Giuseppe D'Argenio

Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation

Direct stimulation of cannabinoid CB1 receptors exerts a protective function in animal models of inflammatory bowel diseases (IBDs). However, it is not known whether endocannabinoids are up‐regulated during IBDs in animals or humans, nor whether pharmacological elevation of endocannabinoid levels can be exploited therapeutically in these disorders. In this study we addressed these questions. Colon inflammation was induced in mice and rats with 2,4dinitrobenzene‐ and 2,4,6‐trinitrobenzene sulfonic acids (DNBS and TNBS), respectively. DNBS‐treated mice were treated chronically (for 3 or 7 days) with inhibitors of anandamide enzymatic hydrolysis (N‐arachidonoyl‐serotonin, AA‐5‐HT) or reuptake (VDM11), 10 or 5 mg/kg, s.c., or with 5‐amino‐salicilic acid (5‐ASA, 1.4 mg/kg, i.r.). Endocannabinoids (anandamide and 2‐arachidonoylglycerol, 2‐AG) were quantified in mouse colon, or in rat colon mucosa and submucosa, and in bioptic samples from the colon of patients with untreated ulcerative colitis, by liquid chromatography‐mass spectrometry. A strong elevation of anandamide, but not 2‐AG, levels was found in the colon of DNBS‐treated mice, in the colon submucosa of TNBS‐treated rats, and in the biopsies of patients with ulcerative colitis. VDM‐11 significantly elevated anandamide levels in the colon of DNBS‐treated mice and concomitantly abolished inflammation, whereas AA‐5‐HT did not affect endocannabinoid levels and was significantly less efficacious at attenuating colitis. 5‐ASA also increased anandamide levels and abolished colitis. Thus, anandamide is elevated in the inflamed colon of patients with ulcerative colitis, as well as in animal models of IBDs, to control inflammation, and elevation of its levels with inhibitors of its cellular reuptake might be used in the treatment of IBDs.

Coffee reduces liver damage in a rat model of steatohepatitis: the underlying mechanisms and the role of polyphenols and melanoidins.

Epidemiological data associate coffee consumption with a lower prevalence of chronic liver disease and a reduced risk of elevated liver enzyme levels (γ glutamyl transpeptidase and alanine aminotransferase), advanced liver disease and its complications, and hepatocellular carcinoma. Knowledge of the mechanisms underlying these effects and the coffee components responsible for these properties is still lacking. In this study, 1.5 mL/day of decaffeinated coffee or its polyphenols or melanoidins (corresponding to approximately 2 cups of filtered coffee or 6 cups of espresso coffee for a 70‐kg person) were added for 8 weeks to the drinking water of rats who were being fed a high‐fat, high‐calorie solid diet (HFD) for the previous 4 weeks. At week 12, HFD + water rats showed a clinical picture typical of advanced nonalcoholic steatohepatitis compared with control rats (normal diet + water). In comparison, HFD + coffee rats showed: (1) reduced hepatic fat and collagen, as well as reduced serum alanine aminotransferase and triglycerides; (2) a two‐fold reduced/oxidized glutathione ratio in both serum and liver; (3) reduced serum malondialdehyde (lipid peroxidation) and increased ferric reducing antioxidant power (reducing activity); (4) reduced expression of tumor necrosis factor α (TNF‐α), tissue transglutaminase, and transforming growth factor β and increased expression of adiponectin receptor and peroxisome proliferator‐activated receptor α in liver tissue; and (5) reduced hepatic concentrations of proinflammatory TNF‐α and interferon‐γ and increased anti‐inflammatory interleukin‐4 and interleukin‐10. Conclusion: Our data demonstrate that coffee consumption protects the liver from damage caused by a high‐fat diet. This effect was mediated by a reduction in hepatic fat accumulation (through increased fatty acid β‐oxidation); systemic and liver oxidative stress (through the glutathione system); liver inflammation (through modulation of genes); and expression and concentrations of proteins and cytokines related to inflammation. (HEPATOLOGY 2010;52:1652‐1661)

Direct evidence of oxidative damage in acute and chronic phases of experimental colitis in rats

During inflammatory colitis in man and experimental animals, the production of free radicals increases. This study evaluated the histological pattern and biochemical parameters of oxidative damage during acute and chronic colitis induced by 2,4,-trinitrobenzenesulfonic acid + ethanol in rats. On the samples of scraped mucosa of six groups of rats, one not treated, one killed after 1 hr, and those killed one, two, four, and eight weeks after the induced-damage, we determined the histological and superoxide dismutase activity and the concentration of lipoperoxides, malonyldialdheyde, and reduced glutathione. After 1 hr, the mucosal damage and superoxide dismutase activity were slight; glutathione, lipoperoxides, and malonyldialdheyde were significantly increased. At one week, the histological damage was severe, decreasing progressively, and significantly correlated to superoxide dismutase activity. Lipoperoxides and malonyldialdheyde were high throughout the study. Glutathione was significantly increased at one and two weeks and dramatically decreased thereafter. Therefore, in experimental colitis the cascade of free-radical production induces a constant self-maintaining lipoperoxidation and consumes the cellular antioxidant capability.

