Indole-3-carbinol and chlorogenic acid combination modulates gut microbiome and attenuates nonalcoholic steatohepatitis in a murine model.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, affecting almost 32% of the population and ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Recent findings indicate that the fast-growing prevalence of NAFLD might be linked to adherence to a Westernized diet (WD), mostly composed of fat/sugar-enriched foods. The WD has been reportedly targeted as a potential driver of gut-liver axis unbalance, suggesting a major role in NASH. On the other hand, bioactive food compounds feature as a potential chemopreventive strategy against NASH, due to their beneficial effects (i.e, anti-inflammatory/oxidant activity and modulation of gut microbiome). Brassicaceae vegetables are known for their high amount of isothiocyanates and polyphenols, as indole-3-carbinol (I3C) and chlorogenic acid (CGA). Thus, we sought to assess the effects of human relevant doses of I3C and CGA isolated or in combination (5/125 mg/Kg of body weight, respectively) on a diet/chemical-induced murine model of NASH. I3C + CGA oral treatment diminished NAFLD activity score (NAS) (p < 0.0001), as well as alleviated the hepatic lipid (p = 0.0011) accumulation, prevented hepatic stellate cell (HSC) activation (p < 0.0001), and subsequent fibrosis (p < 0.0001). The combination also reduced the number of both hepatic CD68-positive macrophages (p < 0.0001) and cleaved caspase-3 hepatocytes (p < 0.0001) and diminished the malondialdehyde levels (p = 0.0155). Additionally, the combination of I3C + CGA restored the relative abundance of Alistipes (p = 0.0299), Allobaculum (p = 0.0014), Bacteroides (p = 0.0046), and Odoribacter (p = 0.0030) bacteria genera on the gut microbiome. Taken together, these findings show that the combination of I3C + CGA at populational-relevant ingestion, rather than the I3C or CGA alone, was able to modulate gut microbiome and attenuate NASH in this hybrid model mouse.

Western diet-induced mouse model of non-alcoholic fatty liver disease associated with metabolic outcomes: Features of gut microbiome-liver-adipose tissue axis.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) has a growing epidemiologic and economic burden. It is associated with Western diet (WD) patterns, and its pathogenesis involves metabolic disorders (obesity, dyslipidemia, hyperglycemia, and diabetes) and gut dysbiosis, features that are usually neglected or not reproduced by most animal models. Thus, we established a 6-mo WD-induced NAFLD mouse model associated with metabolic disorder, investigating its main features at the gut microbiome-liver-adipose tissue axis, also evaluating the correlations of gut dysbiosis to the other disease outcomes. METHODS: Male C57 BL6 mice received a high-fat (30% lard and 0.2% cholesterol, ∼57% calories) and sucrose-rich (20%) chow, and a high-sugar solution (23.1 and 18.9 g/L of D-fructose and D-glucose) for 6 mo. RESULTS: The model featured high serum cholesterol levels, glucose intolerance, and hyperinsulinemia. WD intervention resulted in extensive macro/microvesicular liver steatosis and pericellular fibrosis-resembling human disease-accompanied by hepatic stellate cell activation and CD68+ macrophage infiltration, increased protein levels of proinflammatory p65-nuclear factor-κB, interleukin-6 and tumor necrosis factor-α, with decreased antioxidant regulator Nrf2. Mice showed clear obesity with adipocyte hypertrophy, and CD68+macrophage/mast cell infiltration in adipose tissue while a reduction in number of goblet cells was also observed in the small intestine. Moreover, the pyrosequencing of the 16 S ribosomal RNA of gut cecal content showed decreased bacterial diversity, enriched Firmicutes and Proteobacteria, decreased Bacteroidetes and Fusobacteria, and increased ratio of Firmicutes to Bacteroidetes. Bacteroidetes and Bacteroides had the highest number of significant correlations with liver-adipose tissue axis outcomes. In silico analysis of gut microbiome in NAFLD obese patients revealed a depletion in Bacteroides, which also correlated to disease outcomes. CONCLUSION: This mice model gathered suitable phenotypical alterations in gut-liver-adipose tissue axis that resembled NAFLD associated with metabolic disorders in humans and may be considered for preclinical investigation.

Are isothiocyanates and polyphenols from Brassicaceae vegetables emerging as preventive/therapeutic strategies for NAFLD? The landscape of recent preclinical findings.

