María J. Tuñón

Fruit polyphenols, immunity and inflammation

Flavonoids are a large class of naturally occurring compounds widely present in fruits, vegetables and beverages derived from plants. These molecules have been reported to possess a wide range of activities in the prevention of common diseases, including CHD, cancer, neurodegenerative diseases, gastrointestinal disorders and others. The effects appear to be related to the various biological/pharmacological activities of flavonoids. A large number of publications suggest immunomodulatory and anti-inflammatory properties of these compounds. However, almost all studies are in vitro studies with limited research on animal models and scarce data from human studies. The majority of in vitro research has been carried out with single flavonoids, generally aglycones, at rather supraphysiological concentrations. Few studies have investigated the anti-inflammatory effects of physiologically attainable flavonoid concentrations in healthy subjects, and more epidemiological studies and prospective randomised trials are still required. This review summarises evidence for the effects of fruit and tea flavonoids and their metabolites in inflammation and immunity. Mechanisms of effect are discussed, including those on enzyme function and regulation of gene and protein expression. Animal work is included, and evidence from epidemiological studies and human intervention trials is reviewed. Biological relevance and functional benefits of the reported effects, such as resistance to infection or exercise performance, are also discussed.

The flavonoid quercetin ameliorates liver damage in rats with biliary obstruction

BACKGROUND/AIMS Our aim was to investigate whether the antioxidant quercetin might protect against liver injury in chronically biliary obstructed rats. METHODS Secondary biliary cirrhosis was induced by 28 days of bile duct obstruction. Animals received quercetin at 75, 150 and 300 micromol x kg body wt(-1) x day(-1) i.p. through the experimental period or at 150 micromol x kg body wt(-1) x day(-1) i.p. for the last 2 weeks. RESULTS Bile duct obstruction resulted in a decrease in the activities of antioxidant enzymes. Liver oxidised/reduced (GSSG/GSH) glutathione ratio, hepatic and mitochondrial thiobarbituric acid reactive substances (TBARS) and collagen content were significantly increased and a marked fibrosis and bile ductular proliferation was observed. Quercetin corrected the reduction in glutathione concentration and partially prevented the increase in collagen concentration, TBARS and GSSG/GSH ratio. Treatment resulted in a significant preservation of the activities of antioxidant enzymes, a less pronounced fibrosis and a marked inhibition of bile ductular proliferation. Maximal effects were reached with the intermediate quercetin dose given for 2 or 4 weeks. CONCLUSIONS Quercetin reduces liver oxidative damage, ductular proliferation and fibrosis in biliary-obstructed rats. These effects suggest that it might be a useful agent to preserve liver function in patients with biliary obstruction.

Potential of Flavonoids as Anti-inflammatory Agents: Modulation of Pro- Inflammatory Gene Expression and Signal Transduction Pathways

Flavonoids are a large class of naturally occurring compounds widely present in fruits, vegetables, and beverages derived from plants. Reports have suggested that these compounds might be useful for the prevention of a number of diseases, partly due to their anti-inflammatory properties. It has been demonstrated that flavonoids are able to inhibit expression of isoforms of inducible nitric oxide synthase, ciclooxygenase and lipooxygenase, which are responsible for the production of a great amount of nitric oxide, prostanoids and leukotrienes, as well as other mediators of the inflammatory process such as cytokines, chemokines or adhesion molecules. Modulation of the cascade of molecular events leading to the over-expression of those mediators include inhibition of transcription factors such as nuclear factor kappa B, activator protein 1, signal transducers and activators of transcription, CCAAT/enhancer binding protein and others. Effects on the binding capacity of transcription factors may be regulated through the inhibition of protein kinases involved in signal transduction, such as mitogen activated protein kinases. Although the numerous studies published with in vitro approaches allow identifying molecular mechanisms of flavonoid effects, the limited bioavailability of these molecules makes necessary validation in humans. Whatever the case, the data available make clear the potential utility of dietary flavonoids or new flavonoid-based agents for the possible treatment of inflammatory diseases. The present review summarizes recent research data focusing on the modulation of the expression of different inflammatory mediators by flavonoids and the effects on cell signaling pathways responsible for their anti-inflammatory activity.

Effects of quercetin on liver damage in rats with carbon tetrachloride-induced cirrhosis.

Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free radical-scavenging activities. Evidence of oxidative reactions is often associated with various chronic disease processes characterized by accumulation of connective tissue. This study was aimed to investigate the protective effects of chronic administration of the flavonoid quercetin (150 μmol/kg body wt/day intraperitoneally) in rats with carbon tetrachloride-induced fibrosis. In animals rendered cirrhotic by administration of carbon tetrachloride for 16 weeks, cell necrosis, fibrosis, and inflammatory infiltration were found. Histological abnormalities were accompanied by a higher hepatic content of collagen and thiobarbituric acid-reactive substances. Expression of inducible nitric oxide synthase (iNOS) was significantly increased in the liver. Treatment with quercetin during 3 weeks improved liver histology and reduced collagen content, iNOS expression, and lipid peroxidation. Those effects were associated with an increased total peroxyl radical-trapping antioxidant capacity of liver. We conclude that quercetin is effective in this model of liver damage.

A comparison of the effects of kaempferol and quercetin on cytokine-induced pro-inflammatory status of cultured human endothelial cells.

We investigated the effects of the flavonols kaempferol and quercetin on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), endothelial cell selectin (E-selectin), inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), and on the activation of the signalling molecules NF-kappaB and activator protein-1 (AP-1), induced by a cytokine mixture in cultured human umbilical vein endothelial cells. Inhibition of reactive oxygen and nitrogen species generation did not differ among both flavonols at 1 micromol/l but was significantly stronger for kaempferol at 5-50 micromol/l. Supplementation with increasing concentrations of kaempferol substantially attenuated the increase induced by the cytokine mixture in VCAM-1 (10-50 micromol/l), ICAM-1 (50 micromol/l) and E-selectin (5-50 micromol/l) expression. A significantly inhibitory effect of quercetin on VCAM-1 (10-50 micromol/l), ICAM-1 (50 micromol/l) and E-selectin (50 micromol/l) expression was also observed. Expression of adhesion molecules was always more strongly inhibited in kaempferol-treated than in quercetin-treated cells. The inhibitory effect on iNOS and COX-2 protein level was stronger for quercetin at 5-50 micromol/l. The effect of kaempferol on NF-kappaB and AP-1 binding activity was weaker at high concentrations (50 micromol/l) as compared with quercetin. The present study indicates that differences exist in the modulation of pro-inflammatory genes and in the blockade of NF-kappaB and AP-1 by kaempferol and quercetin. The minor structural differences between both flavonols determine differences in their anti-inflammatory properties and in their efficiency in inhibiting signalling molecules.

Effects of FK506 and rapamycin on generation of reactive oxygen species, nitric oxide production and nuclear factor kappa B activation in rat hepatocytes

We investigated the effect of two immunosuppressant drugs, FK506 and rapamycin, on reactive oxygen species (ROS) generation, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and nuclear factor kappa B (NF-kappaB) activation in lipopolysaccharide (LPS)-activated rat hepatocytes. Primary culture of rat hepatocytes was treated with LPS in the presence and absence of FK506 or rapamycin. LPS increased the release of lactate dehydrogenase (LDH) and nitrite into the culture medium. Western blot and reverse transcription-polymerase chain reaction analyses demonstrated increased levels of iNOS protein and mRNA. Both immunosuppressant agents inhibited the induction of iNOS mRNA and protein stimulated by LPS. ROS generation, assessed by flow cytometry using dichlorodihydrofluorescein diacetate, was significantly decreased by FK506 and rapamycin. Moreover, electrophoretic mobility shift assay experiments indicated that both drugs blocked the LPS-induced activation of NF-kappaB. Inhibitor kappa B protein levels were decreased by LPS and this effect was partly blocked by FK506 or rapamycin. In summary, both immunosuppressant agents decreased the intracellular generation of ROS and inhibited NO production and iNOS expression at mRNA level in association to NF-kappaB activation. In addition to its capacity to reduce acute allograft rejection, this study highlights the anti-inflammatory properties of FK506 and rapamycin.

Melatonin reduces cardiac inflammatory injury induced by acute exercise

Abstract:  Cardiac muscle tissue, when stimulated by acute exercise, presents increased signs of cell damage. This study was designed to investigate whether overexpression of inflammatory mediators induced in the heart by acute exercise could be prevented by melatonin and whether the protective effect of melatonin was related with inhibition of nuclear factor kappa B (NF‐κB) activation. Male Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight 3 min before being exercised for 60 min on a treadmill at a speed of 25 m/min and a 10% slope. Exercise was associated with a significant increase in myeloperoxidase activity and in TNF‐α, IL‐1 and IL‐6 mRNA levels. Both mRNA level and protein concentrations of intercellular adhesion molecule‐1, inducible nitric oxide synthase, and cyclooxygenase‐2 were also significantly elevated. A significant activation of nuclear factor kappa B (NF‐κB) was observed in exercised rats. These effects were totally or partially prevented by melatonin administration. Data obtained indicate that melatonin protects against heart damage caused by acute exercise. Impaired production of noxious mediators involved in the inflammatory process and down‐regulation of the NF‐κB signal transduction pathway appear to contribute to the beneficial effects of melatonin.

Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells.

This study was aimed to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by proinflammatory stimuli in parenchymal liver cells. Chang Liver cells were incubated with a cytokine mixture (CM) supplemented with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin (5-50 microM). Concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were measured. Oxidised glutathione concentration and the oxidised/reduced glutathione ratio were increased by the CM. These effects were significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. Effects of apigenin reached a lesser extent and were not significant at 25 microM. Treatment with quercetin and kaempferol prevented the production of peroxides, superoxide anion and nitric oxide induced by CM. Taxifolin 50 microM and apigenin 25-50 microM caused a significant increase in peroxides and nitric oxide generation. Protein concentration of the different antioxidant enzymes was generally reduced by kaempferol and quercetin in comparison to CM, although quercetin 25 and 50 microM increased Mn SOD protein concentration. GPx protein level was significantly increased by apigenin 25 and 50 microM. Changes in mRNA tended to be parallel to those in protein concentration. Our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells.

Effects of Glutamine on Proinflammatory Gene Expression and Activation of Nuclear Factor Kappa B and Signal Transducers and Activators of Transcription in TNBS-induced Colitis

Background: We investigated the effects of glutamine on proinflammatory gene expression and activation of nuclear factor kappa B (NF‐&kgr;B) and signal transducers and activators of transcription (STAT) in a rat model of experimental colitis. Methods: Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6‐trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/kg) was given by rectal route daily for 7 days. Results: Glutamine significantly reduced gross damage and histopathological scores and prevented the decrease of anal pressure and the elevated myeloperoxidase activity observed in the colon of animals receiving TNBS. TNBS administration induced a marked increase of vascular cell adhesion molecule (VCAM‐1), inducible nitric oxide synthase (iNOS), and cyclooxygenase‐2 (COX‐2) protein levels. These inflammatory events were associated with increased protein level of NF‐&kgr;B p50 and p65 subunits in the nucleus and significant phosphorylation/degradation of the inhibitor I&kgr;B&agr;. Protein levels of the phosphorylated forms of STAT1, STAT5, and Akt were elevated in animals with colonic damage. All these effects were inhibited by administration of glutamine. Increases in the cytosolic concentration of TBARS and hydroperoxide‐initiated chemiluminescence, markers of oxidative stress, and levels of tumor necrosis factor &agr; (TNF&agr;) and interferon &ggr; (IFN&ggr;) were significantly inhibited at 48 hours of TNBS instillation in glutamine‐treated animals. Conclusions: Inhibition of the expression of proinflammatory mediators that are regulated by the NF‐&kgr;B and STAT signaling pathways contribute to the therapeutical effect of glutamine in the TNBS model of experimental colitis. These effects may be brought about by inhibition of oxidative stress and reduced expression of proinflammatory cytokines.

EFFECTS OF AGING ON THE SUSCEPTIBILITY TO THE TOXIC EFFECTS OF CYCLOSPORIN A IN RATS. CHANGES IN LIVER GLUTATHIONE AND ANTIOXIDANT ENZYMES

Free radicals are involved in aging and cyclosporin A-induced toxicity. The age-related changes in the liver oxidative status of glutathione, lipid peroxidation, and the activity of the enzymatic antioxidant defense system, as well as the influence of aging on the susceptibility to the hepatotoxic effects of cyclosporin (CyA) were investigated in rats of different ages (1, 2, 4, and 24 months). The hepatic content of reduced glutathione (GSH) increased with aging, peaked at 4 months, and decreased in senescent rats. By contrast, glutathione disulfide (GSSG) and thiobarbituric acid-reactive substances (TBARS) concentrations and superoxide dismutase, catalase, and glutathione peroxidase activities were higher in the oldest than in the youngest rats. CyA treatment, besides inducing the well-known cholestatic syndrome, increased liver GSSG and TBARS contents and the GSSG/GSH molar ratio, and altered the nonenzymatic and enzymatic antioxidant defense systems. The CyA-induced cholestasis and hepatic depletion of GSH, and the increases in the GSSG/GSH ratio, and in GSSG and TBARS concentrations were higher in the older than the mature rats. Moreover, superoxide dismutase and catalase activities were found to be significantly decreased only in treated senescent rats. The higher CyA-induced oxidative stress, lipoperoxidation, and decreases in the antioxidant defense systems in the aged animals render them more susceptible to the hepatotoxic effects of cyclosporin.