Eicosapentaenoic acid free fatty acid prevents and suppresses colonic neoplasia in colitis-associated colorectal cancer acting on Notch signaling and gut microbiota

Inflammatory bowel diseases are associated with increased risk of developing colitis‐associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω‐3 polyunsaturated fatty acids (ω‐3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid‐free fatty acid (EPA‐FFA) reduces polyp formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA‐FFA are unknown in CAC. We tested the effectiveness of substituting EPA‐FFA, for other dietary fats, in preventing inflammation and cancer in the AOM‐DSS model of CAC. The AOM‐DSS protocols were designed to evaluate the effect of EPA‐FFA on both initiation and promotion of carcinogenesis. We found that EPA‐FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA–FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β‐catenin expression, whilst it increased apoptosis. In both arms, EPA‐FFA treatment led to increased membrane switch from ω‐6 to ω‐3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA‐FFA treated arms and AOM‐DSS controls. Importantly, we found that EPA‐FFA treatment restored the loss of Notch signaling found in the AOM‐DSS control and resulted in the enrichment of Lactobacillus species in the gut microbiota. Taken together, our data suggest that EPA‐FFA is an excellent candidate for CRC chemoprevention in CAC.

Apple polyphenol extracts prevent aspirin-induced damage to the rat gastric mucosa

Aspirin causes gastroduodenal ulcers and complications. Food bioactive compounds could exert beneficial effects in the gastrointestinal tract. We evaluated whether apple polyphenol extract (APE) reduced aspirin-induced injury to the rat gastric mucosa. Rats were treated with APE (10(-4) m catechin equivalent) before oral aspirin (200 mg/kg). Cyclo-oxygenase-2 (COX-2), transforming growth factor-alpha (TGF alpha) and heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA and protein expression were assessed by RT-PCR and Western blot analysis, respectively; malondialdehyde (MDA) was determined by HPLC; gastric secretion was evaluated in pylorus-ligated rats. APE decreased acute and chronic aspirin injury both macroscopically and microscopically (approximately 50 % decrease in lesion score; P < 0.05). Aspirin up-regulated mRNA and protein expression of COX-2 and HB-EGF, but not of TGF alpha; APE reduced aspirin-induced mRNA and protein over-expression of COX-2 and HB-EGF; aspirin significantly increased gastric MDA and this effect was counteracted by APE pre-treatment. APE did not significantly affect gastric acid secretion. In conclusion, APE reduces aspirin-induced gastric injury independently of acid inhibition. We speculate that APE might be of therapeutic use in the prophylaxis of aspirin-related gastropathy.

Recombinant Factor XIII Improves Established Experimental Colitis in Rats

Factor XIII (FXIII) is the plasma-borne transglutaminase involved in fibrin clot stabilization and wound healing. FXIII levels in the plasma of patients with inflammatory bowel diseases are lower than normal and there is a significant inverse correlation of FXIII levels with clinical severity. Moreover, uncontrolled studies report beneficial effects of FXIII supplementation in patients resistant to conventional therapies. We investigated the effects of intravenous recombinant FXIII (rFXIII) treatment in experimentally induced rat colitis to verify that FXIII was the active agent in plasma FXIII concentrates and to better understand the potential therapeutic use of this protein. Colitis was induced by instillation of 12% 2,4,6-trinitrobenzenesulfonic acid (TNBS) in 50% ethanol into the colon of male Wistar rats. Rats were treated with 0.65 mg/kg rFXIII or vehicle (intravenously) daily for 10 days. Treatment was started either immediately after TNBS/EtOH instillation (to evaluate effects on developing lesions) or seven days later (to evaluate effects on established lesions). In both cases rats were killed either immediately after the end of treatment (to evaluate immediate effects) or 17 days later (to evaluate long-lasting effects). The effects of rFXIII were compared to positive (5-amino-2-hydroxybenzoic acid) control over a 35-day time course. The severity of lesions was determined by colon weight and macroscopic and histologic scores. Transglutaminase activity was measured in both colon tissue and serum. rFXIII treatment reduced lesion severity significantly not only in developing but also in established lesions. Improvements in healing persisted at least 18 days after treatment was discontinued. Serum and tissue transglutaminase levels were restored by rFXIII treatment. In conclusion, pure rFXIII is as effective as plasma FXIII concentrates in a rat model of experimental colitis. In addition, rFXIII significantly improves the healing of preexisting lesions, a characteristic useful in treatment of human inflammatory bowel diseases.

