Obeticholic acid alleviates intrauterine growth restriction induced by di-ethyl-hexyl phthalate in pregnant female mice by improving bile acid disorder.

Di-(2-ethylhexyl)-phthalate (DEHP) is a ubiquitous environmental pollutant and is widely used in industrial plastics. Intrahepatic cholestasis of pregnancy (ICP), distinguished by maternal pruritus and elevated serum bile acid levels, is linked to unfavorable pregnancy consequences. Few studies have investigated the potential effect of gestational DEHP exposure on the cholestasis in pregnant female mice, and the underlying mechanisms remain unclear. In the present study, a mouse model of cholestasis during pregnancy was established by DEHP exposure. We found that DEHP induces elevated bile acid levels by affecting bile acid synthesis and transporter receptor expression in the maternal liver and placenta of pregnant female mice, ultimately leading to intrauterine growth restriction (IUGR). In addition, DEHP changed the bile acid composition of maternal serum and liver as well as placenta and amniotic fluid in pregnant female mice; Importantly, we found that DEHP down-regulates the expression of farnesoid X receptor (FXR), which is considered to be a bile acid receptor. FXR agonist obeticholic acid (OCA) effectively alleviated the adverse effects of DEHP on pregnant female mice. While, OCA itself had no adverse effects on normal pregnant female mice. In summary, DEHP could induces bile acid disorder and IUGR in pregnant female mice by affect FXR, which was reversed by OCA.

Establishment of an in vitro cholestasis risk assessment system using two-dimensional cultured HepaRG cells and 12 bile acids.

Drug-induced liver injury (DILI) is a major cause of market withdrawal or drug-development discontinuation because of safety concerns. In this study, we focused on drug-induced cholestasis (DIC) to establish an in vitro cytotoxicity test system and analyze its sensitivity using two-dimensional (2-D) cultured HepaRG cells and 12 types of bile acids (BAs) present in the human serum. First, to detect the cytotoxicity associated with cholestasis effectively, non-toxic BA concentrations were investigated and determined to be 100-fold the human serum value (455 µM total BAs). Next, the cytotoxicity of 31 compounds that can inhibit the bile acid export pump (BSEP) and were categorized as no-DILI-concern, less-DILI-concern, and most-DILI-concern was examined. None of the no-DILI-concern compounds yielded cytotoxicity, whereas almost all less-DILI-concern compounds (with the exception of simvastatin) and most-DILI-concern compounds (with the exception of bosentan) exhibited cytotoxicity. An investigation of the cause of cytotoxicity using 3H-taurocholic acid revealed that most-DILI-concern and less-DILI-concern compounds, but not no-DILI-concern compounds, triggered the accumulation of radioactivity in the cell lysates. Thus, the onset of cytotoxicity seemed to be associated with cholestasis. The established HepaRG cytotoxicity assessment system (sensitivity of 89%, specificity of 100%, and accuracy of 97%) was mostly superior to the Css/BSEP IC50 (> 0.1) assessment system (sensitivity of 83%, specificity of 100%, and accuracy of 72%). Therefore, the assay method using 2-D cultured HepaRG cells and 12 BAs established here can be widely applicable as a model for the in vitro potential assessment of DIC.

Bile acid- and ethanol-mediated activation of Orai1 damages pancreatic ductal secretion in acute pancreatitis.

Regardless of its aetiology, sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevation induces pancreatic ductal cell damage characterized by impaired ion and fluid secretion - essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions - and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1, a store-operated Ca2+ influx channel, is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside predominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protects ductal cell secretory function in vitro and decreases pancreatic ductal cell death. Finally, Orai1 inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. KEY POINTS: Sustained intracellular Ca2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca2+ channel that mediates extracellular Ca2+ influx upon endoplasmic reticulum Ca2+ depletion. Results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo acute pancreatitis models. Orai1 inhibition prevents the bile acid- and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis.

Omega-3 Polyunsaturated Fatty Acid: A Pharmaco-Nutraceutical Approach to Improve the Responsiveness to Ursodeoxycholic Acid.

Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases.

Asociación entre los niveles elevados de ácidos biliares y cáncer digestivo / Association between high bile acid levels and digestive cancer

Introducción: Los ácidos biliares en condiciones no fisiológicas se consideran agentes inflamatorio-carcinógenos endógenos que originan alteraciones en membranas plasmáticas, mitocondrias, el ADN, los genes y, la apoptosis de las células epiteliales. Objetivo: Describir la asociación entre los niveles elevados de ácidos biliares en la luz intestinal y la secuencia inflamación-cáncer, expresados como lesiones inflamatorias, premalignas y malignas del tracto digestivo. Métodos: Revisión sistemática y crítica de las evidencias sobre los mecanismos biomoleculares asociados a niveles altos de ácidos biliares en la luz intestinal y la secuencia inflamación-carcinogénesis, en bases de datos como PubMed, Medline, SciELO, LILACS y Elsevier, publicados entre 2015-2020, que establecen el fundamento teórico y metabolómico de dicha secuencia. Resultados: Los ácidos biliares tienen una acción tóxica en la secuencia inflamación-cáncer del tracto digestivo, al perderse el control de su homeostasis o la integridad anatomo-funcional del sistema hepato-vesículo-bilio-intestinal. Conclusiones: Los mecanismos celulares y biomoleculares desencadenados por los niveles altos de ácidos biliares contextualizan la génesis del proceso secuencial inflamación-cáncer y su interacción con los factores de riesgo clásicos, genéticos y epigenéticos reconocidos como un nuevo paradigma fisiopatológico del cáncer digestivo(AU)

Introduction: In non-physiological conditions, bile acids (BA) are considered to be endogenous inflammatory-carcinogenic agents causing alterations in plasma membranes, mitochondria, DNA, genes and epithelial cell apoptosis. Objective: Describe the association between high bile acid levels in the intestinal lumen and the inflammation-cancer sequence, expressed as inflammatory premalignant and malignant lesions of the digestive tract. Methods: A systematic critical review was conducted of the evidence about biomolecular mechanisms associated to high bile acid levels in the intestinal lumen and the inflammation-carcinogenesis sequence published in the databases PubMed, Medline, SciELO, LILACS and Elsevier in the period 2015-2020, laying the theoretical and metabolomic foundations of that sequence. Results: Bile acids display toxic activity in the inflammation-cancer sequence of the digestive tract, since control is lost of its homeostasis or the anatomical-functional integrity of the hepato-vesicular-biliary-intestinal system. Conclusions: The cellular and biomolecular mechanisms triggered by high bile acid levels provide a context for the genesis of the inflammation-cancer sequential process and its interaction with the classic, genetic and epigenetic risk factors recognized as a new pathophysiological paradigm of digestive cancer(AU)

Hesperidin alleviates cholestasis via activation of the farnesoid X receptor in vitro and in vivo.

Cholestasis causes the intrahepatic accumulation of bile acids leading to hepatobiliary injury. Recently obeticholic acid, a farnesoid X receptor (FXR) agonist, was FDA-approved to treat cholestatic liver diseases, providing a new therapeutic strategy for cholestasis. The purpose of the current study was to characterize a novel FXR agonist and verify the anti-cholestatic effect of hesperidin (HP) in vivo and in vitro. Based on a molecular docking study that predicted that HP would bind to FXR, the hepatoprotective effect of HP against cholestasis and hepatotoxicity was evaluated in mice and in normal and FXR-suppressed HepaRG cells. HP prevented bile acid toxicity in HepaRG cells, and this effect was blocked by FXR silencing. HP appears to activate FXR to prevent cholestatic liver injury. Dynamic change analysis of bile acids revealed that HP promoted bile acid excretion into feces and reduced hepatic accumulation via the regulation of the FXR-target genes bile salt export pump, multi-drug resistance-associated protein 2, and Na+-taurocholate cotransporting polypeptide. Furthermore, HP down-regulated enzymes involved in bile acid synthesis including cholesterol 7α-hydroxylase and sterol 27-hydroxylase. HP produced a protective effect against cholestasis via FXR activation, and may be an effective approach for the prevention and treatment of cholestatic liver diseases.