Nonalcoholic fatty liver disease (NAFLD) is a lipid impairment-related chronic metabolic disease that affects almost 25% of the worldwide population and has become the leading cause of liver transplantation in the United States of America (USA). NAFLD may progress from simple hepatic steatosis (HS) to nonalcoholic steatohepatitis (NASH), which occurs simultaneously in an inflammatory and fibrotic microenvironment and affects approximately 5% of the global population. Recently, NASH has been suggested to be a relevant driver in progressive liver cirrhosis and a population-attributable factor in hepatocellular carcinoma patients. Moreover, predictions show that NAFLD-related annual health costs in the USA have reached ∼$100 bi., but effective therapies are still scarce. Thus, new preventative strategies for this hepatic disease urgently need to be developed. The Brassicaceae vegetable family includes almost 350 genera and 3500 species and these are one of the main types of vegetables harvested and produced worldwide. These vegetables are well-known sources of glucobrassicin-derivative molecules, such as isothiocyanates and phenolic compounds, which have shown antioxidant and antilipogenic effects in preclinical NAFLD data. In this review, we gathered prominent evidence of the in vivo and in vitro effects of these vegetable-derived nutraceutical compounds on the gut-liver-adipose axis, which is a well-known regulator of NAFLD and may represent a new strategy for disease control.

Vitamin D3 supplementation alleviates chemically-induced cirrhosis-associated hepatocarcinogenesis.

Vitamin D3 (VD3) deficiency has been associated with increased risk for cirrhosis and hepatocellular carcinoma, a highly incident malignant neoplasia worldwide. On the other hand, VD3 supplementation has shown some beneficial effects in clinical studies and rodent models of chronic liver disease. However, preventive effects of dietary VD3 supplementation in cirrhosis-associated hepatocarcinogenesis is still unknow. To investigate this purpose, male Wistar rats submitted to a combined diethylnitrosamine- and thioacetamide-induced model were concomitantly supplemented with VD3 (5,000 and 10,000 IU/kg diet) for 25 weeks. Liver samples were collected for histological, biochemical and molecular analysis. Serum samples were used to measure 25-hydroxyvitamin D [25(OH)D] and alanine aminotransferase levels. Both VD3 interventions decreased hepatic collagen deposition and pro-inflammatory p65 protein levels, while increased hepatic antioxidant catalase and glutathione peroxidase activities and serum 25(OH)D, without a clear dose-response effect. Nonetheless, only the highest concentration of VD3 increased hepatic protein levels of VD receptor, while decreased the number of large preneoplastic glutathione-S-transferase- (>0.5 mm²) and keratin 8/18-positive lesions, as well the multiplicity of hepatocellular adenomas. Moreover, this intervention increased hepatic antioxidant Nrf2 protein levels and glutathione-S-transferase activity. In summary, dietary VD3 supplementation - in special the highest intervention - showed antifibrotic and antineoplastic properties in chemically-induced cirrhosis-associated hepatocarcinogenesis. The positive modulation of Nrf2 antioxidant axis may be mechanistically involved with these beneficial effects, and may guide future clinical studies.

Synbiotic sheep milk ice cream reduces chemically induced mouse colon carcinogenesis.

Sheep dairy products containing prebiotic and probiotic ingredients may have health-promoting properties. Thus, this study evaluated the effects of sheep milk ice cream [conventional full-fat (CONV), full-fat enriched with probiotic (PROB, 100 mg % wt/wt of Lacticaseibacillus casei 01), or nonfat synbiotic (SYNB, Lacticaseibacillus casei 01 and inulin, 10% wt/wt)] on carcinogen-induced colonic crypt cytotoxicity and premalignant lesion development. Male Swiss mice received 2 doses of colon carcinogen azoxymethane (AOM, 15 mg/kg of body weight) at wk 3 and 4. Two weeks before and during AOM administrations (4 wk) mice were treated with CONV, PROB, or SYNB by gavage (10 mL/kg). Mice were euthanized at wk 4 or 25 (n = 5 or 10 mice/group, respectively). At wk 4, a significant reduction in micronucleated colonocytes was observed in PROB and SYNB groups, and a significant decrease in both p53 expression and apoptosis indexes in colonic crypts was observed in SYNB group. At wk 25, both PROB and SYNB interventions reduced the mean number of colonic premalignant lesions. However, only SYNB group showed lower incidence and number of high-grade premalignant lesions in the colonic mucosa. These findings indicate that PROB or SYNB sheep milk ice cream, especially SYNB intervention, can reduce chemically induced mouse colon carcinogenesis.

Burdock (Arctium lappa L.) root attenuates preneoplastic lesion development in a diet and thioacetamide-induced model of steatohepatitis-associated hepatocarcinogenesis.