Analysis of breath by proton transfer reaction time of flight mass spectrometry in rats with steatohepatitis induced by high-fat diet†

Breath testing has been largely used as a diagnostic tool, but the difficulties in data interpretation and sample collection have limited its application. We developed a fast (< 20 s), on-line, non-invasive method for the collection and analysis of exhaled breath in awake rats based on proton transfer reaction time of flight mass spectrometry (PTR-ToF-MS) and applied it to investigate possible relationships between pathologies induced by dietary regime and breath composition. As a case study, we investigated rats with dietary induced non-alcoholic steatohepatitis (NASH) and modifications induced by coffee addition to the diet. We considered two different diets (standard and high fat) complemented with two different drinking possibilities (water or decaffeinated coffee) for a total of four groups with four rats each. Several spectrometric peaks were reliable markers for both dietary fat content and coffee supplementation. The high resolution and accuracy of PTR-ToF-MS allowed the identification of related compounds such as methanol, dimethyl sulphide, dimethyl sulphone and ammonia. In conclusion, the rapid and minimally invasive breath analysis of awake rats permitted the identification of markers related to diet and specific pathologic conditions and provided a useful tool for broader metabolic investigations.

Up-regulation of heparin binding epidermal growth factor-like growth factor and amphiregulin expression in Helicobacter pylori-infected human gastric mucosa

BACKGROUND Host response plays a major role in pathogenesis of Helicobacter pylori-induced gastroduodenal disease including adenocarcinoma of distal stomach. Epidermal growth factor-related growth factors are important modulators of gastric homeostasis in normal and damaged gastrointestinal mucosa. AIM To evaluate expression of heparin binding epidermal growth factor and amphiregulin in antral mucosa of Helicobacter pylori-infected and non-infected dyspeptic patients and to correlate levels of heparin binding-epidermal growth factor and amphiregulin mRNA with mitogenic activity of gastric epithelial cells. METHODS A total of 10 Helicobacter pylori-infected and 15 Helicobacter pylori non-infected (10 with and 5 without gastritis) dyspeptic patients were studied. Diagnosis of Helicobacter pylori infection was based on rapid urease test and histology. Heparin binding-epidermal growth factor and amphiregulin mRNA expression in antral mucosa were assessed by reverse transcriptase-polymerase chain reaction. Protein expression and localization of both peptides were determined by immunohistochemistry. Mitogenic activity of antral gastric mucosa was assessed by determination of proliferating cell nuclear antigen labelling index by immunohistochemistry. RESULTS Heparin binding-epidermal growth factor and amphiregulin mRNA expression increased in Helicobacter pylori-infected vs Helicobacter pylori non-infected patients. Heparin binding-epidermal growth factor and amphiregulin immunostaining was more intense and deeper in gastric gland compartment in infected mucosa than in non-infected mucosa. Increase in heparin binding-epidermal growth factor and amphiregulin mRNA expression significantly correlated with increase in proliferating cell nuclear antigen labelling index. CONCLUSIONS Helicobacter pylori gastritis is associated with up-regulation of heparin binding-epidermal growth factor and amphiregulin which correlates with increased mitogenic activity of gastric mucosa. Increased heparin binding-epidermal growth factor and amphiregulin expression is postulated to contribute to reparative response of gastric mucosa to Helicobacter pylori infection.

Nutraceuticals for Protection and Healing of Gastrointestinal Mucosa

Natural medicinal products have been used for millennia for the treatment of several ailments. Although many have been superseded by conventional pharmaceutical approaches, there is currently a resurgence in the interest in natural products by the general public and the use of complementary and alternative medicine is increasing rapidly in developed countries. Also, pharmaceutical industries are more and more interested in examining their potential as sources of novel medicinal compounds which may act as growth factor or show immunomodulatory or anti-microbial activity. The subgroup of natural bioactive compounds that bridge the gap between food products and drugs are termed nutraceuticals or functional foods. In contrast with most standard medicinal compounds, nutraceuticals are generally used to prevent rather than to treat disease. Many of the claims for such products are supported by very limited scientific evidence. However, there has recently been a great interest at evaluating the mechanism by which natural products exert their beneficial effects in the gastrointestinal tract. In particular, a major area of interest is for the use of biologically active chemical components of plants, i.e. phytochemicals, in a number of gastrointestinal disorders. While the major focus of phytochemical research has been on cancer prevention, several products of plant origin are being used and/or under study for a variety of other gastrointestinal problems. In this review we discuss the scientific evidence supporting the potential use of nutraceuticals as agents capable to prevent or accelerate healing of gastrointestinal mucosal damage, with a focus on polyphenol extracts obtained from apple.

Symbiotic formulation in experimentally induced liver fibrosis in rats: intestinal microbiota as a key point to treat liver damage?

Evidence indicates that intestinal microbiota may participate in both the induction and the progression of liver damage. The aim of our research was the detection and evaluation of the effects of chronic treatment with a symbiotic formulation on CCl4‐induced rat liver fibrosis.