Taurodeoxycholic acid-YAP1 upregulates OTX1 in promoting gallbladder cancer malignancy through IFITM3-dependent AKT activation.

Orthodenticle homeobox (OTX1) is reported to be involved in numerous cancers, but the expression level and molecular function of OTX1 in gallbladder cancer (GBC) remain unknown. Here, we found the elevated level of OTX1 associated with poor prognosis in human gallbladder cancer. In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation, whereas the downregulation inhibited it. Additionally, we found a tight correlation between the serum level of taurodeoxycholic acid (TDCA) and OTX1 expression. TDCA-induced activation of YAP1 by phosphorylation inhibition contributed to the transcriptional activation of OTX1. Mechanistically, we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells. Taken together, our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via the AKT signaling pathway. Our experiments also suggested that OTX1 is a novel therapeutic target for GBC.

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.

[Mechanism of Taurohyodeoxycholate-induced Biliary Phospholipid Efflux -Understanding the Function of the ABCB4 Enhancer for Developing Therapeutic Agents against Bile Salt-induced Liver Injury].

Biliary lipids primarily consist of bile salts, phospholipids, and cholesterol. Bile salts have potent detergent properties and deleterious effects on the cell membrane and are cytotoxic to hepatocytes. We have previously reported that phosphatidylcholine (PC), the predominant bile phospholipid, protects hepatocytes from the cytotoxicity of bile salts, whereas cholesterol reverses the cytoprotective effects of PC against bile salts. ABCB4, a member of the ATP-binding cassette transporter family, secretes biliary phospholipids, especially PC, from the hepatocytes into the bile. Using Abcb4 knockout mice and HEK293 cells that stably expressed ABCB4, we examined the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate, and hyodeoxycholate on the ABCB4-mediated efflux of PC. We observed that the biliary secretion of PC in wild-type mice significantly increased following infusion of all the tested bile salts, especially taurohyodeoxycholate. On the other hand, the biliary secretion of PC in Abcb4 knockout mice was not affected by the bile salt infusions. The results also demonstrated that the efflux of PC from ABCB4-expressing HEK293 cells was significantly stimulated by taurohyodeoxycholate, which has a strong potential to form mixed micelles with PC. Furthermore, the results of our study emphasized the possibility that the specific interactions of bile salts with ABCB4 are necessary for the release of PC molecules from the binding pocket of ABCB4 into the aqueous environment. Further understanding of this mechanism will aid in the development of novel therapeutic agents for cholestatic liver diseases.

Inhibitory effects of various solvent extracts from Rhamnus frangula leaves on some inflammatory and metabolic enzymes.

Many enzymes are involved in numerous pathologies which are related to metabolic reactions and inflammatory diseases such as pancreatic lipase, α-amylase, α-glucosidase and xanthine oxidase and secreted phospholipases A2 (Group IIA, V and X), respectively. Therefore, inhibiting these enzymes offer the potential to block production of more inflammatory substances and decrease the risk factors for cardiovascular diseases. The purpose of this study was to investigate some potent, bioavailable and selective inhibitors of some catalytic proteins implicated to metabolic syndrome and their antioxidant effects from various solvent extracts of R. frangula leaves. The anti-inflammatory, obesity, diabete and XO potentials were evaluated through analyses of inhibition activities of corresponding metabolites.The water extract exhibited an important inhibitory effect on human, dromedary and stingray sPLA2-G IIA achieved an IC50 of 0.16±0.06, 0.19±0.05 and 0.07±0.01 mg/mL, respectively. Likewise, the same fraction demonstrated the highest pancreatic lipase inhibitory activity using two different substrates. Indeed, 50% of dromedary pancreatic lipase inhibition was demonstrated for 5 min and 15 min using olive oil and TC4 substrates, respectively. Besides, it was established that methanolic extract had more effective inhibitory lipase activity than ORLISTAT used as a specific inhibitor of gastric, pancreatic and carboxyl ester lipase for treating obesity, with an IC50 of 5.51±0.27 and 91.46±2.3 µg/mL, respectively. In the case of α-amylase, α-glucosidase and xanthine oxidase, the crude methanolic extract showed a potential inhibitory effect with an IC50 of 45±3.45, 3±0.15 and 27±1.71 µg/mL, respectively. Conclusively, R. frangula leaves extracts showed a potential value of some sPLA2, some metabolic enzymes and XO inhibitors as anti-inflammatory and metabolic syndrome drugs.

