Giuseppe D’Argenio

Apple polyphenol extracts prevent damage to human gastric epithelial cells in vitro and to rat gastric mucosa in vivo

Background: Fresh fruit and vegetables exert multiple biological effects on the gastrointestinal mucosa. Aim: To assess whether apple extracts counteract oxidative or indomethacin induced damage to gastric epithelial cells in vitro and to rat gastric mucosa in vivo. Methods: Apple extracts were obtained from freeze dried apple flesh of the “Annurca” variety. Cell damage was induced by incubating MKN 28 cells with xanthine-xanthine oxidase or indomethacin and quantitated by MTT. In vivo gastric damage was induced by indomethacin 35 mg/kg. Intracellular antioxidant activity was determined using the (2,2′-azinobis (3-ethylbenzothiazolin-6-sulfonate) method. Malondialdehyde intracellular concentration, an index of lipid peroxidation, was determined by high pressure liquid chromatography with fluorometric detection. Results: (1) Apple extracts decreased xanthine-xanthine oxidase or indomethacin induced injury to gastric epithelial cells by 50%; (2) catechin or chlorogenic acid (the main phenolic components of apple extracts) were equally effective as apple extracts in preventing oxidative injury to gastric cells; and (3) apple extracts (i) caused a fourfold increase in intracellular antioxidant activity, (ii) prevented its decrease induced by xanthine-xanthine oxidase, (iii) counteracted xanthine-xanthine oxidase induced lipid peroxidation, and (iv) decreased indomethacin injury to the rat gastric mucosa by 40%. Conclusions: Apple extracts prevent exogenous damage to human gastric epithelial cells in vitro and to the rat gastric mucosa in vivo. This effect seems to be associated with the antioxidant activity of apple phenolic compounds. A diet rich in apple antioxidants might exert a beneficial effect in the prevention of gastric diseases related to generation of reactive oxygen species.

Evidence for multipotent endodermal stem/progenitor cell populations in human gallbladder

BACKGROUND & AIMS Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored. METHODS Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis. RESULTS The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice. CONCLUSIONS Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells.

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.

Peptide Hp(2–20) accelerates healing of TNBS-induced colitis in the rat

Background and aims Hp(2–20), a Helicobacter pylori-derived peptide interacting with N-formyl peptide receptors (FPRs), accelerates the healing of gastric injury in rats. Whether Hp(2–20) affects the recovery of inflamed colonic mucosa is unknown. We evaluated whether Hp(2–20) accelerated the healing of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis and explored the mechanism(s) underlying any such effect. Methods Fifteen rats underwent rectal administration of Hp(2–20) 250–500 µg/kg/day, or of its control peptide Hp1 for 10 days, following induction of colitis with TNBS. Macroscopic and histological damage was quantified using predetermined injury scores. FPR1, COX-2, TNF-α, TGF-β, HB-EGF and tissue transglutaminase (t-TG) messenger RNA (mRNA) expression in colonic tissue was determined by quantitative polymerase chain reaction; FPR1, TNF-α and COX-2 protein levels by Western blotting. Results (1) Hp(2–20) accelerated healing of TNBS-induced colitis compared to controls consistently with the expression of FPRs in colonic mucosa; (2) TNBS upregulated mRNA mucosal expression of COX-2, TNF-α, TGF-β, HB-EGF and t-TG and (3) this, with the exception of HB-EGF, was significantly counteracted by Hp(2–20). Conclusions Hp(2–20), an FPR agonist, accelerates the healing of TNBS-induced colitis in the rat. This effect is associated with a significant reduction in colonic tissue levels of COX-2, TGF-β, TNF-α and t-TG. We postulate that FPR-dependent pathways may be involved in the repair of inflamed colonic mucosa.

Dietary supplementation of vitamin D prevents the development of western diet-induced metabolic, hepatic and cardiovascular abnormalities in rats

Background The western diet high in fat and fructose may cause metabolic disorders and cardiovascular diseases. Objective To evaluate whether long-term daily vitamin D3 supplementation prevents hepatic steatosis and cardiovascular abnormalities and restores insulin sensitivity caused by fat diet in rats without vitamin D deficiency. Methods Three groups of rats were fed for 6 months with standard diet (SD), western diet (WD) or WD containing 23 IU/day/rat vitamin D3, respectively. Tail-cuff systolic blood pressure (SBP)measurements in conscious rats and transthoracic echocardiography were performed in basal condition, and after 3 and 6 months of diet. Hepatic steatosis and myocardial fibrosis were assessed in liver and cardiac tissues using standard methods. Serum insulin and 25(OH)D3 concentrations were determined using rat-specific ELISA kits. Insulin resistance was determined according to the homeostasis model assessment of insulin resistance (HOMA-IR) method. Results Sixty-one per cent of hepatocytes in WD rats had steatotic vacuoles compared with just 27% in rats on a WD plus vitamin D3 (p < 0.05).HOMA-IR was reduced in rats with vitamin D supplementation compared with WD alone (19.4 ± 5.2 vs 41.9 ± 8.9, p < 0.05). Rat blood pressure and left ventricular mass were both reduced by vitamin D3 supplementation. Conclusion In animal models of liver and cardiovascular metabolic damage, the supplementation of vitamin D3 shows liver and cardio-protective effects.

Role Of Formyl Peptide Receptors In Gastrointestinal Healing

The wound healing and the barrier restoration of the gastrointestinal (GI) mucosa must be continuously ensured to allow homeostasis of the gastrointestinal tract and of all the surrounding tissues. Several lines of the evidence report a key role of innate immunity, and in particular of Pattern Recognition Receptors (PRRs), in controlling the homeostasis of GI tract by sensing commensal and pathogen bacteria, activating the immune response and regulating epithelial repair, thus guaranteeing the morphological and functional recovery of the injured tissue. We will discuss the role of a particular class of PRRs - the Formyl Peptide Receptors - in the homeostasis of GI mucosa. We here report the results of studies that strongly suggest the possibility that the activation of FPRs is crucial in the maintenance of homeostasis of the GI tract and provide indications of the potential clinical relevance of new treatment regimens involving FPR modulation for several GI disorders.