Induction Of Cholestasis Research Articles

Exploring the remarkable effect of ursodeoxycholic acid and Allium sativum combination on MMP-9 and TIMP-1 levels in cholestatic rat’s model

Background: Cholestasis is a disturbance in the production or flow of bile that causes excessive accumulation of bile fluid and damage in the liver. Chronic liver damage will lead to liver fibrosis. Matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloprotease-1 (TIMP-1) play a critical role in liver fibrosis formation. Although ursodeoxycholic acid (UDCA) and Allium sativum extracts have long been renowned for improving liver function, their combination in alleviating liver fibrosis through the MMP-9 and TIMP-1 inhibitory pathways has yet to be studied.Objective: This study was conducted to assess the efficacy of UDCA and Allium sativum extract combination in altering MMP-9 and TIMP-1 levels, which are the main factors in the progression of liver fibrosis.Methods: We performed an experimental study with a post-test-only control group on 36 Sprague-Dawley rats, which were randomly grouped into healthy, negative, positive, and three treatment groups with UDCA (13.5 mg) and graded doses of Allium sativum extract (3.6 mg; 7.2 mg; and 14.4 mg). Cholestasis induction was done by a choledochal duct ligation, and treatment was given for 14 days. Levels of MMP-9 and TIMP-1 were analyzed after treatment.Results: Combining UDCA and Allium sativum improved the MMP-9 levels significantly in cholestatic rats, compared to administrating UDCA only (p <0.05). Combining UDCA and Allium sativum increased the TIMP-1 level (p < 0.05). Although the MMP-9 level results were under our existing hypothesis, TIMP-1 results showed surprising results. Matrix metalloproteinase-9 levels were strongly negatively correlated with TIMP-1 levels, with a r-value= 0.981.Conclusion: Combining Allium sativum and UDCA alleviates liver fibrosis progression through lowering levels of MMP-9 and increasing levels of TIMP-1.

Kupffer Cells Contested as Early Drivers in the Pathogenesis of Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) is an idiopathic chronic immune-mediated cholestatic liver disease characterized by fibro-inflammatory bile duct strictures, progressive hepatobiliary fibrosis, and gut-liver axis disruption. The pathophysiology of PSC remains insufficiently characterized, which hampers the development of effective therapies. Hepatic macrophages (MFs) such as Kupffer cells (KCs) are implicated in PSC pathogenesis, but their exact role is unclear. Using the latest markers to discriminate resident KCs (ResKCs) from their monocyte-derived counterparts (MoKCs), and two models of intrahepatic and extrahepatic cholestasis, respectively, this study showed that CLEC4F+TIM4+ ResKCs were depleted after chronic cholestatic liver injury. The infiltrating CLEC4F+TIM4- MoKCs were already enriched during the acute phase of PSC. Transcriptional profiling of hepatic MF subsets during early cholestatic injury indicated that ResKCs were indeed activated and that MoKCs expressed higher levels of pro-inflammatory and proliferative markers compared with those of ResKCs. As indicated in experiments with Clec4fDTR transgenic mice, conditional depletion of KCs, before and during early cholestasis induction, had no effect on the composition of the hepatic myeloid cell pool following injury progression and did not affect disease outcomes. Taken together, these results provide new insights into the heterogeneity of the MF pool during experimental PSC and evidence that depletion of resident and activated KCs during sclerosing cholangitis does not affect disease outcome in mice.

Enhanced anxiolytic and analgesic effectiveness or a better safety profile of morphine and tramadol combination in cholestatic and addicted mice.

The involvement of the opioidergic system on anxiolytic and antinociceptive responses induced by cholestasis was investigated in cholestatic and addicted mice. Elevated plus-maze and tail-flick devices were used to assess anxiety and pain levels, respectively. The data indicated that induction of cholestasis and injection of opioid drugs including morphine and tramadol enhanced %OAT and %OAE but naloxone reduced %OAT and %OAE in the sham-operated and bile duct ligation (BDL) mice. Induction of cholestasis and addiction to morphine and tramadol prolonged tail-flick latency, which was reversed by naloxone. Coadministration of morphine and tramadol enhanced anxiolytic and analgesic effects in the sham-operated and BDL mice. It seems (a) cholestasis and addiction affect anxiety and pain behaviors, (b) μ-opioid receptors play a key role in anxiolytic and analgesic effects induced by cholestasis, and (c) cotreatment with morphine and tramadol augmented the effectiveness of them for induction of anxiolytic and analgesic effects both in cholestatic and addicted mice.

