Polyphenol supplementation as a complementary medicinal approach to treating inflammatory bowel disease.

Inflammatory bowel disease (IBD) comprises a group of idiopathic chronic intestinal inflammation syndromes that are very common in developed countries. It is characterized by intermittent episodes of clinical remission and relapse, with recurrent inflammatory injury that can lead to structural damage of the intestine. The uncontrolled intestinal immune response to bacterial antigens leads to the production of abundant cytokines and chemokines, by activated leukocytes and epithelial cells, which trigger inflammatory and oxidative reactions. The current treatment of IBD consists in long-term anti-inflammatory therapy that, however, does not exclude relapses and side effects, frequently resulting in surgical intervention. Polyphenols have been acknowledged to be anti-oxidant and anti-inflammatory and therefore, have been proposed as an alternative natural approach to prevent or treat chronic inflammatory diseases. Most studies have been in animal models of colitis, using chemical inducers or mice defective in anti-inflammatory mediators and in intestinal cell lines treated with pro-inflammatory cytokines or lipid oxidation products. These studies provide evidence that polyphenols can effectively modulate intestinal inflammation. They exert their effects by modulating cell signaling pathways, mainly activated in response to oxidative and inflammatory stimuli, and NF-kB is the principal downstream effector. Polyphenols may thus be considered able to prevent or delay the progression of IBD, especially because they reach higher concentrations in the gut than in other tissues. However, knowledge of the use of polyphenols in managing human IBD is still scanty, and further clinical studies should afford more solid evidence of their beneficial effects.

Design and development of nanovehicle-based delivery systems for preventive or therapeutic supplementation with flavonoids.

To date more than 4000 compounds are recognized to belong to the class of flavonoids. These natural phenolic drugs are poorly soluble in water and are rapidly degraded and metabolized in the human body, but nevertheless are very promising for their potential contribution to the prevention and therapy of major chronic diseases, including cardiovascular and neurodegenerative diseases and cancer. In recent years a number of flavanols (e.g. catechins), flavonols (e.g. quercetin, myricetin) and isoflavones (e.g. genistein, daidzein) have been confirmed to possess strong antioxidant, anti-inflammatory, anti-proliferative and anti-aging activities. Incorporation into lipidic or polymer-based nanoparticles appears to markedly help the oral delivery of flavonoids, as these particles can protect the drug from degradation in the gastrointestinal tract and, by virtue of their unique absorption mechanism through the lymphatic system, also from first-pass metabolism in the liver. In addition, both oral and parenteral administration of flavonoids exploits a pharmacologic delivery route that guarantees sustained release of the active principle at the desired site of action. A comprehensive review of studies currently available on the in vitro and in vivo experimental administration of flavonoids by means of nanovectors may be of use as a foundation for the development of advanced delivery systems for these powerful compounds, in view of their adoption in primary and secondary disease prevention.

4-hydroxynonenal: a membrane lipid oxidation product of medicinal interest.

A comprehensive focus on 4-hydroxynonenal (HNE) as candidate molecule in a variety of pathophysiological conditions occurring in humans is here provided. Despite an active, now well characterized, metabolism in most cells and tissues, HNE can be easily detected and quantified by means of several methods, although with different sensitivity. Measurements of HNE and/or stable metabolites in biological fluids are already applied as lipid peroxidation/oxidative stress markers in a huge number of human disease processes, often sustained by inflammatory reactions. A primary involvement of this aldehydic product of membrane lipid oxidation in inflammation-related events, as well as in regulation of cell proliferation and growth, in necrotic or apoptotic cell death, appears supported by its marked ability to modulate several major pathways of cell signaling and, consequently, gene expression. The actual knowledge of HNE reactivity, metabolism, signaling and modulatory effect in the various human organs should provide a solid background to the investigation of the aldehyde's contribution to the pathogenesis of human major chronic diseases and would likely promote advanced and oriented applications not only in diagnosis and prevention but also in molecular treatment of human diseases.

4-Hydroxynonenal signalling to apoptosis in isolated rat hepatocytes: the role of PKC-delta.

4-Hydroxynonenal, a significant aldehyde end product of membrane lipid peroxidation with numerous biochemical activities, has consistently been detected in various human diseases. Concentrations actually detectable in vivo (0.1-5 microM) have been shown to up-regulate different genes and modulate various enzyme activities. In connection with the latter aspect, we show here that, in isolated rat hepatocytes, 1 microM 4-hydroxynonenal selectively activates protein kinase C-delta, involved in apoptosis of many cell types; it also induces very early activation of Jun N-terminal kinase, in parallel increasing activator protein-1 DNA-binding activity in a time-dependent manner and triggering apoptosis after only 120 min treatment. These phenomena are likely protein kinase C-delta-dependent, being significantly reduced or annulled by cell co-treatment with rottlerin, a selective inhibitor of protein kinase C-delta. We suggest that 4-hydroxynonenal may induce apoptosis through activation of protein kinase C-delta and of Jun N-terminal kinase, and consequent up-regulation of activator protein-1 DNA binding.