Cholesterol Induces Nrf-2- and HIF-1α-Dependent Hepatocyte Proliferation and Liver Regeneration to Ameliorate Bile Acid Toxicity in Mouse Models of NASH and Fibrosis.

Nonalcoholic steatohepatitis (NASH) is currently one of the most common liver diseases worldwide. The toxic effects of lipids and bile acids contribute to NASH. The regenerative pathway in response to damage to the liver includes activation of the inflammatory process and priming of hepatocytes to proliferate to restore tissue homeostasis. However, the effects of cholesterol on bile acid toxicity, inflammation, and fibrosis remain unknown. We have used two mouse models of bile acid toxicity to induce liver inflammation and fibrosis. A three-week study was conducted using wild-type mice receiving an atherogenic diet (1% (w/w) cholesterol and 0.5% (w/w) cholic acid) and its separate constituents. Mdr2-/- mice were fed a high-cholesterol-enriched diet or standard AIN-93 diet for 6 weeks. We measured serum transaminase levels to assess liver tissue necrosis and fibrosis; iNOS, SAA1, SAA2, and F4/80 levels to determine liver inflammation; PCNA and HGF levels to evaluate proliferative response; and Nrf-2, HIF-1α, and downstream gene expression to establish protective responses. In both studies, high bile acid levels increased serum transaminases and liver fibrosis, whereas cholesterol supplementation attenuated these effects. Cholesterol supplementation activated survival and the robustness of HIF-1α and Nrf-2 gene expression in hepatocytes, induced liver inflammation and hepatocyte proliferation, and inhibited stellate cell hyperplasia and fibrosis. In conclusion, our data show for the first time that cholesterol intake protects against bile acid liver toxicity. The balance between hepatic cholesterol and bile acid levels may be of prognostic value in liver disease progression and trajectory.

Actividad biológica y toxicológica de los ácidos biliares en la actualidad / Biological and toxicological activity of bile acids today

Introducción: los ácidos biliares no solo tienen como actividad biológica regular la absorción de vitaminas liposolubles, colesterol y lípidos, sino actúan también como moléculas de señalización, moduladores de la proliferación celular intestinal, de la expresión de genes y del metabolismo energético según estudios in vitro e in vivo; en condiciones fisiológicas mantienen su homeostasis, que al ser interrumpida promueve suacción toxicológica. Objetivo: describir la actualidad de los nuevos conocimientos sobre la actividad biológica y toxicológica de los ácidos biliares en el aparato digestivo, dirigido a cirujanos generales, gastroenterólogos, clínicos y fisiólogos que les permitan contextualizar el proceso inflamación-carcinogénesis relacionado con los efectos toxicológicos de los ácidos biliares. Método: se realizó una revisión sistemática de la actividad biológica y toxicológica de los ácidos biliares para los cirujanos generales, gastroenterólogos, clínicos y fisiólogos, como herramienta útil en la compresión fisiopatológico del metabolismo de los ácidos biliares. Conclusión: los ácidos biliares desempeñan una función clave como moléculas de señalización en la modulación de la proliferación de células epiteliales, la expresión de genes y el metabolismo energético, que cuando se interrumpe su homeostasis se promueve la acción tóxica de estos, lo que se traduce en el proceso inflamación-carcinogénesis digestiva(AU)

