Maria T. Ribecco

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)

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

Background Celiac Disease (CD) is both a frequent disease (1∶100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called “toxic” A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. Methods/Principal Findings Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. Conclusions P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosin Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.

Apple polyphenols extract (APE) improves colon damage in a rat model of colitis

BACKGROUND AND AIM Searching for alternative therapies that are effective, safe and less expensive of those currently used for ulcerative colitis, we investigated the efficacy of a polyphenol extract from apple in rat colitis. METHODS Rats with trinitrobenzensulphonic acid-induced colitis were treated daily with rectal administration of apple polyphenols 10(-4) M for 14 days. COX-2, TNF-α, tissue transglutaminase and calpain in colon mucosa samples were assessed by reverse transcription-polymerase chain reaction and western blot analyses. To ascertain the role of tissue transglutaminase in mucosal healing, wounded rat fibroblasts were incubated with cystamine (a tissue transglutaminase activity inhibitor). RESULTS Colitis was associated with increased COX-2, TNF-α, calpain, and tissue transglutaminase mRNA. The protein expression of COX-2, TNF-α and calpain was increased whilst tissue transglutaminase was decreased. Apple extract treatment reduced the severity of colitis (p<0.05) and restored all the considered biomarkers at the baseline level. Apple polyphenols reduced the degradation of tissue transglutaminase protein occurring through calpain action. Apple polyphenols-treated wounded fibroblast recovered within 24h showing intense immunoreactivity for tissue transglutaminase. CONCLUSION The efficacy of apple extract is mediated by its effects on COX-2 and TNF-α. The unbalance between calpain and tissue transglutaminase may play a role in colonic damage and future therapeutic interventions in ulcerative colitis can target this mechanisms.

Garlic extract prevents CCl4-induced liver fibrosis in rats: The role of tissue transglutaminase

BACKGROUND AND AIM Tissue transglutaminase contributes to liver damage in the development of hepatic fibrosis. In a model of neurodegeneration, the therapeutic benefit of cystamine has been partly attributed to its inhibition of transglutaminase activity. Garlic extract contains many compounds structurally related to cystamine. We investigated the anti-fibrotic effect of garlic extract and cystamine as specific tissue transglutaminase inhibitors. METHODS Rat liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl(4)) for 7 weeks. Cystamine or garlic extract was administrated by daily intraperitoneal injection, starting from the day after the first administration of CCl(4). Hepatic function, histology, tissue transglutaminase immunostaining and image analysis to quantify Red Sirius stained collagen deposition were examined. Reverse transcription-polymerase chain reaction to detect alpha-SMA, IL-1beta and tissue transglutaminase expression and Western blot for tissue transglutaminase protein amount were performed. Transglutaminase activity was assayed on liver homogenates by a radio-enzymatic method. RESULTS Transglutaminase activity was increased in CCl(4) group and reduced by cystamine and garlic extract (p<0.05). Treatment with cystamine and garlic extract reduced the liver fibrosis and collagen deposition, particularly in the garlic extract group (p<0.01). Moreover, the liver damage improved and serum alanine aminotransferase was decreased (p<0.05). Tissue transglutaminase immunolocalised with collagen fibres and is mainly found in the ECM of damaged liver. Alpha-SMA, IL-1beta, tissue transglutaminase mRNA and tissue transglutaminase protein were down-regulated in the cystamine and garlic extract groups compared to controls. CONCLUSION These findings concurrently suggest that transglutaminase may play a pivotal role in the pathogenesis of liver fibrosis and may identify garlic cystamine-like molecules as a potential therapeutic strategy in the treatment of liver injury.

Garlic extract attenuating rat liver fibrosis by inhibiting TGF-β1

BACKGROUND & AIMS We previously demonstrated the efficacy of garlic extract (GE) in the prevention of rat liver fibrosis by inhibiting tissue transglutaminase (tTG) activity. In the present study we aimed to evaluate the potential of GE in the regression of liver fibrosis and the underlining mechanism. METHODS Male Wistar rats were i.p. injected, twice a week, for 7 weeks, with CCl(4) to develop liver fibrosis. Successively, a group was immediately sacrificed, while the remaining two groups received the GE or the vehicle, respectively, over the following 2 wks. A group of normal rats was also included in the study. Liver function, histology, and collagen deposition in parallel with gene and protein expression of α-SMA, tTG, TGF-β1, SEMA-7A, and metalloproteinase inhibitor 1 (TIMP1) as well as measure of active by total TGF-β1 were assessed. RESULTS CCl(4) administration increased alanine-aminotransferase (ALT) activity, hepatic collagen deposition and gene and protein expression of all monitored markers. GE, but not the sole vehicle, restored liver histology and function by decreasing fibrogenesis markers (α-SMA, tTG, TGF-β1, SEMA-7A and TIMP1). Active by total TGF-β1 was significantly reduced (p < 0.05) in GE treated rats compared to the CCl(4) at 7 weeks, and vehicle rats. CONCLUSIONS These findings concurrently suggested that GE elicited therapeutic effect against liver fibrosis. Regression of liver fibrosis occurred by reducing myofibroblasts (through modulation of HSCs activation mechanisms), remodelling extracellular matrix (through increase of its degradation) and regenerating liver tissue and functions: three processes regulated by fine mechanisms where active TGF-β1 and tTG play a central role.