Differential and organ-specific functions of organic solute transporter α and β in experimental cholestasis

Background & AimsOrganic solute transporter (OST) subunits OSTα and OSTβ facilitate bile acid efflux from the enterocyte into the portal circulation. Patients with deficiency of OSTα or OSTβ display considerable variation in the level of bile acid malabsorption, chronic diarrhea, and signs of cholestasis. Herein, we generated and characterized a mouse model of OSTβ deficiency.MethodsOstβ-/- mice were generated using CRISR/Cas9 and compared to wild-type and Ostα-/- mice. OSTβ was re-expressed in livers of Ostβ-/- mice using adeno-associated virus serotype 8 vectors. Cholestasis was induced in both models by bile duct ligation (BDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding.ResultsSimilar to Ostα-/- mice, Ostβ-/- mice exhibited elongated small intestines with blunted villi and increased crypt depth. Increased expression levels of ileal Fgf15, and decreased Asbt expression in Ostβ-/- mice indicate the accumulation of bile acids in the enterocyte. In contrast to Ostα-/- mice, induction of cholestasis in Ostβ-/- mice by BDL or DDC diet led to lower survival rates and severe body weight loss, but an improved liver phenotype. Restoration of hepatic Ostβ expression via adeno-associated virus-mediated overexpression did not rescue the phenotype of Ostβ-/- mice.ConclusionsOSTβ is pivotal for bile acid transport in the ileum and its deficiency leads to an intestinal phenotype similar to Ostα-/- mice, but it exerts distinct effects on survival and the liver phenotype, independent of its expression in the liver. Our findings provide insights into the variable clinical presentation of patients with OSTα and OSTβ deficiencies.Lay summaryOrganic solute transporter (OST) subunits OSTα and OSTβ together facilitate the efflux of conjugated bile acids into the portal circulation. Ostα knockout mice have longer and thicker small intestines and are largely protected against experimental cholestatic liver injury. Herein, we generated and characterized Ostβ knockout mice for the first time. Ostα and Ostβ knockout mice shared a similar phenotype under normal conditions. However, in cholestasis, Ostβ knockout mice had a worsened overall phenotype which indicates a separate and specific role of OSTβ, possibly as an interacting partner of other intestinal proteins.

Inhibition of autotaxin by bile salts and bile salt-like molecules increases its expression by feedback regulation

BackgroundAutotaxin is an enzyme that converts lysophospholipid into lysophosphatidic acid (LPA), a highly potent signaling molecule through a range of LPA receptors. It is therefore important to investigate which factors play a role in regulating ATX expression. Since we have reported that ATX levels increase dramatically in patients with various forms of cholestasis, we embarked on a study to reveal factors that influence the enzyme activity ATX as well as its expression level in vitro and in vivo. MethodsBile from cholestatic patients was fractionated by HPLC and analyzed for modulation of ATX activity. ATX expression was measured in fibroblasts upon stimulation or inhibition of LPA signaling. ResultsSurprisingly, ATX activity was stimulated by most forms of its product LPA, but it was inhibited by bile salts and bile salt-like molecules, particularly by 3-OH sulfated bile salts and sulfated progesterone metabolites that are known to accumulate during chronic cholestasis and cholestasis of pregnancy, respectively. Activation of fibroblasts by LPA decreased ATX expression by 72%. Conversely, inhibition of LPA signaling increased ATX expression 3-fold, indicating strong feedback regulation by LPA signaling. In fibroblasts, we could verify that inhibition of ATX activity by bile salts induces its expression. Furthermore, induction of cholestasis in mice causes increased plasma ATX activity. ConclusionsMultiple biliary compounds that accumulate in the systemic circulation during cholestasis inhibit ATX activity and thereby increase ATX expression through feedback regulation. This mechanism may contribute to increased serum ATX activity in patients with cholestasis.

