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 ß-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.

Coffee and liver health.

Coffee is one of the most widely used beverages in the world. It includes a wide array of components that can have potential implications for health. Several epidemiological studies associate coffee consumption with a reduced incidence of various chronic diseases such as diabetes, cardiovascular diseases, and neurodegenerative diseases. Over the past 20 years, an increasing number of epidemiological and experimental studies have demonstrated the positive effects of coffee on chronic liver diseases. Coffee consumption has been inversely associated with the activity of liver enzymes in subjects at risk, including heavy drinkers. Coffee favours an improvement in hepatic steatosis and fibrosis, and a reduction in cirrhosis and the risk of hepatocellular carcinoma. The mechanisms of action through which it exerts its beneficial effects are not fully understood. Experimental studies show that coffee consumption reduces fat accumulation and collagen deposition in the liver and promotes antioxidant capacity through an increase in glutathione as well as modulation of the gene and protein expression of several inflammatory mediators. Animal and in vitro studies indicate that cafestol and kahweol, 2 diterpens, can operate by modulating multiple enzymes involved in the detoxification process of carcinogens causing hepatocellular carcinoma. It is unclear whether the benefits are significant enough to "treat" patients with chronic liver disease. While we await clarification, moderate daily unsweetened coffee use is a reasonable adjuvant to therapy for these patients.

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

AIM: 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. RESULTS: CCl4 significantly increased gastric permeability in respect to basal values, and the treatment with symbiotic significantly decreased it. CCl4 per se induced a decrease in intestinal permeability. This effect was also seen in fibrotic rats treated with symbiotic and was still evident when normal rats were treated with symbiotic alone (P < 0.001 in all cases). Circulating levels of pro-inflammatory cytokine TNF-α were significantly increased in rats with liver fibrosis as compared with normal rats, while symbiotic treatment normalized the plasma levels of TNF-α and significantly enhanced anti-inflammatory cytokine IL 10. TNF-α, TGF-ß, TLR4, TLR2, iNOS and α-SMA mRNA expression in the liver were up-regulated in rats with CCl4 -induced liver fibrosis and down-regulated by symbiotic treatment. Moreover, IL-10 and eNOS mRNA levels were increased in the CCL4 (+) symbiotic group. Symbiotic treatment of fibrotic rats normalized serum ALT, AST and improved histology and liver collagen deposition. DGGE analysis of faecal samples revealed that CCl4 administration and symbiotic treatment either alone or in combination produced modifications in faecal profiles vs controls. CONCLUSIONS: Our results provide evidence that in CCl4 -induced liver fibrosis, significant changes in gastro-intestinal permeability and in faecal flora occur. Treatment with a specific symbiotic formulation significantly affects these changes, leading to improvement in both liver inflammation and fibrosis.

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

UNLABELLED: 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 ß 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 ß-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.

The Combination of Berberine, Tocotrienols and Coffee Extracts Improves Metabolic Profile and Liver Steatosis by the Modulation of Gut Microbiota and Hepatic miR-122 and miR-34a Expression in Mice.

Non-alcoholic-fatty liver disease (NAFLD) is spreading worldwide. Specific drugs for NAFLD are not yet available, even if some plant extracts show beneficial properties. We evaluated the effects of a combination, composed by Berberis Aristata, Elaeis Guineensis and Coffea Canephora, on the development of obesity, hepatic steatosis, insulin-resistance and on the modulation of hepatic microRNAs (miRNA) levels and microbiota composition in a mouse model of liver damage. C57BL/6 mice were fed with standard diet (SD, n = 8), high fat diet (HFD, n = 8) or HFD plus plant extracts (HFD+E, n = 8) for 24 weeks. Liver expression of miR-122 and miR-34a was evaluated by quantitativePCR. Microbiome analysis was performed on cecal content by 16S rRNA sequencing. HFD+E-mice showed lower body weight (p < 0.01), amelioration of insulin-sensitivity (p = 0.021), total cholesterol (p = 0.014), low-density-lipoprotein-cholesterol (p < 0.001), alanine-aminotransferase (p = 0.038) and hepatic steatosis compared to HFD-mice. While a decrease of hepatic miR-122 and increase of miR-34a were observed in HFD-mice compared to SD-mice, both these miRNAs had similar levels to SD-mice in HFD+E-mice. Moreover, a different microbial composition was found between SD- and HFD-mice, with a partial rescue of dysbiosis in HFD+E-mice. This combination of plant extracts had a beneficial effect on HFD-induced NAFLD by the modulation of miR-122, miR-34a and gut microbiome.

