Taurocholate uptake by Caco-2 cells is inhibited by pro-inflammatory cytokines and butyrate.

Inflammatory bowel disease (IBD) is a group of chronic and life-threating inflammatory diseases of the gastrointestinal tract. The active intestinal absorption of bile salts is reduced in IBD, resulting in higher luminal concentrations of these agents that contribute to the pathophysiology of IBD-associated diarrhea. Butyrate (BT) is a short-chain fatty acid produced by colonic bacterial fermentation of dietary fibers. BT utilization is impaired in the intestinal inflamed mucosa of IBD patients. Our aim was to investigate the link between IBD and bile acid absorption, by testing the effect of the pro-inflammatory cytokines TNF-α and IFN-γ and of BT upon 3H-TC uptake by Caco-2 cells. The proinflammatory cytokines TNF-α and IFN-γ inhibit Na+-independent, non-ASBT (sodium-dependent bile acid transporter)-mediated 3H-TC uptake by Caco-2 cells. The inhibitory effect of these cytokines on Na+-independent 3H-TC uptake is PI3K- and JAK/STAT1-mediated. These two compounds upregulate ASBT expression levels, but no corresponding increase in Na+-dependent component of 3H-TC is observed. Moreover, BT was also found to inhibit 3H-TC uptake and showed an additive effect with IFN-γ in reducing 3H-TC uptake. We conclude that an interaction between BT and bile acids appears to exist in IBD, which may participate in the link between diet, microbiota and IBD.

Bile components affect the functions and transcriptome of the rainbow trout intestinal epithelial cell line RTgutGC.

The concomitant increase in cultivation of fish and decrease in supply of marine ingredients, have greatly increased the demand for new nutrient sources. This also regards so-called functional ingredients which may benefit health and welfare of the fish. In vitro cell line-based intestinal epithelial barrier models may serve as tools for narrowing down the broad range of ingredient options, to identify the most promising candidates before in vivo feeding trials are run. In vivo, differentiation of the various epithelial cells in the fish intestine, from the multipotent stem cells, takes place in the presence of a variety of substances from dietary and endogenous origin. Among these, bile salts have recently received attention as regulators of epithelial function in health and disease but have not, until now, been included in the medium when culturing fish gut epithelial cells in vitro. As bile salts are present at high levels in the chyme of the fish intestine, in particular in salmon and rainbow trout, mostly as taurocholate (>90%), their role for effects of diet ingredients on the in vitro gut cell model should be understood. With this study, we wanted to investigate whether inclusion of bile from rainbow trout or pure taurocholate in the culture media would modulate functions of the RTgutGC epithelial cells. Here, we demonstrated that the rainbow trout intestinal epithelial cell line RTgutGC responded significantly to the presence of bile components. Treatment with rainbow trout bile taken from the gall bladder (RTbile) or pure taurocholate (TC) at taurocholate concentrations of ≤0.5 mg/mL retained normal cell morphology, cell viability as in cell oxidation-reduction metabolic activity and membrane integrity, and barrier features, while high concentrations of bile salts (≥1 mg/mL) were cytotoxic to the cells. After long-term (4 days) bile treatment, transcriptome responses showed how bile salts play important roles in intestinal epithelial cell metabolism. qPCR data demonstrated that barrier function genes, brush border enzyme genes and immune genes were significantly affected. Although similar trends were seen, treatment with bile salt as a component of rainbow trout bile or pure taurocholate, induced somewhat different effects. In conclusion, this study clearly indicates that bile salts should be included in the cell medium when running in vitro studies of gut cell functions, not at least immune functions, preferably at the level of ∼0.5 mg/mL supplemented as pure taurocholate to ensure reproducibility.

Inhibition of canalicular and sinusoidal taurocholate efflux by cholestatic drugs in human hepatoma HepaRG cells.