Evaluation of Serum Alkaline Phosphatase Changes and TGF- β Expression in the Liver of Cholestatic Rats Treated with Ethanolic Extract of Plantago Ovata

BACKGROUND: Induction of cholestasis is one of the methods of liver fibrosis which causes the development of oxidative stress, increased expression of fibrogenic markers, excessive deposition of extracellular matrix, and finally the incidence of fibrosis. Plantago ovata is known as a rich source of various secondary metabolites such as phenolic compounds, flavonoids, alkaloids, trypanoids, and ascorbic acid. OBJECTIVES: the present study, the expression of TGF- β as a fibrotic marker and serum alkaline phosphatase (ALP) changes in cholestatic rats treated with P. ovata extract were evaluated. METHODS: In this study, 48 adult Wistar rats were used. The rats were randomly divided into eight groups of six animals each as follows: (1) healthy control group without bile duct ligation (BDL) surgery and treatment; (2–4) three healthy experimental plus P. ovata groups: rats without BDL, treated with P. ovata at dose levels of 100, 200, and 400 mg/kg body weight, respectively; (5) the BDL group: rats with BDL and treated with distilled water; and (6–8) the BDL plus P. ovata groups: rats with BDL and treated with P. ovata at dose levels of 100, 200, and 400 mg/kg body weight, respectively. The rats were treated with P. ovata extract for 45 consecutive days (once per day). After euthanasia and serum isolation, ALP enzyme level was measured. Moreover, the rat liver was fixed in 10% formalin buffer solution. The immunohistochemical study was performed by TGF-β antibody. Data analysis was performed using the One-way ANOVA and Kruskal-Wallis test and the Prism statistical program (p <0.0001). RESULTS: The results showed a significant increase in the serum levels of ALP enzyme and TGF-β expression in BDL group. Treatment with P. ovata extract was able to significantly improve these changes in a dose-dependent manner. CONCLUSIONS: The results of this study showed that P. ovata extract probably due to its phenolic compounds and its antioxidant effect has a protective effect on the liver and subsequently improves the increased serum ALP level and also reduced TGF-β expression

Probiotic intervention mitigates the metabolic disturbances of perfluorobutanesulfonate along the gut-liver axis of zebrafish

Probiotic supplementation is effective to modulate the metabolic disorders caused by perfluorobutanesulfonate (PFBS). However, the underlying mechanisms remain unclear. To this end, the present study exposed adult zebrafish to PFBS (0 and 10 μg/L), probiotics, or their binary combinations for 40 days. After the exposure, the nutritional stores, intestinal organization, and metabolic activities along the gut-liver axis were investigated. The results showed that PFBS exposure decreased the nutrient reserves significantly, especially the lipid content, which was alleviated by the probiotic administration. Intestinal mucus secretion was promoted remarkably in the presence of the probiotic, which enhanced epithelial protection against PFBS damage. Metagenomic analysis showed that PFBS alone induced gut microbial dysbiosis, which was efficiently antagonized by the probiotic bacteria. Intestinal metabolomic profiling revealed that ferroptosis occurred because of the unrestricted lipid peroxidation following PFBS exposure. However, probiotic administration prevented the ferroptotic symptoms induced by PFBS, further highlighting the beneficial effects of the probiotic on the host. In PFBS-exposed livers, high levels of bile acid metabolites (e.g., taurochenodeoxycholic acid) accumulated, implying the induction of cholestasis. Notably, probiotic addition recovered the metabolomic homeostasis under PFBS stress, probably resulting from the activation of detoxification pathways based on the pentose and glucuronate interconversion. Overall, the present study provides systematic evidence of the antagonistic interaction between PFBS and the probiotic regarding the metabolic activities along the microbe, gut and liver axis, highlighting the application values of probiotic recipe in aquaculture industry and ecological reservation.

Evaluation of the protective effect of curcumin on encephalopathy caused by intrahepatic and extrahepatic damage in male rats.