Introduction: bile acids not only have as a regular biological activity the absorption of fat-soluble vitamins, cholesterol and lipids, but also act as signaling molecules, modulators of intestinal cell proliferation, gene expression and energy metabolism according to in vitro studies and in vivo; under physiological conditions they maintain their homeostasis, which when interrupted promotes their toxicological action. Objective: to describe the news of the new knowledge about the biological and toxicological activity of bile acids in the digestive system, aimed at general surgeons, gastroenterologists, clinicians and physiologists that allow them to contextualize the inflammation-carcinogenesis process related to the toxicological effects of bile acids. Method: A systematic review of the biological and toxicological activity of bile acids was performed for general surgeons, gastroenterologists, clinicians and physiologists, as a useful tool in the pathophysiological compression of bile acid metabolism. Conclusion: bile acids play a key role as signaling molecules in the modulation of epithelial cell proliferation, gene expression and energy metabolism, which when their homeostasis is interrupted, their toxic action is promoted, which translates in the inflammation-digestive carcinogenesis process(AU)

Facial Amphiphilicity-Induced Polymer Nanostructures for Antimicrobial Applications.

New antimicrobial agents are needed to address ever-increasing antimicrobial resistance and a growing epidemic of infections caused by multidrug resistant pathogens. We design nanostructured antimicrobial copolymers containing multicyclic natural products that bear facial amphiphilicity. Bile acid based macromolecular architectures of these nanostructures can interact preferentially with bacterial membranes. Incorporation of polyethylene glycol into the copolymers not only improved the colloidal stability of nanostructures but also increased the biocompatibility. This study investigated the effects of facial amphiphilicity, polymer architectures, and self-assembled nanostructures on antimicrobial activity. Advanced nanostructures such as spheres, vesicles, and rod-shaped aggregates are formed in water from the facial amphiphilic cationic copolymers via supramolecular interactions. These aggregates were particularly interactive toward Gram-positive and Gram-negative bacterial cell membranes and showed low hemolysis against mammalian cells.

Microbial Metabolites Determine Host Health and the Status of Some Diseases.

The gastrointestinal (GI) tract is a highly complex organ composed of the intestinal epithelium layer, intestinal microbiota, and local immune system. Intestinal microbiota residing in the GI tract engages in a mutualistic relationship with the host. Different sections of the GI tract contain distinct proportions of the intestinal microbiota, resulting in the presence of unique bacterial products in each GI section. The intestinal microbiota converts ingested nutrients into metabolites that target either the intestinal microbiota population or host cells. Metabolites act as messengers of information between the intestinal microbiota and host cells. The intestinal microbiota composition and resulting metabolites thus impact host development, health, and pathogenesis. Many recent studies have focused on modulation of the gut microbiota and their metabolites to improve host health and prevent or treat diseases. In this review, we focus on the production of microbial metabolites, their biological impact on the intestinal microbiota composition and host cells, and the effect of microbial metabolites that contribute to improvements in inflammatory bowel diseases and metabolic diseases. Understanding the role of microbial metabolites in protection against disease might offer an intriguing approach to regulate disease.

Development of an in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells in sandwich configuration.

Toxicological approaches in screening drugs that cause drug-induced liver injury (DILI) are urgently needed to reduce the risk of developing DILI and avoid immense costs resulting from late-stage drug withdrawal from clinical trials. Cholestatic DILI is characterized by bile acid (BA) accumulation in hepatocytes, typically caused by drug-induced inhibition of important bile transporters, such as bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4). Therefore, NTCP expression is essential for construction of an in vitro hepatocellular toxicity evaluation system. Here, we investigated whether sandwich-cultured HepG2-hNTCP-C4 (SCHepG2-hNTCP-C4) cells were applicable for evaluation of cholestatic DILI. In SCHepG2-hNTCP-C4 cells, NTCP and MRP2/4 expression levels were comparable to those in human primary hepatocytes; however, BSEP expression was low. In addition, the substrates tauro-nor-THCA-24 DBD and CDF confirmed the functionality of NTCP and MRP2, respectively. When 22 known hepatotoxins were exposed to BAs to evaluate cholestatic DILI, cytotoxicity in SCHepG2-hNTCP-C4 cells was more frequent than that in SCHepG2 cells. Thus, SCHepG2-hNTCP-C4 cells may be useful preclinical screening tools to predict the risk of cholestatic DILI induced by drug candidates. However, further studies are needed to determine why the cholestatic cytotoxicity of some compounds would be still insufficient in SCHepG2-hNTCP-C4 cells.