HepaRG cells are highly-differentiated human hepatoma cells, which are increasingly recognized as a convenient cellular model for in vitro evaluation of hepatic metabolism, transport, and/or toxicity of drugs. The present study was designed to evaluate whether HepaRG cells can also be useful for studying drug-mediated inhibition of canalicular and/or sinusoidal hepatic efflux of bile acids, which constitutes a major mechanism of drug-induced liver toxicity. For this purpose, HepaRG cells, initially loaded with the bile acid taurocholate (TC), were reincubated in TC-free transport assay medium, in the presence or absence of calcium or drugs, before analysis of TC retention. This method allowed us to objectivize and quantitatively measure biliary and sinusoidal efflux of TC from HepaRG cells, through distinguishing cellular and canalicular compartments. In particular, time-course analysis of the TC-free reincubation period of HepaRG cells, that is, the efflux period, indicated that a 20 min-efflux period allowed reaching biliary and sinusoidal excretion indexes for TC around 80% and 60%, respectively. Addition of the prototypical cholestatic drugs bosentan, cyclosporin A, glibenclamide, or troglitazone during the TC-free efflux phase period was demonstrated to markedly inhibit canalicular and sinusoidal secretion of TC, whereas, by contrast, incubation with the noncholestatic compounds salicylic acid or flumazenil was without effect. Such data therefore support the use of human HepaRG cells for in vitro predicting drug-induced liver toxicity (DILI) due to the inhibition of hepatic bile acid secretion, using a biphasic TC loading/efflux assay.

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators.

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin-Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Bile acid treatment and FXR agonism lower postprandial lipemia in mice.

Postprandial dyslipidemia is a common feature of insulin-resistant states and contributes to increased cardiovascular disease risk. Recently, bile acids have been recognized beyond their emulsification properties as important signaling molecules that promote energy expenditure, improve insulin sensitivity, and lower fasting lipemia. Although bile acid receptors have become novel pharmaceutical targets, their effects on postprandial lipid metabolism remain unclear. Here, we investigated the potential role of bile acids in regulation of postprandial chylomicron production and triglyceride excursion. Healthy C57BL/6 mice were given an intraduodenal infusion of taurocholic acid (TA) under fat-loaded conditions, and circulating lipids were measured. Targeting of bile acid receptors was achieved with GW4064, a synthetic agonist to the farnesoid X receptor (FXR), and deoxycholic acid (DCA), an activator of the Takeda G-protein-coupled receptor 5. TA, GW4064, and DCA treatments all lowered postprandial lipemia. FXR agonism also reduced intestinal triglyceride content and activity of microsomal triglyceride transfer protein, involved in chylomicron assembly. Importantly, TA (but not DCA) effects were largely lost in FXR knockout mice. These bile acid effects are reminiscent of the antidiabetic hormone glucagon-like peptide-1 (GLP-1). Although the GLP-1 receptor agonist exendin-4 retained its ability to acutely lower postprandial lipemia during bile acid sequestration and FXR deficiency, it did raise hepatic expression of the rate-limiting enzyme for bile acid synthesis. Bile acid signaling may be an important mechanism of controlling dietary lipid absorption, and bile acid receptors may constitute novel targets for the treatment of postprandial dyslipidemia.NEW & NOTEWORTHY We present new data suggesting potentially important roles for bile acids in regulation of postprandial lipid metabolism. Specific bile acid species, particularly secondary bile acids, were found to markedly inhibit absorption of dietary lipid and reduce postprandial triglyceride excursion. These effects appear to be mediated via bile acid receptors, farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5). Importantly, bile acid signaling may trigger glucagon-like peptide-1 (GLP-1) secretion, which may in turn mediate the marked inhibitory effects on dietary fat absorption.

Taurocholic acid inhibits features of age-related macular degeneration in vitro.