Coffee prevents fatty liver disease induced by a high-fat diet by modulating pathways of the gut-liver axis.

Coffee consumption is inversely associated with the risk of non-alcoholic fatty liver disease (NAFLD). A gap in the literature still exists concerning the intestinal mechanisms that are involved in the protective effect of coffee consumption towards NAFLD. In this study, twenty-four C57BL/6J mice were divided into three groups each receiving a standard diet, a high-fat diet (HFD) or an HFD plus decaffeinated coffee (HFD+COFFEE) for 12 weeks. Coffee supplementation reduced HFD-induced liver macrovesicular steatosis (P < 0·01) and serum cholesterol (P < 0·001), alanine aminotransferase and glucose (P < 0·05). Accordingly, liver PPAR- α (P < 0·05) and acyl-CoA oxidase-1 (P < 0·05) as well as duodenal ATP-binding cassette (ABC) subfamily A1 (ABCA1) and subfamily G1 (ABCG1) (P < 0·05) mRNA expressions increased with coffee consumption. Compared with HFD animals, HFD+COFFEE mice had more undigested lipids in the caecal content and higher free fatty acid receptor-1 mRNA expression in the duodenum and colon. Furthermore, they showed an up-regulation of duodenal and colonic zonulin-1 (P < 0·05), duodenal claudin (P < 0·05) and duodenal peptide YY (P < 0·05) mRNA as well as a higher abundance of Alcaligenaceae in the faeces (P < 0·05). HFD+COFFEE mice had an energy intake comparable with HFD-fed mice but starting from the eighth intervention week they gained significantly less weight over time. Data altogether showed that coffee supplementation prevented HFD-induced NAFLD in mice by reducing hepatic fat deposition and metabolic derangement through modification of pathways underpinning liver fat oxidation, intestinal cholesterol efflux, energy metabolism and gut permeability. The hepatic and metabolic benefits induced by coffee were accompanied by changes in the gut microbiota.

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.

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.

Garlic extract attenuating rat liver fibrosis by inhibiting TGF-ß1.

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-ß1, SEMA-7A, and metalloproteinase inhibitor 1 (TIMP1) as well as measure of active by total TGF-ß1 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-ß1, SEMA-7A and TIMP1). Active by total TGF-ß1 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-ß1 and tTG play a central role.

Rapid "breath-print" of liver cirrhosis by proton transfer reaction time-of-flight mass spectrometry. A pilot study.

UNLABELLED: The aim of the present work was to test the potential of Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) in the diagnosis of liver cirrhosis and the assessment of disease severity by direct analysis of exhaled breath. Twenty-six volunteers have been enrolled in this study: 12 patients (M/F 8/4, mean age 70.5 years, min-max 42-80 years) with liver cirrhosis of different etiologies and at different severity of disease and 14 healthy subjects (M/F 5/9, mean age 52.3 years, min-max 35-77 years). Real time breath analysis was performed on fasting subjects using a buffered end-tidal on-line sampler directly coupled to a PTR-ToF-MS. Twelve volatile organic compounds (VOCs) resulted significantly differently in cirrhotic patients (CP) compared to healthy controls (CTRL): four ketones (2-butanone, 2- or 3- pentanone, C8-ketone, C9-ketone), two terpenes (monoterpene, monoterpene related), four sulphur or nitrogen compounds (sulfoxide-compound, S-compound, NS-compound, N-compound) and two alcohols (heptadienol, methanol). Seven VOCs (2-butanone, C8-ketone, a monoterpene, 2,4-heptadienol and three compounds containing N, S or NS) resulted significantly differently in compensate cirrhotic patients (Child-Pugh A; CP-A) and decompensated cirrhotic subjects (Child-Pugh B+C; CP-B+C). ROC (Receiver Operating Characteristic) analysis was performed considering three contrast groups: CP vs CTRL, CP-A vs CTRL and CP-A vs CP-B+C. In these comparisons monoterpene and N-compound showed the best diagnostic performance. CONCLUSIONS: Breath analysis by PTR-ToF-MS was able to distinguish cirrhotic patients from healthy subjects and to discriminate those with well compensated liver disease from those at more advanced severity stage. A breath-print of liver cirrhosis was assessed for the first time.

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 CCl(4)-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.