Oxidative stress and cell signalling.

An increasing body of evidence from animal models, human specimens and cell lines points to reactive oxygen species as likely involved in the pathways, which convey both extracellular and intracellular signals to the nucleus, under a variety of pathophysiological conditions. Indeed, reactive oxygen species (ROS), in a concentration compatible with that detectable in human pathophysiology, appear able to modulate a number of kinases and phosphatases, redox sensitive transcription factors and genes. This type of cell signalling consistently implies the additional involvement of other bioactive molecules that stem from ROS reaction with cell membrane lipids. The present review aims to comprehensively report on the most recent knowledge about the potential role of ROS and oxidised lipids in signal transduction processes in the major events of cell and tissue pathophysiology. Among the lipid oxidation products of ROS-dependent reactivity, which appear as candidates for a signalling role, there are molecules generated by oxidation of cholesterol, polyunsaturated fatty acids and phospholipids, as well as lysophosphatidic acid and lysophospholipids, platelet activating factor-like lipids, isoprostanes, sphingolipids and ceramide.

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents.

Among the oxidative breakdown products of omega-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor beta1 (TGFbeta1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFbeta1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFbeta1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Physiological amounts of ascorbate potentiate phorbol ester-induced nuclear-binding of AP-1 transcription factor in cells of macrophagic lineage.

The aim of the reported research was to assess the potential modulatory effect exerted by physiological amounts of ascorbate complexed or not to iron on activator protein 1 (AP-1) nuclear binding. The metal-vitamin complex was shown able to strongly potentiate AP-1 binding as induced by phorbol 12-myristate 13-acetate (PMA). Such enhancing activity by ascorbate was not observed on PMA-dependent induction of another redox-sensitive transcription factor nuclear factor kappaB (NF-kappaB). Experiments performed in the presence of the metal chelator desferrioxamine (DFO) clearly indicated that ascorbate rather than iron was responsible for the potentiation of PMA effect. The composition of AP-1 heterodimers revealed c-Jun, Jun D, and c-Fos as the major subunits upon PMA +/- ascorbate stimulation. The change in AP-1 components consequent to such stimuli was mainly dependent upon new synthesis. In fact, protein synthesis inhibitor cycloheximide (CHX) prevented the stimulation of AP-1 nuclear binding due to PMA and ascorbate plus PMA. Further, the vitamin was able to amplify the PMA-dependent induction of p38 and pJNK. Thus, a fine modulation of critical thiols by the vitamin along the MAPK pathway is conceivable.

Cholesterol oxidation products and fibrogenesis.

Oxidatively modified low density lipoproteins (oxLDL) are known to affect various cellular processes by modulating molecular transduction pathways and signaling nuclear transcription. In particular, the proinflammatory and proatherosclerotic effects of oxLDL are increasingly supported by a multitude of independent but consistent experimental studies. LDL oxidation might be a sequencial process where their lipid moieties are progressively but discretely oxidized, preceding the oxidation/modification of the apolipoprotein domain, an effect that can ultimately result in the uncontrolled uptake of these particles by cells, such as macrophages, and conversion of them to foam cells which is a hallmark of early atherogenesis. These lipoproteins appear to trigger a variety of events which are strongly implicated in the atherogenesis, the pathological process underlying vascular disease.

Lipid oxidation products in cell signaling.

The recent research on the impact that oxidative changes of biolipids could have in pathophysiology serves to explain how free radical-driven reactions not only are considered as mere toxicologic events, but also modulators of cell activity and function. Oxidatively modified low-density lipoproteins are known to affect various cellular processes by modulating various molecular pathways and signaling nuclear transcription. Among the lipid oxidation products detectable in ox-LDLs, and also in the atherosclerotic plaques, 4-hydroxynonenal has been widely investigated. This aldehyde was shown to upregulate AP-1 transcription factor, signaling through the MAP kinase pathway, with eventual nuclear localization and induction of a series of genes. Further, oxidation products of cholesterol and cholesterol esters, in ox-LDL are of similar interest, especially in relation to the pathogenesis of fibrosclerotic lesions of the arterial wall.

Liver AP-1 activation due to carbon tetrachloride is potentiated by 1,2-dibromoethane but is inhibited by alpha-tocopherol or gadolinium chloride.