Previous metabolomics studies from our lab found altered plasma levels of bile acids in patients with age-related macular degeneration (AMD) compared to controls. In this study, we investigated the ability of the bile acid taurocholic acid (TCA) to inhibit features of AMD modeled in vitro. Paraquat was used to induce oxidative stress in HRPEpiC primary retinal pigment epithelial (RPE) cells. Cells were treated with 300 µM paraquat alone or with TCA (10, 50, 100, 200, or 500 µM). RPE tight junction integrity was assessed via ZO-1 immunofluorescence and transepithelial electrical resistance (TEER) measurements. RF/6A macaque choroidal endothelial cells were treated with 100 ng/mL vascular endothelial growth factor (VEGF) to induce angiogenesis. The effect of TCA on VEGF-induced angiogenesis was evaluated with cell proliferation, cell migration, and tube formation assays. Addition of TCA at 100 (P = 8.6 × 10-4), 200 (P = 0.0035), and 500 (P = 2.1 × 10-4) µM resulted in significant preservation of TEER in paraquat treated cells. In RF/6A cells, TCA did not significantly affect VEGF-induced cell proliferation. VEGF-induced migration of RF/6A cells was significantly inhibited at TCA concentrations of 100 (P = 0.010), 200 (P = 0.023) and 500 (P = 0.0049) µM. VEGF-induced tube formation was significantly inhibited when treated with 200 (P = 0.014) and 500 (P = 7.1 × 10-4) µM TCA. In vitro, TCA promoted RPE cell integrity and diminished VEGF-induced choroidal endothelial cell migration and tube formation. This suggests that TCA may have protective effects against both degenerative and neovascular AMD.

In Vitro Functional Characterization and in Silico Prediction of Rare Genetic Variation in the Bile Acid and Drug Transporter, Na+-Taurocholate Cotransporting Polypeptide (NTCP, SLC10A1).

Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1) is a key hepatic uptake transporter for bile acids and drugs and is the main functional receptor for hepatitis B and D viruses. Next-generation sequencing has revealed that a large number of rare SLC10A1 variants exist in the population. Little data exist regarding head-to-head comparison of in silico algorithms to predict functional effects of pharmacogenetic variants when compared to direct in vitro functional assessment. This study aimed at characterizing rare SLC10A1 variants in vitro and to assess the performance of seven in silico algorithms to predict the observed functional impacts. Thirty-five previously uncharacterized, rare, missense SLC10A1 variants were transiently expressed in human embryonic kidney 293 type T (HEK293T) cells. NCTP protein expression as well as uptake of substrates taurocholic acid (TCA) and rosuvastatin were assessed. Substrate-specific effects were observed for NTCP G191R, with TCA and rosuvastatin transport observed at 89 and 8% of wild-type (WT) uptake, respectively. Significantly reduced transport of TCA and rosuvastatin was observed for 19 variants (p < 0.05), with seven variants displaying decreased protein expression and marked reduction in transport of both substrates (0-13% of WT uptake, p < 0.0001). Performance of in silico algorithms to predict in vitro uptake, assessed using the area under the receiver operating characteristic curves (AUCROC), ranged from 0.69 to 0.97 and 0.72 to 0.84 for TCA and rosuvastatin uptake, respectively. In conclusion, we identified rare variants with significantly reduced NTCP expression and function. We demonstrated that no algorithm performed robustly enough to replace functional study in vitro, particularly given the broad substrate specificity of many pharmacogenes.

Long-term intervention of taurocholic acid over-expressing in cholestatic liver disease inhibits the growth of hepatoma cells.

Bile acids usually build up in patients with cholestatic liver disease. It was found that the concentration of taurocholic acid (TCA), one of the taurine conjugates of primary bile acids in serum, was elevated the most. While the role played by TCA in the disease is unclear, there is concern whether TCA contributes to the development of hepatocarcinoma from cholestasis. In the present study, the cell viability, flow cytometry, real-time polymerase chain reaction, intracellular ROS measurement, and intracellular Ca2+ measurement were used to investigate the effects of TCA on THLE-2 and HepG2 cells. The results showed that TCA is capable of inhibiting HepG2 cell growth whereas it has relatively little or no impact on that of THLE-2 cells until later stages of 16-day treatment. The growth inhibition is a result of cell apoptosis induced by the increase of Ca2+ and ROS level, and also associated with the increased expression of c-Myc, CEBPα, TNF-α, ICAM-1, VCAM-1, CXCL-2, Egr-1. HepG2 growth inhibition could contribute to the research on the treatment methods of patients already with hepatocarcinoma.