Nonalcoholic steatohepatitis (NASH) is considered growing risk factor for hepatocellular carcinoma development in high-income countries. Diet- and chemically induced rodent models have been applied for the translational study of NASH-associated hepatocarcinogenesis due to their morphological and molecular similarities to the corresponding human disease. Arctium lappa L. (burdock) root tea has been extensively consumed in Traditional Chinese Medicine due to its potential therapeutic properties. Indeed, the bioactive compounds of A. lappa root, as the polyphenols, have already showed antioxidant and anti-inflammatory properties in different in vivo and in vitro bioassays. In this study, we investigated whether burdock root ethanolic extract (BRE) administration attenuates NASH-associated hepatocarcinogenesis. Eight-week-old male Wistar rats received choline-deficient high-fat diet for 8 weeks and multiple thioacetamide doses for 4 weeks in order to induce NASH and preneoplastic glutathione-S-transferase pi (GST-P)+ preneoplastic foci. Subsequently, rats were treated with BRE (100 or 200 mg/kg body weight) or vehicle by oral gavage for 2 weeks. BRE displayed high levels of chlorogenic and caffeic acids and BRE administration reduced total fatty acid and lipid hydroperoxide levels, while increasing the activities of antioxidant superoxide dismutase and catalase enzymes in the liver. Furthermore, burdock intervention diminished the size of GST-P+ remodeling preneoplastic lesions (PNLs) and displayed a trend on reducing hepatocyte proliferation (Ki-67) inside them. These findings suggest that short-term exposure to BRE alleviated remodeling PNL development in NASH-associated hepatocarcinogenesis.

Lyophilized açaí pulp (Euterpe oleracea Mart) attenuates colitis-associated colon carcinogenesis while its main anthocyanin has the potential to affect the motility of colon cancer cells.

This study evaluated the possible protective effects of lyophilized açaí pulp (AP) in a colitis-associated carcinogenesis (CAC) rat model and the modifying effect of cyanidin 3-rutinoside (C3R) on the motility of RKO colon adenocarcinoma cells, using the wound healing assay. Male Wistar rats were induced to develop CAC using 1,2-dimethylhydrazine (DMH) and 2,4,6-trinitrobenzene acid (TNBS). Animals were randomly assigned to different groups that received basal diet or basal diet supplemented with 5.0% or 7.5% lyophilized AP. The findings indicate: 1) C3R (25 µM) has the potential to reduce RKO cell motility in vitro; 2) ingestion of lyophilized AP reduces the total number of aberrant crypt foci (ACF), ACF multiplicity, tumor cell proliferation and incidence of tumors with high grade dysplasia; 3) AP increases the gene expression of negative regulators of cell proliferation such as Dlc1 and Akt3, as well as inflammation (Ppara). Thus, lyophilized AP could exert a potential antitumor activity.

TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice.

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

Vitamin D3 supplementation attenuates the early stage of mouse hepatocarcinogenesis promoted by hexachlorobenzene fungicide.

Hexachlorobezene (HCB), a fungicide widely distributed in the environment, promotes the development of hepatocellular preneoplastic lesions (PNL) and tumors in rodents. In contrast, vitamin D3 (VD3) supplementation presents a potential role for the prevention/treatment of chronic liver diseases. Thus, we investigated whether VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis. Female Balb/C mice were injected a single dose of diethylnitrosamine (DEN, 50 mg/kg) at postnatal day 15. From day 40 onwards, mice were fed with a standard diet containing 0.02% HCB alone or supplemented with VD3 (10,000 or 20,000 IU/Kg diet) for 20 weeks. Untreated mice were fed just standard diet. After this period, mice were euthanized and liver and serum samples were collected. Compared to the untreated group, DEN/HCB treatment decreased total hepatic glutathione levels and glutathione peroxidase (GSH-Px) activity while increased lipid peroxidation, p65 protein expression, cell proliferation/apoptosis and the PNL development. In contrast, dietary VD3 supplementation enhanced vitamin D receptor (VDR) protein expression, total glutathione levels and GSH-Px activity while diminished lipid hydroperoxide levels. Also, VD3 supplementation decreased p65 protein expression, hepatocyte proliferation, the size and the liver area occupied by PNL. Therefore, our findings indicate that VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis.

Açai (Euterpe oleracea Mart.) feeding attenuates dimethylhydrazine-induced rat colon carcinogenesis.

This study investigated the protective effect of spray-dried açaí powder (AP) intake on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats. After 4 weeks of DMH administrations, the groups were fed with standard diet, a diet containing 2.5% or 5.0% AP or a diet containing 0.2% N-acetylcysteine (NAC) for 10 weeks, using aberrant crypt foci (ACF) as the endpoint. Additionally, two groups were fed with standard diet or a diet containing 5.0% AP for 20 weeks, using colon tumors as the endpoint. In ACF assay, a reduction in the number of aberrant crypts (ACs) and ACF (1-3 AC) were observed in the groups fed with 5.0% AP (37% AC and 47% ACF inhibition, p=0.036) and 0.2% NAC (39% AC and 41% ACF inhibition, p=0.042). In tumor assay, a reduction in the number of invasive tumors (p<0.005) and tumor multiplicity (p=0.001) was observed in the group fed with 5.0% AP. Also, a reduction in tumor Ki-67 cell proliferation (p=0.003) and net growth index (p=0.001) was observed in the group fed with 5.0% AP. Therefore the findings of this study indicate that AP feeding may reduce the development of chemically-induced rat colon carcinogenesis.