Biliary reflux as a causal factor in hypopharyngeal carcinoma: New clinical evidence and implications.

BACKGROUND: Recent preclinical explorations strongly support the tumorigenic potential of bile on laryngopharyngeal mucosa. Herein, the authors describe, in bile-related human hypopharyngeal squamous cell carcinoma (HSCC), NF-κB-related messenger RNA (mRNA) and microRNA (miRNA) oncogenic phenotypes similar to those previously identified in acidic bile-exposed premalignant murine hypopharyngeal mucosa. METHODS: In this pilot study, the authors included human HSCC specimens paired with their adjacent normal tissue (ANT) derived from 3 representative patients with documented biliary laryngopharyngeal reflux (bile[+]) compared with 5 control patients without signs of bile reflux disease (bile[-]). Immunohistochemical, quantitative polymerase chain reaction, and miRNA analyses were used to detect the levels of activated NF-κB and expression levels of STAT3, EGFR, BCL2, WNT5A, IL-6, IL-1B, ΔNp63, cREL, TNF-α, TP53, NOTCH1, NOTCH2, NOTCH3, miR-21, miR-155, miR-192, miR-34a, miR-375, miR-451a, miR-489, miR-504, and miR-99a. RESULTS: Bile(+) HSCC demonstrated an intense NF-κB activation accompanied by significant overexpression of RELA(p65), EGFR, STAT3, BCL-2, cREL, ΔNp63, WNT5A, IL-6, and IL1B; upregulation of oncomir miR-21; and downregulation of tumor suppressor miR-375 compared with their respective ANTs. Bile(+) HSCC demonstrated significantly higher mRNA levels of all the analyzed genes, particularly RELA(p65), IL-6, EGFR, and TNF-α compared with bile(-) tumors. The miR-21/miR-375 ratio, which previously has been linked to tumor aggressiveness, was found to be >260-fold and >30-fold higher, respectively, in bile(+) HSCCs compared with their ANTs and bile(-) tumors. CONCLUSIONS: Although limitations apply to this pilot study due to the small number of patients with HSCC, the novel findings suggest that a history of bile as a component of esophageal reflux disease may represent an independent risk factor for hypopharyngeal carcinogenesis.

Differential toxic effects of bile acid mixtures in isolated mitochondria and physiologically relevant HepaRG cells.

Bile acids (BAs) are recognised as the causative agents of toxicity in drug-induced cholestasis (DIC). Research in isolated mitochondria and HepG2 cells have demonstrated BA-mediated mitochondrial dysfunction as a key mechanism of toxicity in DIC. However, HepG2 cells are of limited suitability for DIC studies as they do not express the necessary physiological characteristics. In this study, the mitotoxic potentials of BA mixtures were assessed in isolated mitochondria and a better-suited hepatic model, HepaRG cells. BAs induced structural alterations and a loss of mitochondrial membrane potential (MMP) in isolated mitochondria however, this toxicity did not translate to HepaRG cells. There were no changes in oxygen consumption rate, MMP or ATP levels in glucose and galactose media, indicating that there was no direct mitochondrial toxicity mediated via electron transport chain dysfunction in HepaRG cells. Assessment of key biliary transporters revealed that there was a time-dependent reduction in the expression and activity of multi-drug resistance protein 2 (MRP2), which was consistent with the induction of cytotoxicity in HepaRG cells. Overall, the findings from this study have demonstrated that mitochondrial dysfunction is not a mechanism of BA-induced toxicity in HepaRG cells.

Bile salts regulate CYP7A1 expression and elicit a fibrotic response and abnormal lipid production in 3D liver microtissues.

