Effects of Egr1 on pancreatic acinar intracellular trypsinogen activation and the associated ceRNA network.

Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response protein 1 (Egr1), and the effect of competing endogenous (ce)RNA network on trypsinogen activation. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP; however, the underlying mechanisms remain unclear. The present study used taurolithocholic acid 3­sulfate (TLC­S)­treated AR42J cells (pancreatic cell line) to establish a PAITA model. A gene microarray and bioinformatics analysis was performed to identify the potential key targets in PAITA. The results demonstrated that Egr1, an important transcription factor, was significantly overexpressed in PAITA. In Egr1 small interfering (si)RNA­transfected cells, Egr1 expression was decreased and trypsinogen activation was significantly decreased compared with negative control siRNA­transfected cells, indicating that in TLC­S­induced PAITA, overexpression of Egr1 enhanced trypsinogen activation. A ceRNA network [mRNA­microRNA (miRNA/miR)­long non­coding (lnc)RNA] generated using the PAITA model revealed that the effects of Egr1 on PAITA may be regulated by multiple ceRNA pairs, and the lncRNAs (including NONRATT022624 and NONRATT031002) and miRNAs [including Rattus norvegicus (rno)­miR­214­3p and rno­miR­764­5p] included in the ceRNA pairs may serve roles in PAITA by regulating the expression of Egr1. The results of the present study may provide novel targets for researching the underlying mechanisms of, and developing treatments for AP.

Acinar injury and early cytokine response in human acute biliary pancreatitis.

Clinical acute pancreatitis (AP) is marked by an early phase of systemic inflammatory response syndrome (SIRS) with multiorgan dysfunction (MODS), and a late phase characterized by sepsis with MODS. However, the mechanisms of acinar injury in human AP and the associated systemic inflammation are not clearly understood. This study, for the first time, evaluated the early interactions of bile acid induced human pancreatic acinar injury and the resulting cytokine response. We exposed freshly procured resected human pancreata to taurolithocolic acid (TLCS) and evaluated for acinar injury, cytokine release and interaction with peripheral blood mononuclear cells (PBMCs). We observed autophagy in acinar cells in response to TLCS exposure. There was also time-dependent release of IL-6, IL-8 and TNF-α from the injured acini that resulted in activation of PBMCs. We also observed that cytokines secreted by activated PBMCs resulted in acinar cell apoptosis and further cytokine release from them. Our data suggests that the earliest immune response in human AP originates within the acinar cell itself, which subsequently activates circulating PBMCs leading to SIRS. These findings need further detailed evaluation so that specific therapeutic targets to curb SIRS and resulting early adverse outcomes could be identified and tested.

Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway.

Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1-40 µM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth.

Conjugation is essential for the anticholestatic effect of NorUrsodeoxycholic acid in taurolithocholic acid-induced cholestasis in rat liver.

UNLABELLED: NorUDCA (24-norursodeoxycholic acid), the C23-homolog of ursodeoxycholic acid (UDCA), showed remarkable therapeutic effects in cholestatic Mdr2 (Abcb4) (multidrug resistance protein 2/ATP-binding cassette b4) knockout mice with sclerosing/fibrosing cholangitis. In contrast to UDCA, norUDCA is inefficiently conjugated in human and rodent liver, and conjugation has been discussed as a key step for the anticholestatic action of UDCA in cholestasis. We compared the choleretic, anticholestatic, and antiapoptotic properties of unconjugated and taurine-conjugated UDCA (C24) and norUDCA (C23) in isolated perfused rat liver (IPRL) and in natrium/taurocholate cotransporting polypeptide (Ntcp)-transfected human hepatoma (HepG2) cells. Taurolithocholic acid (TLCA) was used to induce a predominantly hepatocellular cholestasis in IPRL. Bile flow was determined gravimetrically; bile acids determined by gas chromatography and liquid chromatography/tandem mass spectrometry; the Mrp2 model substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP) was determined spectrophotometrically; and apoptosis was determined immunocytochemically. The choleretic effect of C23-bile acids was comparable to their C24-homologs in IPRL. In contrast, TnorUDCA, but not norUDCA antagonized the cholestatic effect of TLCA. Bile flow (percent of controls) was 8% with TLCA-induced cholestasis, and unchanged by coinfusion of norUDCA (14%). However, it was increased by TnorUDCA (83%), UDCA (73%) and TUDCA (136%). Secretion of GS-DNP was markedly reduced by TLCA (5%), unimproved by norUDCA (4%) or UDCA (17%), but was improved modestly by TnorUDCA (26%) or TUDCA (58%). No apoptosis was observed in IPRL exposed to low micromolar TLCA, but equivalent antiapoptotic effects of TUDCA and TnorUDCA were observed in Ntcp-HepG2 cells exposed to TLCA. CONCLUSION: Conjugation is essential for the anticholestatic effect of norUDCA in a model of hepatocellular cholestasis. Combined therapy with UDCA and norUDCA may be superior to UDCA or norUDCA monotherapy in biliary disorders in which hepatocyte as well as cholangiocyte dysfunction contribute to disease progression.

Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1.

BACKGROUND & AIMS: The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor. METHODS: Acute pancreatitis was induced in wild-type and Gpbar1(-/-) mice by either retrograde ductal infusion of taurolithocholic acid-3-sulfate (TLCS) or supramaximal secretagogue stimulation with caerulein. In vitro experiments were performed in which acini obtained from wild-type and Gpbar1(-/-) mice were exposed to either submicellar concentrations of TLCS (200-500 microM) or a supramaximally stimulating concentration of caerulein (10 nM). RESULTS: Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in TLCS-induced, but not caerulein-induced, pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to TLCS, but not when they are induced by exposure to caerulein. CONCLUSIONS: Gpbar1 may play a critical role in the evolution of bile-acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra-acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a "receptor-mediated" disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of biliary acute pancreatitis.

Impaired localisation and transport function of canalicular Bsep in taurolithocholate induced cholestasis in the rat.

BACKGROUND: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. AIMS: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. METHODS: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of (14)C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of (14)C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. RESULTS: In vivo pharmacokinetic studies revealed that taurolithocholate (3 micro mol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of (14)C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 micro M). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of (14)C taurocholate. CONCLUSIONS: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.

Tauroursodeoxycholate prevents taurolithocholate-induced cholestasis and toxicity in rat liver.

Ursodeoxycholate has been advocated for the treatment of cholestatic liver diseases. The coinfusion of tauroursodeoxycholate with taurolithocholate in the perfused rat liver completely prevented the decrease of bile flow and the increase of oxygen uptake found with taurolithocholate only. Bile flow and bile salt secretion were increased with the coinfusion of both bile acids as compared with the infusion of tauroursodeoxycholate only (+4.30 microliters/g liver per 30 min) with 16 and 32 mumol/l tauroursodeoxycholate (+1.55 microliters/g liver per 30 min with 80 and 160 mumol/l). Morphological examination revealed a 50% decrease of the number of necrotic cells in the periportal area. Tauroursodeoxycholate did not inhibit the uptake of taurolithocholate, but increased its transcellular passage and biotransformation. Thus, tauroursodeoxycholate prevents taurolithocholate-induced cholestasis and liver cell toxicity probably by an intracellular mechanism.