Topical protection of mice laryngeal mucosa using the natural product cashew gum.

OBJECTIVES/HYPOTHESIS: Evaluate the effect of in vitro exposure of mice laryngeal mucosa to solutions that simulated human gastric juice and to assess the topical protective effect of cashew gum on mice laryngeal mucosal integrity in vitro. STUDY DESIGN: Animal study. METHODS: Murine (Swiss) laryngeal samples were mounted in Ussing chambers. The luminal side of biopsies was exposed to solutions of different acidity with or without pepsin and/or taurodeoxycholic acid (TDC). Transepithelial electrical resistance (TER) was continuously recorded. The topical protective effect of cashew gum solution was evaluated by precoating the biopsies before the exposure with a solution at pH 5 containing 5 mM TDC. Changes in TER and mucosal permeability to fluorescein were measured. RESULTS: Exposure of laryngeal mucosa to acidic solutions containing pepsin and TDC provoked a pH-dependent drop in TER with the maximal effect at pH 1, but still present at pH 5 (weakly acidic). The exposure of the laryngeal mucosa to a solution of pH 5 with TDC, but not with pepsin, produced a dose-dependent decrease in TER. Precoating the mucosa with cashew gum prevented the reduction of TER and increased transepithelial permeability by exposure to a solution at pH5 containing TDC. CONCLUSIONS: Weakly acidic solutions containing bile acids can produce impairment of laryngeal epithelial barrier, which may be protected by topical treatment with cashew gum. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:1157-1162, 2018.

Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors.

Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca(2+). In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca(2+) response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber-mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.

Buccal mucosal delivery of a potent peptide leads to therapeutically-relevant plasma concentrations for the treatment of autoimmune diseases.

Stichodactyla helianthus neurotoxin (ShK) is an immunomodulatory peptide currently under development for the treatment of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis by parenteral administration. To overcome the low patient compliance of conventional self-injections, we have investigated the potential of the buccal mucosa as an alternative delivery route for ShK both in vitro and in vivo. After application of fluorescent 5-Fam-ShK to untreated porcine buccal mucosa, there was no detectable peptide in the receptor chamber using an in vitro Ussing chamber model. However, the addition of the surfactants sodium taurodeoxycholate hydrate or cetrimide, and formulation of ShK in a chitosan mucoadhesive gel, led to 0.05-0.13% and 1.1% of the applied dose, respectively, appearing in the receptor chamber over 5h. Moreover, confocal microscopic studies demonstrated significantly enhanced buccal mucosal retention of the peptide (measured by mucosal fluorescence associated with 5-Fam-ShK) when enhancement strategies were employed. Administration of 5-Fam-ShK to mice (10mg/kg in a mucoadhesive chitosan-based gel (3%, w/v) with or without cetrimide (5%, w/w)) resulted in average plasma concentrations of 2.6-16.2nM between 2 and 6h, which were substantially higher than the pM concentrations required for therapeutic activity. This study demonstrated that the buccal mucosa is a promising administration route for the systemic delivery of ShK for the treatment of autoimmune diseases.

Developmental competence of bovine early embryos depends on the coupled response between oxidative and endoplasmic reticulum stress.

