Distinct structural and dynamic components of portal hypertension in different animal models and human liver disease etiologies.

BACKGROUND AND AIMS: Liver fibrosis is the static and main (70%-80%) component of portal hypertension (PH). We investigated dynamic components of PH by a three-dimensional analysis based on correlation of hepatic collagen proportionate area (CPA) with portal pressure (PP) in animals or HVPG in patients. APPROACH AND RESULTS: Different animal models (bile duct ligation: n = 31, carbon tetrachloride: n = 12, thioacetamide: n = 12, choline-deficient high-fat diet: n = 12) and patients with a confirmed single etiology of cholestatic (primary biliary cholangitis/primary sclerosing cholangitis: n = 16), alcohol-associated (n = 22), and metabolic (NASH: n = 19) liver disease underwent CPA quantification on liver specimens/biopsies. Based on CPA-to-PP/HVPG correlation, potential dynamic components were identified in subgroups of animals/patients with lower-than-expected and higher-than-expected PP/HVPG. Dynamic PH components were validated in a patient cohort (n = 245) using liver stiffness measurement (LSM) instead of CPA. CPA significantly correlated with PP in animal models (Rho = 0.531; p < 0.001) and HVPG in patients (Rho = 0.439; p < 0.001). Correlation of CPA with PP/HVPG varied across different animal models and etiologies in patients. In models, severity of hyperdynamic circulation and specific fibrosis pattern (portal fibrosis: p = 0.02; septa width: p = 0.03) were associated with PH severity. In patients, hyperdynamic circulation (p = 0.04), vascular dysfunction/angiogenesis (VWF-Ag: p = 0.03; soluble vascular endothelial growth factor receptor 1: p = 0.03), and bile acids (p = 0.04) were dynamic modulators of PH. The LSM-HVPG validation cohort confirmed these and also indicated IL-6 (p = 0.008) and hyaluronic acid (HA: p < 0.001) as dynamic PH components. CONCLUSIONS: The relative contribution of "static" fibrosis on PH severity varies by type of liver injury. Next to hyperdynamic circulation, increased bile acids, VWF-Ag, IL-6, and HA seem to indicate a pronounced dynamic component of PH in patients.

Covered vs bare stent for distal malignant biliary obstruction due to primary common biliary cancer.

ABSTRACT: This study was designed as a means of comparing the clinical efficacy and long-term outcomes of covered vs bare stent insertion as a treatment for distal malignant biliary obstruction (DMBO) caused by primary common biliary cancer (PCBC).This retrospective study was designed using data collected between January 2012 and December 2019 to assess the short- and long-term outcomes in patients with DMBO caused by PCBC treated by inserting either bare or covered stents were compared.Ninety two patients with DMBO caused by PCBC were divided between bare (n = 51) or covered (n = 41) stent groups. Technical success rates in both groups were 100%. Clinical success of bare vs covered stent use were 96.1% and 97.6% (P = 1.00). Stent dysfunction was seen in 17 and 6 patients in the bare and covered stent groups, respectively (P = .04). The median stent patency for bare and covered stents was 177 and 195 days, respectively (P = .51). The median survival was 188 and 200 days in the bare and covered stent groups, respectively (P = .85).For patients with DMBO caused by PCBC, using bare vs covered stents yields similar clinical efficacy and long term outcomes.

[Mechanism of gut-microbiota-liver axis in the pathogenesis of intestinal failure-associated liver disease].

Intestinal failure (IF) is defined as the critical reduction of functional intestines below the minimum needed to absorb nutrients and fluids, so that intravenous supplementation with parenteral nutrition (PN) is required to maintain health and/or growth. Although the benefits are evident, patients receiving PN can suffer from serious cholestasis due to lack of enteral feeding and small intestinal bacterial overgrowth (SIBO). One such complication that may arise is intestinal failure-associated liver disease (IFALD). Evidences from recent studies suggest that alterations in the intestinal microbiota, as well as intraluminal bile acid driven signaling, may play a critical role in both hepatic and intestinal injury. Since Marshall first proposed the concept of the gut-liver axis in 1998, the role of gut-liver axis disorders in the development of IFALD has received considerable attention. The conversation between gut and liver is the key to maintain liver metabolism and intestinal homeostasis, which influences each other and is reciprocal causation. However, as a "forgotten organ" , intestinal microbiota on the pathogenesis of IFALD has not been well reflected. As such, we propose, for the first time, the concept of gut-microbiota-liver axis to emphasize the importance of intestinal microbiota in the interaction of gut-liver axis. Analysis and research on gut-microbiota-liver axis will be of great significance for understanding the pathogenesis of IFALD and improving the prevention and treatment measures.