Release of endogenous hydrogen sulfide in enteric nerve cells suppresses intestinal motility during severe acute pancreatitis.

Previous studies have shown that during severe acute pancreatitis (SAP) attacks, hydrogen sulfide (H2S) is released in the colon. However, the roles played by H2S in regulating enteric nerves remain unclear. In this study, we examined the association between SAP-induced H2S release and loss of intestinal motility, and also explored the relevant mechanism in enteric nerve cells. A rat SAP model was constructed and enteric nerve cells were prepared. Intestinal mobility was evaluated by measuring the number of bowel movements at indicated time points and by performing intestinal propulsion tests. The production of inflammatory cytokines during a SAP attack was quantified by ELISA, and the levels of cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS) were examined by immunohistochemistry and western blot analysis. In vivo studies showed that PI3K/Akt/Sp1 signaling in enteric nerve cells was blocked, confirming the mechanism of endogenous H2S formation by western blot analysis and immunofluorescence. Our results also showed that rats with SAP symptoms had reduced intestinal motility. Furthermore, PI3K/Akt/Sp1 signaling was triggered and CSE expression was up-regulated, and these changes were associated with H2S formation in the colon. In addition, propargylglycine reduced the levels of inflammatory cytokines and suppressed the release of H2S. Enteric nerve cells that were incubated with LY294002 and transfected with a Sp1-knockdown vector displayed decreased levels of CSE production, which led to a decrease in H2S production. These results suggest that SAP symptoms suppressed the intestinal motility of rats via the release of H2S in enteric nerve cells, which was dependent on the inflammation-induced PI3K/Akt/Sp1 signaling pathway.

Transbuccal Delivery of Isoniazid: Ex Vivo Permeability and Drug-Surfactant Interaction Studies.

The oral administration of isoniazid (INH) may lead to discontinuation of tuberculosis treatment due to drug-related hepatotoxicity events, and thus, the transbuccal delivery of this drug was investigated, for the first time, as an alternative administration route. Ex vivo permeability assays were performed in Franz-type diffusion chambers, applying INH alone and in combination with sodium dodecyl sulfate (SDS) and sodium taurocholate (ST). After confirming the formation of micelle structures by dynamic light scattering analysis, UV-visible spectroscopy and zeta potential analyses were used to investigate drug-micelle interactions. In zeta potential analyses, no electrostatical interactions were identified for both surfactants in saliva buffer pH 6.8. Spectrophotometric analyses, in turn, indicated chemical interactions between INH and SDS in both pH values (2.0 and 6.8) whereas no interaction between the drug and ST was observed. Despite the interaction between SDS and drug, this surfactant increased the buccal transport rate of INH by approximately 11 times when compared with the control. In contrast, ST did not increase the drug permeability. The INH retention in SDS-treated mucosa was significantly higher when compared with the control and an effect on intercellular lipids was suggested. In vivo studies are needed to confirm the high INH absorption found here. Grapical abstract.

Systemic bile acids induce insulin resistance in a TGR5-independent manner.