The stress produced by the coupling of reactive oxygen species (ROS) and endoplasmic reticulum (ER) has been explored extensively, but little is known regarding their roles in the early development of mammalian embryos. Here, we demonstrated that the early development of in vitro-produced (IVP) bovine embryos was governed by the cooperative action between ROS and ER stress. Compared with the tension produced by 5% O2, 20% O2 significantly decreased the blastocyst formation rate and cell survival, which was accompanied by increases in ROS and in levels of sXBP-1 transcript, which is an ER stress indicator. In addition, treatment with glutathione (GSH), a ROS scavenger, decreased ROS levels, which resulted in increased blastocyst formation and cell survival rates. Importantly, levels of sXBP-1 and ER stress-associated transcripts were reduced by GSH treatment in developing bovine embryos. Consistent with this observation, tauroursodeoxycholate (TUDCA), an ER stress inhibitor, improved blastocyst developmental rate, trophectoderm proportion, and cell survival. Moreover, ROS and sXBP-1 transcript levels were markedly decreased by supplementation with TUDCA, suggesting a possible mechanism governing the mutual regulation between ROS and ER stress. Interestingly, knockdown of XBP-1 transcripts resulted in both elevation of ROS and decrease of antioxidant transcripts, which ultimately reduced in vitro developmental competence of bovine embryos. Based on these results, in vitro developmental competence of IVP bovine embryos was highly dependent on the coupled response between oxidative and ER stresses. These results increase our understanding of the mechanism(s) governing early embryonic development and may improve strategies for the generation of IVP embryos with high developmental competence.

Role of endoplasmic reticular stress in aortic endothelial apoptosis induced by intermittent/persistent hypoxia.

BACKGROUND: Accumulated evidence shows that hypoxia can induce endothelial apoptosis, however the mechanism is still unknown. We hypothesized whether intermittent or persistent hypoxia could induce endoplasmic reticular stress, leading to endothelial apoptosis. METHODS: Twenty-four 8-week male Sprague Dawley (SD) rats were divided into three groups: normoxia (NC) group, intermittent hypoxia (IH) group and persistent hypoxia (PH) group. TUNEL staining was performed to detect aortic arch endotheliar apoptosis, and immunohistochemistry for BIP, CHOP and caspase12 to test protein expression; human umbilical vein endothelial cells (HUVECs) of the line ECV304 were cultured (with or without taurodeoxycholic acid (TUDCA) 10 mmol/L, 100 mmol/L) and divided into four groups: NC group (20.8% O2 for 4 hours), PH1 group (5% O2 for 4 hours), PH2 group (5% O2 for 12 hours) and IH group (20.8% O2 and 5% O2 alternatively for 8 hours). Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in each group. The expressions of GRP78, CHOP and caspase12 were detected by real-time quantitative reverse-transcription PCR. Result Intermittent and persistent hypoxia could increase the rate of endothelium apoptosis and the expressions of GRP78, CHOP and caspase12 compared with the control, induction by intermittent hypoxia was slightly higher than persistent hypoxia. In the HUVEC experiment, TUDCA significantly reduced apoptosis and the expressions of GRP78, CHOP and caspase12. CONCLUSION: Hypoxia, especially intermittent, can induce endothelial cell apoptosis possibly through endoplasmic reticulum stress pathway, which can be attenuated by taurodeoxycholic acid.

Isolation and biochemical analysis of vesicles from taurohyodeoxycholic acid-infused isolated perfused rat livers.

AIM: To isolate biliary lipid-carrying vesicles from isolated perfused rat livers after taurohyodeoxycholic acid (THDC) infusion. Biliary lipid vesicles have been implicated in hepatic disease and THDC was used since it increases biliary phospholipid secretion. METHODS: Rat livers were isolated and perfused via the hepatic portal vein with THDC dissolved in Krebs Ringer Bicarbonate solution, pH 7.4, containing 1 mmol/L CaCl2, 5 mmol/L glucose, a physiological amino acid mixture, 1% bovine serum albumin and 20% (v/v) washed human erythrocytes at a rate of 2000 nmol/min for 2 h. The livers were then removed, homogenized and subjected to centrifugation, and the microsomal fraction was obtained and further centrifuged at 350000 g for 90 min to obtain subcellular fractions. These were analyzed for total phospholipid, cholesterol, protein and alkaline phosphodiesterase I (PDE). RESULTS: No significant changes were observed in the total phospholipid, cholesterol and protein contents of the gradient fractions obtained from the microsomal preparation. However, the majority of the gradient fractions (ρ= 1.05-1.07 g/mL and ρ = 1.95-1.23 g/mL) obtained from THDC-infused livers had significantly higher PDE activity compared to the control livers. The low density gradient fraction (ρ = 1.05-1.07 g/mL) which was envisaged to contain the putative vesicle population isolated from THDC-perfused livers had relatively small amounts of phospholipids and protein when compared to the relevant control fractions; however, they displayed an increase in cholesterol and PDE activity. The phospholipids were also isolated by thin layer chromatography and subjected to fractionation by high performance liquid chromatography; however, no differences were observed in the pattern of the fatty acid composition of the phospholipids isolated from THDC and control perfused livers. The density gradient fractions (ρ = 1.10-1.23 g/mL) displayed an increase in all the parameters measured from both control and THDC-infused livers. CONCLUSION: No significant changes in biliary lipids were observed in the fractions from THDC-infused livers; however, PDE activity was significantly increased compared to the control livers.