Objective(s):Along with increased intracranial pressure (ICP) and brain damage, brain edema is the most common cause of death in patients with hepatic encephalopathy. Curcumin can pass the blood-brain barrier and possesses anti-inflammatory and anti-oxidant properties. This study focuses on the curcumin protective effect on intrahepatic and extrahepatic damage in the brain. Materials and Methods:One hundred and forty-four male Albino N-Mary rats were randomly divided into 2 main groups: intrahepatic injury group and extrahepatic cholestasis group. In intra-hepatic injury group intrahepatic damage was induced by intraperitoneal (IP) injection of acetaminophen (500 mg/kg) [19] and included four subgroups: 1. Sham, 2. Acetaminophen (APAP), 3. Normal saline (Veh) which was used as curcumin solvent, and 4. Curcumin (CMN). In extrahepatic cholestasis group intrahepatic damage was caused by common bile duct litigation (BDL) and included four subgroups: 1. Sham, 2. BDL, 3. Vehicle (Veh), and 4. Curcumin (CMN). In both groups, 72 hr after induction of cholestasis, brain water content, blood-brain barrier permeability, serum ammonia, and histopathological indicators were examined and ICP was measured every 24 hr for three days.Results:The results showed that curcumin reduced brain edema, ICP, serum ammonia, and blood-brain barrier permeability after extrahepatic and intrahepatic damage. The maximum effect of curcumin on ICP was observed 72 hr after the injection.Conclusion:According to our findings, it seems that curcumin is an effective therapeutic intervention for treating encephalopathy caused by extrahepatic and intrahepatic damage.

The Food Contaminants Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis Structure-Dependently in the Human Hepatoma Cell Line HepaRG.

Pyrrolizidine alkaloids (PAs) are a group of secondary plant metabolites being contained in various plant species. The consumption of contaminated food can lead to acute intoxications in humans and exert severe hepatotoxicity. The development of jaundice and elevated bile acid concentrations in blood have been reported in acute human PA intoxication, indicating a connection between PA exposure and the induction of cholestasis. Additionally, it is considered that differences in toxicity of individual PAs is based on their individual chemical structures. Therefore, we aimed to elucidate the structure-dependent disturbance of bile acid homeostasis by PAs in the human hepatoma cell line HepaRG. A set of 14 different PAs, including representatives of all major structural characteristics, namely, the four different necine bases retronecine, heliotridine, otonecine and platynecine and different grades of esterification, was analyzed in regard to the expression of genes involved in bile acid synthesis, metabolism and transport. Additionally, intra- and extracellular bile acid levels were analyzed after PA treatment. In summary, our data show significant structure-dependent effects of PAs on bile acid homeostasis. Especially PAs of diester type caused the strongest dysregulation of expression of genes associated with cholestasis and led to a strong decrease of intra- and extracellular bile acid concentrations.

Cholestasis Alters miR-34c-5p Expression in the Testes of Male Wistar Rats

Background: Cholestasis is a pathophysiological condition, significantly reducing spermatozoa production. MiR-34c is highly expressed in adult male testicles and controls different stages of spermatogenesis. Objectives: Here, we aimed to investigate miR-34c expression in the testes of rat models of cholestasis. The expressions of THY-1, FGF-2, and CASP-3 genes, that are targeted by mirR-34c were also investigated. Methods: Cholestasis was induced in six adult rats via bile duct ligation. Four weeks after cholestasis induction, sera and testicular tissues were collected for further examinations. The levels of liver enzymes were measured using the ELISA. The structure of the testes was evaluated by histological examination. Total RNA was extracted from testes using a special kit and converted to cDNA. The expressions of miR-34c-5p, THY-1, FGF-2, and CASP-3 genes were determined by Real-Time PCR. Results: The serum levels of ALP, AST, and ALT were significantly elevated in the rat models of cholestasis (P &lt; 0.001). Real-Time PCR revealed that the expressions of miR-34c-5p, THY-1, and FGF-2 genes decreased while CASP-3 gene was upregulated in the testes of cholestatic animals (all differences were significant at P &lt; 0.05). Conclusions: Our study indicated that cholestasis was associated with reduced expression of miR-34c and altered expression of its target genes in the testis. Our results highlight the potential effects of cholestasis, a hepatobiliary disease, on testicular tissue function and male fertility.

