to the putative anti-inflammatory effects of fish oils in humans.     

Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.

In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.     

Interaction between nitric oxide and prostanoids in arterioles of rat cremaster muscle in vivo

We studied in vivo interactions of nitric oxide (NO), oxidative stress, and prostanoids derived from the cyclooxygenase pathway in the arterioles studied by intravital microscopy in peripheral muscle. Topical administration of NO synthase (NOS) inhibitor Nomega-nitro-l-arginine (l-NNA) or cyclooxygenase inhibitor mefenamic acid (MA) alone leads to vasoconstriction. We found that l-NNA after MA induced an additional constriction, whereas MA after l-NNA induced a relative dilation. Therefore, an additional constriction was found when MA was administered after l-NNA in the presence of the thromboxane A2 synthase-PGH2 (TP) receptor antagonist SQ-29548. We also found a relative dilation when the TP receptor antagonist was administered after NOS inhibition by l-NNA. In the presence of superoxide dismutase and catalase, l-NNA-induced vasoconstriction is reduced, and the dilation observed after addition of MA in presence of the reactive oxygen species is no longer present. Taken together, these results showed that NO inhibition induced a shift in the synthesis or in the effects of cyclooxygenase products, in favor of constrictor prostanoids. This effect of NO inhibition disappears when reactive oxygen species are scavenged by superoxide dismutase and catalase.     

Vascular effects of oxygen-derived free radicals

This review attempts to summarize the available data regarding the vascular actions of free oxygen radicals. Studies on blood vessels in situ and in vitro demonstrate that free oxygen radicals can evoke both vasodilation and vasoconstriction. Free oxygen radicals can modulate the tone of vascular smooth muscle by acting directly on the smooth muscle cells, and also via indirect mechanisms by changes in the production or biological activity of vasoactive mediators. The individual oxygen radicals may have different (sometimes opposite) vascular effects. Superoxide anion inactivates endothelium-derived relaxing factor and the adrenergic neurotransmitter norepinephrine. Hydrogen peroxide and the hydroxyl radical evoke vasodilation by acting directly on vascular smooth muscle and also by stimulating the synthesis/release of endothelium-derived relaxing factor. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. Production of oxygen-derived free radicals by activated neutrophils may be responsible for vasodilation and increased permeability of capillary membrane during the acute inflammatory process. Free oxygen radicals also play an important role in reperfusion injury of various organs, and vascular actions of the free radicals may contribute to the damage of parenchymal tissues.     

Lipid droplets in inflammation and cancer

Accumulation of lipid droplets (also known as lipid bodies or adiposomes) within leukocytes, epithelial cells, hepatocytes and other non-adipocytic cells is a frequently observed phenotype in infectious, neoplastic and other inflammatory conditions. Lipid droplet biogenesis is a regulated cellular process that culminates in the compartmentalization of lipids and of an array of enzymes, protein kinases and other proteins, suggesting that lipid droplets are inducible organelles with roles in cell signaling, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Enzymes involved in eicosanoid synthesis are localized at lipid droplets and lipid droplets are sites for eicosanoid generation in cells during inflammation and cancer. In this review, we discuss the current evidence related to the biogenesis and function of lipid droplets in cell metabolism and signaling in inflammation and cancer. Moreover, the potential of lipid droplets as markers of disease and targets for novel anti-inflammatory and antineoplastic therapies will be discussed.     

Conversion of the 2,2,6,6-tetramethylpiperidine moiety to a 2,2-dimethylpyrrolidine by cytochrome P450: evidence for a mechanism involving nitroxide radicals and heme iron

Earlier we described a novel cytochrome P450 (CYP) catalyzed metabolism of the 2,2,6,6-tetramethylpiperidine (2,2,6,6-TMPi) moiety in human liver microsomes to a ring-contracted 2,2-dimethylpyrrolidine (2,2-DMPy) [Yin, W., et al. (2003) Drug Metab. Dispos. 31, 215-223]. In the current report, evidence is provided for the involvement of 2,2,6,6-TMPi hydroxylamines and their one-electron oxidation products, the nitroxide radicals, as intermediates in this pathway. Nitroxide radicals could be converted to their corresponding 2,2-DMPy metabolites by "inactivated CYP3A4", as well as by a number of other heme proteins and hemin, suggesting that this is a heme-catalyzed process. The conversion of nitroxide radicals to the 2,2-DMPy products by CYP3A4 or hemin was accompanied by the generation of acetone in incubations, providing evidence that the three-carbon unit from 2,2,6,6-TMPi was lost as acetone. With one model 2,2,6,6-TMPi nitroxide radical, evidence for an alternate pathway, which resulted in the formation of an intermediate that incorporated two oxygen atoms from water of the incubation medium before collapsing to the 2,2-DMPy product, was also obtained. To account for both pathways, a mechanism involving interaction of the nitroxide radicals with heme iron (Fe(III)), followed by a homolytic scission of the N-O bond and transfer of the nitroxide oxygen to heme iron to form a perferryl-oxygen complex, is proposed. The nitrogen-centered 2,2,6,6-TMPi radical thus formed then precipitates the contraction of the piperidine ring via C2-C3 bond cleavage, and the resulting product further oxidizes to an exocyclic iminium ion (by the perferryl-oxygen complex); the latter may undergo capture by water from the incubation medium and eliminate the three-carbon unit via N-dealkylation. It remains to be determined whether this novel interaction of nitroxide radicals with heme iron has any relevance in regard to the known biological properties of these stable radical species.     

诱导型一氧化氮合酶抑制剂研发现状

一氧化氮(NO)对炎症性疾病的治疗作用近来引起了广泛的重视。诱导型一氧化合成酶(iNOS)被发现广泛地参与趋炎因子表达和反应性氧化产物(ROS)/反应性氮化产物(RNS)的产生,从而进一步证明了一氧化氮在炎症病理发生发展中的关键作用。由于传统的抗炎药物环氧合酶-2(COX-2)抑制剂被报导有较多副作用,新型抑制炎症药物的研究开发势在必行。本文分别介绍了化学来源、生物来源、植物来原性iNOS抑制剂阻的开发、研究现状,阐述了其在断炎症信息传递通道中的作用。表明了iNOS抑制剂防止炎症损害的相关机理,提出iNOs不仅能在初始阶段影响炎症的发生,也对抑制和终结炎症有作用。最后进一步介绍了用中草药研发iNOs抑制剂的可能性,展望了于中药在该领域内的巨大前景。