Disrupted regulation and accumulation of bile salts (BS) in the liver can contribute towards progressive liver damage and fibrosis. Here, we investigated the role of BS in the progression of cholestatic injury and liver fibrosis using 3D scaffold-free multicellular human liver microtissues (MTs) comprising the cell lines HepaRG, THP-1 and hTERT-HSCs. This in vitro model has been shown to recapitulate cellular events leading to fibrosis including hepatocellular injury, inflammation and activation of HSCs, ultimately leading to increased deposition of extracellular matrix (ECM). In order to better differentiate the contribution of individual cells during cholestasis, the effects of BS were evaluated either on each of the three cell types individually or on the multicellular MTs. Our data corroborate the toxic effects of BS on HepaRG cells and indicate that BS exposure elicited a slight increase in cytokines without causing stellate cell activation. Contrarily, using the MTs, we could demonstrate that low concentrations of BS led to cellular damage and triggered a fibrotic response. This indicates that cellular interplay is required to achieve BS-triggered activation of HSC. Moreover, BS were capable of down-regulating CYP7A1 expression in MTs and elicited abnormal lipid production (accumulation) concordant with clinical cases where chronic cholestasis results in hypercholesterolemia.

Safety, functional properties and technological performance in whey-based media of probiotic candidates from human breast milk

We aimed at isolating and characterising microorganisms present in human breast milk with probiotic potential. In an 8-week postpartum sampling period, two strains of bifidobacteria (Bifidobacterium longum LM7a and Bifidobacterium dentium LM8a') and four strains of lactobacilli were isolated, all during the first 4-week postpartum. B. longum LM7a and B. dentium LM8a', together with four strains previously isolated from breast milk (Bifidobacterium lactis INL1, INL2, INL4 and INL5), were considered for further studies. Susceptibility of the strains to tetracycline, erythromycin, clindamycin, streptomycin, vancomycin and chloramphenicol was evaluated and the isolates exhibited, in general, the same properties as previously reported for bifidobacteria. All isolates showed low hydrophobicity and B. lactis and B. longum strains had satisfactory resistance to gastric digestion and bile shock, but not to pancreatin. B. lactis INL1, B. longum LM7a and B. dentium LM8a' were selected for some comparative technological studies. In particular, B. lactis INL1 displayed technological potential, with satisfactory growth in cheese whey-based media in biofermentor and resistance to freeze-drying, accelerated storage conditions and simulated gastric digestion

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Molecular Mechanisms for Protection of Hepatocytes against Bile Salt Cytotoxicity.

Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.

Safety, functional properties and technological performance in whey-based media of probiotic candidates from human breast milk.

We aimed at isolating and characterising microorganisms present in human breast milk with probiotic potential. In an 8-week postpartum sampling period, two strains of bifidobacteria (Bifidobacterium longum LM7a and Bifidobacterium dentium LM8a') and four strains of lactobacilli were isolated, all during the first 4-week postpartum. B. longum LM7a and B. dentium LM8a', together with four strains previously isolated from breast milk (Bifidobacterium lactis INL1, INL2, INL4 and INL5), were considered for further studies. Susceptibility of the strains to tetracycline, erythromycin, clindamycin, streptomycin, vancomycin and chloramphenicol was evaluated and the isolates exhibited, in general, the same properties as previously reported for bifidobacteria. All isolates showed low hydrophobicity and B. lactis and B. longum strains had satisfactory resistance to gastric digestion and bile shock, but not to pancreatin. B. lactis INL1, B. longum LM7a and B. dentium LM8a' were selected for some comparative technological studies. In particular, B. lactis INL1 displayed technological potential, with satisfactory growth in cheese whey-based media in biofermentor and resistance to freeze-drying, accelerated storage conditions and simulated gastric digestion.

Bile acids induce visceral hypersensitivity via mucosal mast cell-to-nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis.