Experimental acute intoxication by prooxidant haloalkanes produces marked stimulation of hepatic lipid peroxidation and cytolysis, which is followed by tissue regeneration. Our aim was to clarify the role of oxidative imbalance in the activation of the redox-sensitive transcription factor, activator protein-1 (AP-1), which is involved in tissue repair. Rats were poisoned with a very low concentration of carbon tetrachloride, given alone or in combination with another hepatotoxin, 1,2-dibromoethane, to provide varying extents of oxidative damage. The level of AP-1-DNA binding was analyzed by electrophoretic mobility shift assay on liver extracts, obtained from rats killed 6 h after poisoning. Stimulation of lipid peroxidation and AP-1 upregulation were already established when the hepatic damage due to carbon tetrachloride +/-1,2-dibromoethane was beginning to appear. Rat supplementation with the antioxidant vitamin E completely inhibited AP-1 upregulation, thus supporting a causative role of membrane lipid oxidation in the observed modulation of the transcription factor. Moreover, activation of Kupffer cells appears to be a crucial step in the increased AP-1 binding to DNA, the latter being largely prevented by gadolinium chloride, a macrophage-specific inhibitor.

Expression of monocyte chemotactic protein-1 precedes monocyte recruitment in a rat model of acute liver injury, and is modulated by vitamin E.

BACKGROUND: Increased expression of monocyte chemotactic protein-1 (MCP-1) has been indicated as a mechanism underlying leukocyte recruitment after liver injury. In this study we examined the temporal relationship between MCP-1 expression and the appearance of monocyte infiltration during acute liver injury. In addition, we tested the effects of vitamin E, a well known antioxidant, on these parameters. Rats were intoxicated with a single intragastric administration of CCl4 with or without pretreatment with vitamin E (atocopherol). METHODS: Monocyte chemotactic protein-1 expression was analyzed by northern blotting and in situ hybridization and monocyte infiltration was determined by ED-1 immunostaining. The results were quantitated by computerized image analysis. Expression of MCP-1 mRNA was significantly increased as early as 12 hours following injury, and progressively increased thereafter. In contrast, a significant increase in the number of ED-1 positive cells, an index of monocyte infiltration, was observed only 24 and 48 hours after injury. RESULTS: Vitamin E markedly reduced MCP-1 expression at the mRNA and protein levels, and caused a significant reduction in the number of monocyte/macrophages, indicating a role for oxidative stress in the induction of MCP-1 expression in vivo. Accordingly, in cultured hepatic stellate cells, different oxidative stress-related molecules increased MCP-1 mRNA. CONCLUSIONS: These data suggest the existence of a direct relationship between MCP-1 expression and monocyte infiltration after acute liver injury, and that preventing the generation of oxidative stress-related molecules results in decreased expression and release of this chemokine.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor beta1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis.

An increasing number of reports underscore the frequent association of fibrosclerotic diseases of lung, liver, arterial wall, brain, etc., with the accumulation of oxidatively modified lipids and proteins. A cause-and-effect relationship has been proposed between cellular oxidative damage and increased fibrogenesis based on the fact that experimental treatment with antioxidants either prevents or quenches the fibrotic process. With some peculiarities in the different organs, fibrosclerosis is essentially the result of the interaction of macrophages and extracellular matrix-producing cells. The cross-talk is mediated by fibrogenic cytokines, among which the most important appears to be transforming growth factor beta1 (TGF-beta1). This report describes treatment of different types of macrophage, of both human and murine origin, with 4-hydroxy-2,3-nonenal (HNE) a major aldehyde end product of membrane lipid oxidation found consistently to induce both mRNA expression and synthesis of TGF-beta1. Since increased HNE levels have been demostrated in the cirrhotic liver and in the oxidatively modified low-density human lipoproteins associated with atherosclerosis, the up-regulation of macrophage TGF-beta1 by HNE appears to be involved in the pathogenesis of these and similar diseases characterized by fibrosclerosis.

Detection of cytochrome P4503A (CYP3A) in human hepatic stellate cells.

In this study we have investigated the occurrence of cytochrome P450 isoforms and of related cytochrome P450 reductase in human hepatic stellate cells (hHSC), a type of cell having relevant roles in physiopathological conditions of the liver. By performing immunoblotting of hHSC microsomes and immunofluorescence analysis associated to confocal laser microscopy we detected only P450 enzymes belonging to the cytochrome P450 3A subfamily (CYP3A) as well as cytochrome P450 reductase. The presence of CYP3A was further indicated by detection of testosterone 6beta-hydroxylase activity in hHSC microsomes. Other important human P450 forms were either undetectable (CYP1A2, CYP2E1, CYP2C8/9/19 and CYP4A) or bearly detectable (CYP1A1) in hHSC. This is the first study showing existence of active cytochrome P450 isoforms in human HSC.

Oxidative damage and transforming growth factor beta 1 expression in pretumoral and tumoral lesions of human intestine.