Bile acids are involved in the emulsification and absorption of dietary fats, as well as acting as signaling molecules. Recently, bile acid signaling through farnesoid X receptor and G protein-coupled bile acid receptor (TGR5) has been reported to elicit changes in not only bile acid synthesis but also metabolic processes, including the alteration of gluconeogenic gene expression and energy expenditure. A role for bile acids in glucose metabolism is also supported by a correlation between changes in the metabolic state of patients (i.e., obesity or postbariatric surgery) and altered serum bile acid levels. However, despite evidence for a role for bile acids during metabolically challenging settings, the direct effect of elevated bile acids on insulin action in the absence of metabolic disease has yet to be investigated. The present study examines the impact of acutely elevated plasma bile acid levels on insulin sensitivity using hyperinsulinemic-euglycemic clamps. In wild-type mice, elevated bile acids impair hepatic insulin sensitivity by blunting the insulin suppression of hepatic glucose production. The impaired hepatic insulin sensitivity could not be attributed to TGR5 signaling, as TGR5 knockout mice exhibited a similar inhibition of insulin suppression of hepatic glucose production. Canonical insulin signaling pathways, such as hepatic PKB (or Akt) activation, were not perturbed in these animals. Interestingly, bile acid infusion directly into the portal vein did not result in an impairment in hepatic insulin sensitivity. Overall, the data indicate that acute increases in circulating bile acids in lean mice impair hepatic insulin sensitivity via an indirect mechanism.

Regulation of Plasma Membrane Localization of the Na⁺-Taurocholate Co-Transporting Polypeptide by Glycochenodeoxycholate and Tauroursodeoxycholate.

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.

Conjugated Bile Acids Promote Invasive Growth of Esophageal Adenocarcinoma Cells and Cancer Stem Cell Expansion via Sphingosine 1-Phosphate Receptor 2-Mediated Yes-Associated Protein Activation.

Esophageal adenocarcinoma (EAC) is the sixth leading cause of cancer deaths worldwide and has been dramatically increasing in incidence over the past decade. Gastroesophageal reflux and Barrett esophagus are well-established risk factors for disease progression. Conjugated bile acids (CBAs), including taurocholate (TCA), represent the major bile acids in the gastroesophageal refluxate of advanced Barrett esophagus and EAC patients. Our previous studies suggested that CBA-induced activation of sphingosine 1-phosphate receptor 2 (S1PR2) plays a critical role in promoting cholangiocarcinoma cell invasive growth. However, the role of CBAs in EAC development and underlying mechanisms remains elusive. In the current study, we identified that the expression level of S1PR2 is correlated to invasiveness of EAC cells. TCA significantly promoted cell proliferation, migration, invasion, transformation, and cancer stem cell expansion in highly invasive EAC cells (OE-33 cells), but had less effect on the lower invasive EAC cells (OE-19 cells). Pharmacologic inhibition of S1PR2 with specific antagonist JTE-013 or knockdown of S1PR2 expression significantly reduced TCA-induced invasive growth of OE-33 cells, whereas overexpression of S1PR2 sensitized OE-19 cells to TCA-induced invasive growth. Furthermore, TCA-induced activation of S1PR2 was closely associated with YAP and ß-catenin signaling pathways. In conclusion, CBA-induced activation of the S1PR2 signaling pathway is critically involved in invasive growth of EAC cells and represents a novel therapeutic target for EAC.

Commensal Escherichia coli Aggravates Acute Necrotizing Pancreatitis through Targeting of Intestinal Epithelial Cells.

An increase of Escherichia-Shigella was previously reported in acute necrotizing pancreatitis (ANP). We investigated whether Escherichia coli MG1655, an Escherichia commensal organism, increased intestinal injury and aggravated ANP in rats. ANP was induced by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct. Using gut microbiota-depleted rats, we demonstrated that gut microbiota was involved in the pancreatic injury and intestinal barrier dysfunction in ANP. Using 16S rRNA gene sequencing and quantitative PCR, we found intestinal dysbiosis and a significant increase of E. coli MG1655 in ANP. Afterward, administration of E. coli MG1655 by gavage to gut microbiota-depleted rats with ANP was performed. We observed that after ANP induction, E. coli MG1655-monocolonized rats presented more severe injury in the pancreas and intestinal barrier function than gut microbiota-depleted rats. Furthermore, Toll-like receptor 4 (TLR4)/MyD88/p38 mitogen-activated protein (MAPK) and endoplasmic reticulum stress (ERS) activation in intestinal epithelial cells were also increased more significantly in the MG1655-monocolonized ANP rats. In vitro, the rat ileal epithelial cell line IEC-18 displayed aggravated tumor necrosis factor alpha-induced inflammation and loss of tight-junction proteins in coculture with E. coli MG1655, as well as TLR4, MyD88, and Bip upregulation. In conclusion, our study shows that commensal E. coli MG1655 increases TLR4/MyD88/p38 MAPK and ERS signaling-induced intestinal epithelial injury and aggravates ANP in rats. Our study also describes the harmful potential of commensal E. coli in ANP.IMPORTANCE This study describes the harmful potential of commensal E. coli in ANP, which has not been demonstrated in previous studies. Our work provides new insights into gut bacterium-ANP cross talk, suggesting that nonpathogenic commensals could also exhibit adverse effects in the context of diseases.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