Increased colonic bile acid (BA) exposure, frequent in diarrhea-predominant irritable bowel syndrome (IBS-D), can affect gut function. Nerve growth factor (NGF) is implicated in the development of visceral hypersensitivity (VH). In this study, we tested the hypothesis that BAs cause VH via mucosal mast cell (MMC)-to-nociceptor signaling, which involves the farnesoid X receptor (FXR)/NGF/transient receptor potential vanilloid (TRPV)1 axis. BAs were intracolonically administered to rats for 15 d. Visceral sensitivity to colorectal distention and colonic NGF expression were examined. BAs caused VH, an effect that involved MMC-derived NGF and was accompanied by enhanced TRPV1 expression in the dorsal root ganglia. Anti-NGF treatment and TRPV1 antagonism inhibited BA-induced VH. BAs induced NGF mRNA and protein expression and release in cultured mast cells. Colonic supernatants from patients with IBS-D with elevated colonic BA content transcriptionally induced NGF expression. In FXR-/- mice, visceral sensitivity and colonic NGF expression were unaltered after BA treatment. Pharmacological antagonism and FXR silencing suppressed BA-induced NGF expression and release in mast cells. Mitogen-activated protein kinase kinase (MKK) 3/6/p38 MAPK/NF-κB signaling was mechanistically responsible for FXR-mediated NGF expression and secretion. The findings show an MMC-dependent and FXR-mediated pronociceptive effect of BAs and identify the BA/FXR/NGF/TRPV1 axis as a key player in MMC-to-neuron communication during pain processing in IBS.-Li, W.-T., Luo, Q.-Q., Wang, B., Chen, X., Yan, X.-J., Qiu, H.-Y., Chen, S.-L. Bile acids induce visceral hypersensitivity via mucosal mast cell-to-nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis.

Acidic Bile Salts Induce Epithelial to Mesenchymal Transition via VEGF Signaling in Non-Neoplastic Barrett's Cells.

BACKGROUND & AIMS: Metaplastic glands buried under squamous epithelium are frequently detected in patients with Barrett esophagus (BE). This subsquamous intestinal metaplasia might be responsible for cancers that develop despite endoscopic surveillance and for metaplasia recurrences after endoscopic ablation. To determine whether reflux induces BE cells to undergo an epithelial-to-mesenchymal transition (EMT) that produces subsquamous intestinal metaplasia, we assessed EMT in BE cells exposed to acidic bile salts and in rat and human esophageal tissues. METHODS: We compared markers of EMT and cell motility in trans-well and 3-dimensional organotypic culture systems among dysplastic BE epithelial cell lines, nondysplastic telomerase-immortalized BE cell lines (BAR-T), and BAR-T cells exposed acutely or for 20 weeks to acidic bile salts. Vascular endothelial growth factor (VEGF) A was inhibited with a neutralizing antibody or CRISPR-Cas9n and VEGF receptor 2 was inhibited with SU1498 or shRNA, and cells were analyzed by immunohistochemistry, quantitative polymerase chain reaction, or immunoblotting for markers of VEGF signaling and EMT; cell motility was assessed by trans-well assay. We used immunohistochemistry and quantitative polymerase chain reaction to assess EMT markers in the columnar-lined esophagus of rats with surgically induced reflux esophagitis and in esophagectomy specimens from patients with BE. RESULTS: We detected features of EMT (decreased cadherin 1 [CDH1]; increased fibronectin 1, vimentin, and matrix metalloproteinase 2; and increased motility) in dysplastic BE epithelial cell lines and in BAR-T cells exposed for 20 weeks, but not in unexposed BAR-T cells. Acute acidic bile salt exposure induced expression of zinc finger E-box binding homeobox 1 and 2 (ZEB1/2) in BAR-T cells, which decreased their expression of CDH1 and increased motility; inhibitors of VEGF signaling blocked these effects. Columnar-lined esophagus of rats with reflux esophagitis had increased expression of ZEB1/2 and decreased expression of CDH1 compared with controls. Dysplastic BE tissues also had significantly increased levels of ZEB1 and significantly decreased levels of CDH1 compared with nondysplastic BE tissues. CONCLUSIONS: In BE cell lines, acidic bile salts induce EMT by VEGF signaling, which increases expression of ZEB1/2, repressors of CDH1. These observations suggest that reflux induces EMT in metaplastic BE tissues, which promotes development of subsquamous intestinal metaplasia.