The aim of this study was to evaluate a possible relationship between oxidative stress and transforming growth factor beta 1 (TGF beta 1) expression in human colon adenocarcinoma. Crohn's disease, an inflammatory pathology of the intestine often regarded to as precancerous, was also examined. Indices of impaired redox balance were monitored in blood and in bioptic samples from 10 adult patients with adenocarcinoma of the colon and from five patients with Crohn's disease. On tissue samples TGF beta 1 mRNA expression was also determined. Ten healthy adults provided normal reference values for plasma indices of oxidative stress, and normal tissue distant from the lesions was used for comparative analysis. Fluorescent adducts with plasma proteins of malonaldehyde (MDA) and 4-hydroxynonenal (HNE) were significantly lower than controls in the plasma from cancer patients and significantly higher in the plasma from Crohn's patients. In adenocarcinoma biopsies, susceptibility to lipid peroxidation processes and TGF beta 1 expression were below the relative control; in Crohn's disease, lipid peroxidation and cytokine expression were both above the relative control. The findings obtained suggest the existence of an association between oxidative damage and fibrogenic cytokine expression in the human intestine. Further studies are needed to conclusively prove the correlation between the two events.

Activation of human immunodeficiency virus long terminal repeat by arachidonic acid.

Arachidonic acid is the precursor of highly reactive mediators, including prostaglandins and leukotrienes, and the most abundant n-6 polyunsaturated fatty acid in mammalian cell membranes. It is released from phospholipids upon many inflammatory stimuli. In this study, a chloramphenicol acyltransferase reporter gene, under control of the human immunodeficiency virus-1 long terminal repeat, was strongly induced upon treating human promonocytes with arachidonic acid. The n-3 fatty acid eicosapentenoic, found in abundance in fish oil, had no effect. HIV-1 long terminal repeat activation by arachidonic acid was suppressed by inhibitors of both lipoxygenase and cyclooxygenase pathways, suggesting that metabolites, rather than arachidonic acid itself, mediated the stimulatory effect. This is the first report linking HIV-1 expression to the metabolism of arachidonic acid.

Biogenic 4-hydroxy-2-nonenal activates transcription factor AP-1 but not NF-kappa B in cells of the macrophage lineage.

A large spectrum of pro-oxidant agents, including molecules with lipoperoxidative effect, can modulate gene expression through modification of the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). In this study the effect on these redox-sensitive factors by 4-hydroxy-2-nonenal (HNE), a major aldehydic product of lipid peroxidation, was examined in two cell lines of the macrophage type. Incubation in the presence of microM concentrations of the aldehyde led to a rapid increase of AP-1 binding with a transient maximum 30 min from HNE addition to the culture medium in both cell lines. On the contrary, HNE did not stimulate nuclear translocation of NF-kappa B. The diverging effect of HNE on the two transcription factors is likely related to the demonstrated differential activation pathway of AP-1 and NF-kappa B in macrophages. The HNE-induced activation of AP-1 suggests the aldehyde's involvement in the regulatory mechanisms of cell proliferation and differentiation.

Nuclear factor kB is activated by arachidonic acid but not by eicosapentaenoic acid.

The omega-6 arachidonic acid supplementation of the human promonocytic cell line U937 strongly stimulates the nuclear translocation of the transcription factor NF-kB. Inhibitors of arachidonate oxidative metabolism prevent NF-kB activation, indirectly indicating a role for prostaglandin and leukotriene metabolites in the genesis of this phenomenon. Of note, omega-3 eicosapentaenoic acid does not exert any effect on NF-kB DNA binding. In subsequent experiments, prostaglandin E2 consistently showed the ability to activate NF-kB in U937 promonocytic cells, as well as in J774 macrophages. NF-kB activation by arachidonate, together with the lack of effect by eicosapentaenoic acid, suggests a way to modulate the expression of certain genes by means of a suitable dietary n-6/n-3 fatty acid ratio.

Induction of procollagen type I gene expression and synthesis in human hepatic stellate cells by 4-hydroxy-2,3-nonenal and other 4-hydroxy-2,3-alkenals is related to their molecular structure.

4-Hydroxy-2,3-nonenal (HNE) has been shown to induce procollagen type I gene expression and synthesis in hepatic stellate cells (HSC), i.e. the cells responsible for deposition of collagen and other extracellular matrix proteins in fibrotic liver. Here we report that the stimulatory effect of HNE mostly depends on the contemporary presence of the hydroxyl group in position C4 and of the double bond between position C2 and C3 since equimolar concentrations of 2,3-nonenal as well as of nonenal did not procollagen type I synthesis either at mRNA or at protein levels. Accordingly to this concept, all the other 4-hydroxy-2,3-alkenals of different chain length tested on cultured human HSC (4-hydroxy-2,3-hexenal, 4-hydroxy-2,3-octenal and 4-hydroxy-2,3-undecenal) strongly induced procollagen type I gene expression and synthesis. The stimulatory effect of 4-hydroxy-2,3-alkenals may depend on the well known ability of these aldehydes to react with either SH-groups or NH2-groups of functional proteins.