In silico toxicity evaluation of Salubrinal and its analogues

This paper reports on a comprehensive in silico toxicity assessment of Salubrinal and its analogues containing a cinnamic acid residue or quinoline ring using the online servers admetSAR, ADMETlab, ProTox, ADVERPred, Pred-hERG and Vienna LiverTox. Apart from rare exceptions, in all 55 studied structures, mild or practical absence of acute toxicity was predicted for rats (III or IV toxicity class). Cardiotoxic, hepatotoxic and immunotoxic effects were predicted for Salubrinal and its analogues. We constructed models of the main predicted anti-targets hERG, BSEP, MRP3, MRP4 and AhR using the principle of homologous modeling. Molecular docking studies were carried out with the obtained models. We carried out molecular docking for all targets using AutoDock Vina, implemented in the PyRx 0.8 software package. According to the results of molecular docking, the compounds analyzed are potential moderate or weak hERG blockers. Induction of cholestasis and, as a consequence, liver damage by these drugs, directly related to inhibition of BSEP, MRP3 and MRP4, most likely will not be observed. Interaction with AhR for the studied compounds is impossible for steric reasons and, as a consequence, toxic effects on the immune and other organ systems associated with the activation of the AhR signaling pathway are excluded.

Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats.

Paternal metabolic status is an important factor in the health status of offspring. Cholestasis, as a metabolic disorder, significantly disrupts spermatogenesis. Spermatogonial stem cells (SSCs) are considered the dividing germ cells, which maintain spermatogenesis throughout the lifespan. Here, we investigated the in vivo and in vitro effect(s) of cholestasis on SSCs. Cholestasis was induced in rats by bile duct ligation. Four weeks after the cholestasis induction, testicular tissues were analysed using histopathological examinations. The expression of SSC markers, including Plzf and Thy-1, was determined using the immunofluorescent technique. Also, SSCs were isolated from animals, and their proliferation was examined in vitro. The histological examinations revealed that cholestasis caused irregularities in the structure of seminal tubes. Immunostaining showed that the total number of Thy-1- and Plzf-expressing cells was declined in the cholestasis group compared with the control group. In vitro culture of SSCs indicated that the number of SSC colonies and those expressing Plzf were significantly reduced in the culture medium of the cholestasis group. Our results indicated that cholestasis affects the functionality of SSCs and reduces the number and proliferation of them. This finding may be of interest to the effect of metabolic diseases such as cholestasis on spermatogenesis.

Effect of sodium butyrate on gastric ulcer aggravation and hepatic injury inflicted by bile duct ligation in rats

Background and AimCholestasis is positively associated with an increased risk of peptic ulceration. The present study investigated the aggravating effect of cholestasis on piroxicam-induced gastric ulceration. The study also evaluated the effect of sodium butyrate (SoB) on piroxicam-induced gastric ulceration in cholestatic animals and its effect on hepatic tissues and both effects were compared to ursodeoxycholic acid (UDCA) as a standard anticholestatic drug. MethodsBile duct ligation was adopted for induction of cholestasis in rats. The cholestatic animals received saline, SoB (P.O, 400 mg/kg, twice daily) or UDCA (P.O, 30 mg/kg/day) for 4 days starting from the first day of surgery. On the 4th day, blood samples were collected for determination of serum hepatic markers, then gastric ulcers were induced by piroxicam administration (P.O, 50 mg/kg) and 4 h later, the stomach was isolated and gastric mucosa was collected for biochemical determinations. The ulcer indices for the investigated drugs were compared to omeprazole as a standard acid suppressive drug. ResultsPiroxicam-induced ulceration was exacerbated in cholestatic rats. Gastric mucosa showed a significant elevation of MDA and TNF-α together with a significant decrease in GSH &VEGF levels. SoB treatment significantly attenuated ulcer development. The afforded protection was higher than that provided by UDCA and was not significantly different from that afforded by omeprazole. SoB significantly decreased gastric mucosal MDA and TNF-α level, whereas UDCA failed to alter these parameters. Both drugs significantly elevated GSH, VEGF and IL10 levels. Similar to UDCA, SoB showed a significant reduction in AST, ALT, GGT, ALP and bilirubin level. Histopathological examination confirmed the attenuating effect of SoB on gastric and hepatic injury. ConclusionsSodium butyrate effectively protected gastric and hepatic tissues against cholestasis-induced damage. Gastroprotection was mediated through antioxidant, anti-inflammatory and angiogenic activities.