Ion pairing with bile salts modulates intestinal permeability and contributes to food-drug interaction of BCS class III compound trospium chloride.

In the current study the involvement of ion pair formation between bile salts and trospium chloride (TC), a positively charged Biopharmaceutical Classification System (BCS) class III substance, showing a decrease in bioavailability upon coadministration with food (negative food effect) was investigated. Isothermal titration calorimetry provided evidence of a reaction between TC and bile acids. An effect of ion pair formation on the apparent partition coefficient (APC) was examined using (3)H-trospium. The addition of bovine bile and bile extract porcine led to a significant increase of the APC. In vitro permeability studies of trospium were performed across Caco-2-monolayers and excised segments of rat jejunum in a modified Ussing chamber. The addition of bile acids led to an increase of trospium permeation across Caco-2-monolayers and rat excised segments by approximately a factor of 1.5. The addition of glycochenodeoxycholate (GCDC) was less effective than taurodeoxycholate (TDOC). In the presence of an olive oil emulsion, a complete extinction of the permeation increasing effects of bile salts was observed. Thus, although there are more bile acids in the intestine in the fed state compared to the fasted state, these are not able to form ion pairs with trospium in fed state, because they are involved in the emulsification of dietary fats. In conclusion, the formation of ion pairs between trospium and bile acids can partially explain its negative food effect. Our results are presumably transferable to other organic cations showing a negative food effect.

Expression of bile acid receptor TGR5 in gastric adenocarcinoma.

Bile reflux is a risk factor in the development of intestinal metaplasia in the stomach and is believed to function as an initiator of gastric carcinogenesis. However, whether the G protein-coupled bile acid receptor TGR5 is expressed in this tumor is not known. In this study, we determined the expression of TGR5 in gastric adenocarcinoma and examined the role of TGR5 in cell proliferation. Strong TGR5 staining was present in 12% of cases of intestinal metaplasia but in no cases of normal gastric epithelium (P < 0.01). Moderate to strong TGR5 membranous and cytoplasmic staining was present in 52% of the intestinal but in only 25% of the diffuse subtype of adenocarcinomas (P < 0.001). Kaplan-Meier univariate survival analysis revealed that moderate to strong TGR5 staining was associated with decreased patient survival (P < 0.05). Treatment with taurodeoxycholic acid (TDCA, a bile acid) significantly increased thymidine incorporation in the AGS gastric adenocarcinoma cell line, suggesting that bile acids may increase cell proliferation. This increase was significantly decreased by knockdown of TGR5 with TGR5 small-interfering RNA (siRNA). In addition, overexpression of TGR5 significantly enhanced TDCA-induced increases in thymidine incorporation. TGR5 is coupled with G(q)α and Gα(i-3) proteins. TDCA-induced increase in thymidine incorporation was significantly decreased by knockdown of G(q)α and Gα(i-3) with their siRNAs. We conclude that TGR5 is overexpressed in most gastric intestinal-type adenocarcinomas, and moderate to strong TGR5 staining is associated with decreased patient survival in all gastric adenocarcinomas. Bile acids increase cell proliferation via activation of TGR5 receptors and G(q)α and Gα(i-3) proteins.