Organic Anion-Transporting Polypeptide 1a4 (Oatp1a4/Slco1a4) at the Blood-Arachnoid Barrier is the Major Pathway of Sulforhodamine-101 Clearance from Cerebrospinal Fluid of Rats.

The blood-arachnoid barrier (BAB), which is formed by arachnoid epithelial cells linked by tight junctions, has generally been considered impermeable to water-soluble substances. However, we recently demonstrated that organic anion transporters 1 and 3 (Oat1 and Oat3) play roles in drug clearance at the BAB. Here, we examined whether an organic anion-transporting polypeptide (Oatp) also plays a role, using the fluorescent organic anion sulforhodamine-101 (SR-101) as a model substrate. SR-101 was injected into the cisterna magna of rats in order to minimize the contribution of choroid plexus transport. The in vivo cerebrospinal fluid (CSF) elimination clearance of SR-101 after intracisternal administration was ninefold greater than that of fluorescein-labeled inulin, a bulk flow marker. In the case of pre-administration of taurocholate, a broad-spectrum inhibitor of Oatps, or digoxin, a strong substrate/inhibitor for Oatp1a4 but not for Oatp1a1, Oat1, and Oat3, the CSF elimination of SR-101 was significantly reduced, becoming similar to that of inulin, and thus indicating complete inhibition of SR-101 clearance from the CSF. The distribution of SR-101 fluorescence was restricted to the arachnoid mater in the absence of inhibitor, whereas the fluorescence was increased in the parenchyma of the spinal cord after co-injection of taurocholate or digoxin. Immunostaining confirmed the localization of Oatp1a4 in the arachnoid mater. These results indicate that Oatp1a4 at the BAB acts as an avid clearance pathway of SR-101 in the CSF to the blood. Thus, Oatp1a4 appears to play a major role in CSF detoxification by limiting the distribution of organic anions to the brain and spinal cord.

Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis.

BACKGROUND Gut microbiota dysbiosis plays a key role in pathogenesis of severe acute pancreatitis (SAP). In this study, we explored the protective effects of the p38 MAPK inhibitor, SB203580, against gut inflammation and microbiota dysbiosis induced by pancreatic duct injection with 3.5% sodium taurocholate in an SAP rat model. MATERIAL AND METHODS Ninety male Sprague-Dawley rats were randomly assigned to sham-operated, SAP model, and SAP plus SB203580 groups (n=30/group). Histological examination was conducted to assess gut and pancreatitis injury. The levels of amylase, D-lactate, diamine oxidase, tumor necrosis factor alpha, IL-6, IL-1ß, and phospho-p38MAPK in the plasma and intestine were evaluated at 3, 6, or 12 h after SAP induction. The gut microbiome was investigated based on16S rDNA gene sequencing at 12 h after SAP induction. RESULTS Histological examination revealed edema and inflammatory infiltrations in the pancreas and distal ileum. The expression of tumor necrosis factor alpha, IL-1ß, and IL-6 in plasma and distal ileum was increased in the SAP group, which were restored after treatment with SB203580. Significantly lower bacterial diversity and richness was found in the SAP group. In the SAP group, the abundance of Bacteroidetes and Firmicutes was decreased, and there was a higher proportion of Proteobacteria at the phylum level. The SAP plus SB203580 group exhibited significantly less damage to the gut microbiota, with higher bacterial diversity and a more normal proportion of intestinal microbiota. CONCLUSIONS SB203580 mediated suppression of the p38 MAPK signaling pathway via reduced gut inflammatory response and microbiota dysbiosis.