Genetically engineered animal models of biliary tract cancers

Biliary tract cancers which include intrahepatic and extrahepatic cholangiocarcinomas and gallbladder cancer, are characterized by poor outcome. Therefore, identifying the molecular mechanisms of the disease has become a priority. However, such identification has to cope with extreme heterogeneity of the disease, which results from the variable anatomical location, the numerous cell types of origin and the high number of known genetic alterations. Animal models can develop invasive and metastatic tumours that recapitulate as faithfully as possible the molecular features of the human tumours. To generate animal models of cholangiocarcinoma, investigators resorted to the administration of carcinogens, induction of cholestasis, grafting of tumour cells and induction of genetic modifications. Here, we summarize the currently available genetically engineered animal models, and focus on mice and zebrafish. The experimental strategies that were selected to induce cholangiocarcinoma in a time-controlled and cell-type-specific manner are critically examined. We discuss their strengths and limitations while considering their relevance to human pathophysiology.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

MicroRNA-29a mitigation of endoplasmic reticulum and autophagy aberrance counteracts in obstructive jaundice-induced fibrosis in mice.

Hepatic fibrosis was caused by a number of signaling pathways that damage liver integrity. We have previously shown that microRNA-29a (miR-29a) protects against liver fibrosis. Aberrant endoplasmic reticulum (ER) and autophagy function reportedly exaggerate hepatic disorders. The aim of this study was to characterize the biological influence of miR-29a on ER function in injured livers with bile duct ligation (BDL). We performed BDL on miR-29a transgenic mice (miR-29aTg) and wild-type mice to induce cholestatic liver injury. Rat T6 cells were transfected with miR-29a mimic and tunicamycin. Compared to the wild-type mice, the BDL deterioration of liver function in terms of total bilirubin, alanine transaminase, and aspartate transaminase activity in the miR-29aTg mice was significantly reduced. Affected livers in the miR-29aTg mice demonstrated a slight fibrotic matrix formation. miR-29a over-expression reduced the BDL disturbance of the expressions of inositol-requiring kinase 1alpha, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase, spliced-X-box binding protein 1 (sXBP1), CCAAT/enhancer-binding protein homologous protein (CHOP), ULK, LC3BII, p62, and cleaved caspase-8, 9 and 3. In vitro, T6 cells exposed to tunicamycin by increasing abundances of CHOP, sXBP1, cleaved caspase-3, and LC3BII were diminished in the cell cultures transfected with the miR-29a mimic. On the other hand, we observed that miR-29a signaling protected liver tissues from BDL-mediated metabolic dysfunction and excessive fibrosis histopathology. This study provides new molecular insight into the miR-29a stabilization of ER integrity that slows the progression of cholestatic liver deterioration. Impact statement Long-term hepatic damage caused by hepatitis and cholestasis can accelerate fibrosis matrix over-production, which is a harmful process attributed to the dysregulation of a number of cellular and molecular events. The purpose of this study is to characterize the biological influence of miR-29a on endoplasmic reticulum (ER) function in bile duct ligation (BDL)-injured livers. To the best of our knowledge, this report is the first demonstration that miR-29a over-expression diminishes BDL provocation of ER stress (unfolded protein response, UPR) effector protein expression. This work also demonstrates that miR-29a decreased caspases protein expression in cholestatic livers, while an increase in miR-29a function reduced sXBP1 and CHOP expressions in T6 cells in mice. Analyses of this study highlight that controlling miR-29a signaling can serve as an innovative strategy in the future for microRNA regulation of ER homeostasis to combat cholestasis induction hepatic disorders.