Short-term feedback regulation of bile salt uptake by bile salts in rodent liver.

UNLABELLED: The sodium taurocholate cotransporting polypeptide (Ntcp) is the major bile salt uptake transporter at the sinusoidal membrane of hepatocytes. Short-term feedback regulation of Ntcp by primary bile salts has not yet been investigated in vivo. Subcellular localization of Ntcp was analyzed in Ntcp-transfected HepG2-cells by flow cytometry and in immunofluorescence images from tissue sections by a new automated image analysis method. Net bile salt uptake was investigated in perfused rat liver by a pulse chase technique. In Flag-Ntcp-EGFP (enhanced green fluorescent protein) expressing HepG2-cells, taurochenodeoxycholate (TCDC), but not taurocholate (TC), induced endocytosis of Ntcp. TCDC, but not TC, caused significant internalization of Ntcp in perfused rat livers, as shown by an increase in intracellular Ntcp immunoreactivity, whereas Bsep distribution remained unchanged. These results correlate with functional studies. Rat livers were continuously perfused with 100 µmol/L of TC. 25 µmol/L of TCDC, taurodeoxycholate (TDC), tauroursodeoxycholate (TUDC), or TC were added for 30 minutes, washed out, followed by a pulse of (3) [H]-TC. TCDC, but not TDC, TUDC, or TC significantly increased the amount of (3) [H]-TC in the effluent, indicating a reduced sinusoidal net TC uptake. This effect was sensitive to chelerythrine (protein kinase C inhibitor) and cypermethrin (protein phosphatase 2B inhibitor). Phosphoinositide 3-kinase (PI3K) inhibitors had an additive effect, whereas Erk1/2 (extracellular signal activated kinase 1/2), p38MAPK, protein phosphatase 1/2A (PP1/2A), and reactive oxygen species (ROS) were not involved. CONCLUSION: TCDC regulates bile salt transport at the sinusoidal membrane by protein kinase C- and protein phosphatase 2B-mediated retrieval of Ntcp from the plasma membrane. During increased portal bile salt load this mechanism may adjust bile salt uptake along the acinus and protect periportal hepatocytes from harmful bile salt concentrations.

Bile salts increase epithelial cell proliferation through HuR-induced c-Myc expression.

BACKGROUND: Bile salts increase intestinal mucosal proliferation through an increase in c-Myc, a transcription factor that controls the expression of numerous translation regulatory proteins. HuR is an RNA-binding protein that regulates translation of target mRNAs. RNA-binding proteins can control mRNA stability by binding to AU- and U-rich elements located in the 3'-untranslated regions (3'-UTRs) of target mRNAs. AIM: To determine how bile salt-induced c-Myc stimulates enterocyte proliferation. METHODS: Enterocyte proliferation was measured both in vivo using C57Bl6 mice and in vitro using IEC-6 cells after taurodeoxycholate (TDCA) supplementation. HuR and c-Myc protein expression was determined by immunoblot. c-Myc mRNA expression was determined by PCR. HuR expression was inhibited using specific small interfering RNA. HuR binding to c-Myc mRNA was determined by immunoprecipitation. RESULTS: TDCA increased enterocyte proliferation in vivo and in vitro. TDCA stimulates translocation of HuR from the nucleus to the cytoplasm. Cytoplasmic HuR regulates c-Myc translation by HuR binding to the 3'-UTR of c-Myc mRNA. Increased TDCA-induced c-Myc increases enterocyte proliferation. CONCLUSIONS: Bile salts have beneficial effects on the intestinal epithelial mucosa, which are important in maintaining intestinal mucosal integrity and function. These data further support an important beneficial role of bile salts in regulation of mucosal growth and repair. Decreased enterocyte exposure to luminal bile salts, as occurs during critical illness, liver failure, starvation, and intestinal injury, may have a detrimental effect on mucosal integrity.

Bile acids acutely stimulate insulin secretion of mouse ß-cells via farnesoid X receptor activation and K(ATP) channel inhibition.

Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic ß-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and ß-cell ATP-dependent K(+) (K(ATP)) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of ß-cells and enhanced cytosolic Ca(2+) concentration ([Ca(2+)](c)). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca(2+)](c) and insulin secretion. FXR activation by TCDC is suggested to inhibit K(ATP) current. The decline in K(ATP) channel activity by TCDC was only observed in ß-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on ß-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and K(ATP) channel inhibition.

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells.

Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.

Protective effect of taurohyodeoxycholic acid from Pulvis Fellis Suis on trinitrobenzene sulfonic acid induced ulcerative colitis in mice.

Ulcerative colitis is a nonspecific inflammatory disorder characterized by oxidative and nitrosative stress, leucocyte infiltration and up-regulation of pro-inflammatory cytokines. The aim of this study is to evaluate the effect of taurohyodeoxycholic acid (THDCA) isolated from Pulvis Fellis Suis on acute ulcerative colitis model induced by trinitrobenzene sulfonic acid (TNBS) in mice. The efficacy of THDCA was studied by macroscopical and histological scoring systems as well as myeloperoxidase (MPO) activity. Serum levels, including tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed by enzyme-linked immunoassay. The expression of cyclooxygenase (COX)-2 in the colons was assessed by immunohistochemical analysis. Treatment with THDCA in doses of 25, 50 and 100mg/kg/day and sulfasalazine in a dose of 500 mg/kg/day used as reference for 7 consecutive days after the induction of colitis, significantly decreased colonic MPO activity, TNF-α, IL-6 serum levels and the expression of COX-2 in colon compared with TNBS induced ulcerative colitis model group. Moreover, THDCA attenuated the macroscopic colonic damage and the histopathological changes induced by TNBS. All the effects of these parameters were comparable to that of the standard sulfasalazine, especially at the highest dose level. The results suggested that THDCA from Pulvis Fellis Suis has a protective effect in TNBS-induced ulcerative colitis which might be due to its anti-inflammatory activities, and that it may have therapeutic value in the setting of inflammatory bowel disease.

Bile salt supplementation acts via the farnesoid X receptor to alleviate lipopolysaccharide-induced intestinal injury.

BACKGROUND: Intestinal barrier integrity may be disrupted in many conditions allowing for bacterial invasion and ensuing systemic illness. We investigated the efficacy and mechanism of bile salts in protecting the intestinal mucosa integrity after injury through stimulation of cell proliferation and an increased resistance to apoptosis. METHODS: Over 7 days, wild-type C57Bl/6J and Nr1h4(tm1Gonz)/J (farnesoid X receptor [FXR] knockout) male mice received either liquid rodent chow alone (for control animals) or with added 50 mg/kg per day of taurodeoxycholic acid (TDCA; for experimental animals). On day 6, all mice received 10 mL/kg of lipopolysaccharide intraperitoneally. On day 7, small intestines were harvested. After immunohistochemistry with hematoxylin and eosin, activated caspase-3, and 5-bromo2'-deoxy-uridine (BrdU), mean proliferating and apoptotic cells were determined with light microscopy. In vitro, FXR proteins were immunoblotted from cultured cells after exposure to TDCA. FXR expression was then inhibited in the presence and absence of TDCA. Intestinal epithelial proliferation along with c-Myc and FXR protein expressions were determined. RESULTS: C57Bl/6J mice exhibited significant mucosal enterocyte proliferation and decreased mucosal enterocyte apoptosis when provided with supplemental TDCA in their diet. Inhibition of FXR, both in vivo and in vitro, prevented the bile salt-induced enterocyte proliferation and resistance to apoptosis. TDCA exposure stimulated nuclear translocation of FXR resulting in increased expression of c-Myc. CONCLUSION: A diet supplemented with bile salts, especially in patients who have decreased luminal bile salt, may prove beneficial and therapeutic in critical illness where intestinal injury is part of the spectrum.

Study on improvement of extracellular production of recombinant Thermobifida fusca cutinase by Escherichia coli.

Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl ß-D: -1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.