Paneth Cell Ablation Aggravates Pancreatic and Intestinal Injuries in a Rat Model of Acute Necrotizing Pancreatitis after Normal and High-Fat Diet.

We previously reported that acute necrotizing pancreatitis (ANP) after normal or high-fat diet is associated with a decreased number of Paneth cells in ileal crypts. Here, we ablated Paneth cells in a rat model of ANP after normal and high-fat diet to investigate the effects on disease symptoms. Adult male Sprague-Dawley rats received standard rat chow or a high-fat diet for 2 weeks, after which they were treated with dithizone to deplete Paneth cells. Six hours later, ANP was established by retrograde injection of sodium taurocholate into the biliopancreatic duct. Rats were sacrificed at 6, 12, and 24 h for assessment. We found dithizone aggravated ANP-associated pathological injuries to the pancreas and ileum in rats on high-fat or standard diets. Lysozyme expression in ileal crypts was decreased, while serum inflammatory cytokines (TNFα, IL-1ß, and IL-17A) and intestinal permeability (serum DAO activity and D-lactate) were increased. Expression of tight junction proteins (claudin-1, zo-1, and occludin) was decreased. Using high-throughput 16S rRNA sequencing, we found dithizone reduced microbiota diversity and altered microbiota composition in rats on high-fat or standard diets. Dithizone decreased fecal short-chain fatty acids (SCFAs) in rats on high-fat or standard diets. Changes in intestinal microbiota correlated significantly with SCFAs, lysozyme, DAO activity, D-lactate, inflammatory cytokines, and pathological injury to the pancreas and ileum in rats on high-fat or standard diets. In conclusion, ablation of Paneth cells exacerbates pancreatic and intestinal injuries in ANP after normal and high-fat diet. These symptoms may be related to changes in the intestinal microbiota.

Host factors influence Barrett's carcinogenesis: findings from a mouse gastroduodenal reflux model.

BACKGROUND: Rat gastroduodenal reflux models have been used for analyzing Barrett's carcinogenesis. Mice seem to be more useful than rats for studies targeting genes. METHODS: We induced gastroduodenal contents reflux by esophagojejunostomy using C57BL/6J mice. Mice were divided into a standard diet and high-fat diet groups and kept for 60 weeks. Bile was sampled from the gallbladder to analyze bile acid fractions, and the esophagus was removed for a histological investigation. Human esophagogastric junction adenocarcinoma cells (OE19) were exposed to taurocholic acid (TCA), after which cell proliferative activity was measured. Rat esophageal cancer cell lines, ESCC-DR and ESCC-DRtca with higher malignant potential induced by continuous TCA exposure, were used to perform comprehensive genetic analysis (CGH). RESULTS: Barrett's epithelium onset occurred in all mice, and no differences in histological changes were noted between the standard diet and high-fat diet groups. However, no development of adenocarcinoma was noted. Most of the mouse bile acid was taurine conjugates. In the experiment using OE-19 cells, TCA promotes cell proliferation in a dose-dependent manner. Array CGH analysis revealed a large number of chromosomal abnormalities in the ESCC-DR, in addition to genetic abnormalities such as in the UGT2B gene, the substrate of which is bile acid. TCA administration resulted in more chromosomal abnormalities being detected. CONCLUSIONS: We showed the effects of TCA in cancer progression in vitro. However, Barrett's adenocarcinoma onset rates differ between mice and rats despite undergoing similar reflux stimulation including taurine-conjugated bile acids being detected in mouse bile juice. These results suggest that host factors seem to influence Barrett's carcinogenesis.