Cholestasis induced antinociception and decreased gene expression of MOR1 in rat brain

We examined antinociception and gene expression of mu-opioid receptor 1 (MOR1) in some brain areas of cholestatic rats, 21days after common bile duct ligation (BDL). Cholestasis was induced in male Wistar rats during laparotomy and common BDL. Pain behavior was assessed on days 7, 14 or 21 of BDL using a hotplate test in control, sham and cholestatic groups. On day 21 of BDL, other groups of rats were sacrificed, whole brains were extracted, and the hypothalamus, prefrontal cortex (PFC), hippocampus and striatum in control, sham and cholestatic rats were dissected. We used a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for evaluating MOR1 gene expression. The results revealed that cholestatic rats showed significant antinociception on days 14 and 21 of ligation with the most significant effect on day 21, which was prevented by naloxone (1mg/kg). On the other hand, the expression of MOR1 gene compared to the sham group was decreased by 42% in the hypothalamus, 41% in the PFC, and 67% in the hippocampus after 21days of BDL, while no significant change in its expression in the striatum was observed. It can be concluded that a change in endogenous opioid levels and its subsequent influence on the gene expression of MOR in some areas of the rat brain may underlie the altered nociception and other possible pathological changes such as pruritus after induction of cholestasis.

Gene expression markers in the zebrafish embryo reflect a hepatotoxic response in animal models and humans

The zebrafish embryo (ZFE) is a promising non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatotoxic responses. Here, we hypothesize that the detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of new compounds and to the reduction of rodents used for screening. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of cholestasis, steatosis and necrosis, and two non-hepatotoxic controls. Histopathology revealed various specific morphological changes in the ZFE hepatocytes indicative of cell injury. Gene expression profiles of the individual compounds were generated using microarrays. Regulation of single genes and of pathways could be linked to hepatotoxic responses in general, but phenotype-specific responses could not be distinguished. Hepatotoxicity-associated pathways included xenobiotic metabolism and oxidoreduction related pathways. Overall analysis of gene expression identified a limited set of potential biomarkers specific for a common hepatotoxicity response. This set included several cytochrome P450 genes (cyp2k19, cyp4v7, cyp2aa3), genes related to liver development (pklr) and genes important in oxidoreduction processes (zgc:163022, zgc:158614, zgc:101858 and sqrdl). In conclusion, the ZFE model allows for identification of hepatotoxicants, without discrimination into specific phenotypes.

Effects of dopamine receptor agonist and antagonists on cholestasis-induced anxiolytic-like behaviors in rats

Dysfunctions in the dopamine transmission system have been suggested to contribute to the pathogenesis of hepatic encephalopathy. In an experimental animal model, cholestasis induction through bile duct ligation may present several main pathological features of hepatic encephalopathy. Dopaminergic systems are shown to play pivotal roles in regulation of anxiety-like behaviors. The main bile duct in male Wistar rats, weighing 220–240g, was ligated using two ligatures plus duct transection in between. Anxiety-like behaviors were measured using the elevated plus maze task. Cholestasis increased the open arm time percentage (%OAT), 13 but not 10 days after bile duct ligation, indicating an anxiolytic-like effect. Sole intraperitoneal injection of apomorphine (dopamine D1/D2 receptor agonist, 0.25mg/kg), SCH23390 (dopamine D1 receptor antagonist, 0.005, 0.01 and 0.02mg/kg) or sulpiride (dopamine D2 receptor antagonist, 0.125, 0.25 and 0.5mg/kg) did not alter %OAT, open arm entries percentage (%OAE) and locomotor activity in the sham-operated rats. Meanwhile, the higher dose apomorphine (0.5mg/kg) induced anxiolytic-like behaviors in this group. The subthreshold dose injection of SCH23390 or sulpiride, partially reversed the anxiolytic-like behaviors induced by cholestasis (13 days after bile duct ligation). On the other hand, subthreshold dose of apomorphine in cholestatic rats (10 days post bile duct ligation) induced anxiolytic-like effects which could be blocked by SCH23390 or sulpiride. The effective doses of above drugs did not alter locomotor activity, number of rearings, groomings and defections. These findings suggested that the dopaminergic system may potentially be involved in the modulation of cholestasis-induced anxiolytic-like behaviors in rats.

Liver-specific p38α deficiency causes reduced cell growth and cytokinesis failure during chronic biliary cirrhosis in mice

p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor β, tumor necrosis factor-α, interleukin-1β, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice. Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.