Protective effects of n-Butanol extract and iridoid glycosides of Veronica ciliata Fisch. Against ANIT-induced cholestatic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch. is a traditional medical herb that present in more than 100 types of Tibetan medicine prescriptions, most of which are used for liver disease therapy. Iridoid glycosides have been identified as the major active components of V.ciliata with a variety of biological activities. AIMS OF THE STUDY: The aim of this study is to explore the protective effect and potential mechanism of n-Butanol extract (BE) and iridoid glycosides (IG) from V.ciliata against ɑ-naphthyl isothiocyanate (ANIT)-induced hepatotoxicity and cholestasis in mice. MATERIALS AND METHODS: Mice were intragastrically (i.g.) given BE and IG at different dose or positive control ursodeoxycholic acid (UCDA) once a day for 14 consecutive days, and were treated with ANIT to cause liver injury on day 12th. Serum levels of hepatic injury markers and cholestasis indicators, liver index and liver histopathology were measured to evaluate the effect of BE and IG on liver injury caused by ANIT. The protein levels of tumor necrosis factor-α (TNF-α), nuclear factor kappa B(NF-κB), interleukin-6 (IL-6), Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and the levels of oxidative stress indicators in liver tissue were investigated to reveal the underlying protective mechanisms of BE and IG against ANIT-induced hepatotoxicity and cholestasis. RESULTS: The n-Butanol extract (BE) and iridoid glycosides (IG) isolated from V.ciliata significantly decreased serum level of cholestatic liver injury markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) in ANIT-treated mice. Histopathology of the liver tissue showed that pathological damages were relieved upon BE and IG treatment. Meanwhile, the results indicated BE and IG notably restored relative liver weights, inhibited oxidative stress induced by ANIT through increasing hepatic level of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) and decreasing hepatic content of malondialdehyde (MDA). Western blot revealed that BE and IG inhibited the expression of pro-inflammatory factors TGF-α, IL-6 and NF-κB. Furthermore, the decreased protein expression of bile acid transporters NTCP, BSEP, MRP2 were upregulated by BE and IG in a dose-dependent manner. CONCLUSION: The results have demonstrated that BE and IG exhibited a dose-dependently protective effect against ANIT-induced liver injury with acute intrahepatic cholestasis in mice, which might be related to the regulation of oxidative stress, inflammatory response and bile acid transport. In addition, these findings pointed out that iridoid glycosides as main active components of V.ciliata play a critical role in hepatoprotective effect of V.ciliata.

Cholestasis Differentially Affects Liver Connexins.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Mammalian Target of Rapamycin Complex 2 Signaling Is Required for Liver Regeneration in a Cholestatic Liver Injury Murine Model.

Cholestatic liver injury may lead to a series of hepatobiliary syndromes, which can progress to cirrhosis and impaired liver regeneration, eventually resulting in liver-related death. Mammalian target of rapamycin complex 2 (mTORC2) is a major regulator of liver metabolism and tumor development. However, the role of mTORC2 signaling in cholestatic liver injury has not been characterized to date. In this study, we generated liver-specific Rictor knockout mice to block the mTORC2 signaling pathway. Mice were treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce cholestatic liver injury. DDC feeding induced cholestatic liver injury and ductular reaction as well as activation of the mTORC2/Akt signaling pathway in wild-type mice. Loss of mTORC2 led to significantly decreased oval cell expansion after DDC feeding. Mechanistically, this phenotype was independent of mTORC1/fatty acid synthase cascade (Fasn) or yes-associated protein (Yap) signaling. Notch pathway was instead strongly inhibited during DDC-induced cholestatic liver injury in liver-specific Rictor knockout mice. Furthermore, mTORC2 deficiency in adult hepatocytes did not inhibit ductular reaction in this cholestatic live injury mouse model. Our results indicated that mTORC2 signaling effectively regulates liver regeneration by inducing oval cell proliferation. Liver progenitor cells or bile duct cells, rather than mature hepatocytes, would be the major source of ductular reaction in DDC-induced cholestatic liver injury.

Picroside II protects against cholestatic liver injury possibly through activation of farnesoid X receptor.

BACKGROUD: Cholestasis, accompanied by the accumulation of bile acids in body, may ultimately cause liver failure and cirrhosis. There have been limited therapies for cholesteric disorders. Therefore, development of appropriate therapeutic drugs for cholestasis is required. Picroside II is a bioactive component isolated from Picrorhiza scrophulariiflora Pennell, its mechanistic contributions to the anti-cholestasis effect have not been fully elucidated, especially the role of picroside II on bile acid homeostasis via nuclear receptors remains unclear. PURPOSE: This study was designed to investigate the hepatoprotective effect of picroside II against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury and elucidate the mechanisms in vivo and in vitro. METHODS: The ANIT-induced cholestatic mouse model was used with or without picroside II treatment. Serum and bile biochemical indicators, as well as liver histopathological changes were examined. siRNA, Dual-luciferase reporter, quantitative real-time PCR and Western blot assay were used to demonstrate the farnesoid X receptor (FXR) pathway in the anti-cholestasis effects of picroside II in vivo and in vitro. RESULTS: Picroside II exerted hepatoprotective effect against ANIT-induced cholestasis by impaired hepatic function and tissue damage. Picroside II increased bile acid efflux transporter bile salt export pump (Bsep), uptake transporter sodium taurocholate cotransporting polypeptide (Ntcp), and bile acid metabolizing enzymes sulfate transferase 2a1 (Sult2a1) and UDP-glucuronosyltransferase 1a1 (Ugt1a1), whereas decreased the bile acid synthesis enzymes cholesterol 7α-hydroxylase (Cyp7a1) and oxysterol 12α-hydroxylase (Cyp8b1). In addition, expression of FXR and the target gene Bsep was increased, whereas aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARα) and their corresponding target genes were not significantly influenced by picroside II under cholestatic conditions. Furthermore, regulation of transporters and enzymes involved in bile acid homeostasis by picroside II were abrogated by FXR silencing in mouse primary cultured hepatocytes. Dual-luciferase reporter assay performed in HepG2 cells demonstrated FXR activation by picroside II. CONCLUSION: Our findings demonstrate that picroside II exerts protective effect on ANIT-induced cholestasis possibly through FXR activation that regulates the transporters and enzymes involved in bile acid homeostasis. Picroside II might be an effective approach for the prevention and treatment of cholestatic liver diseases.

Bone Health in Patients With Liver Diseases.

Osteoporosis is the most common bone disease in chronic liver disease (CLD) resulting in frequent fractures and leading to significant morbidity in this population. In addition to patients with cirrhosis and chronic cholestasis, patients with CLD from other etiologies may be affected in the absence of cirrhosis. The mechanism of osteoporosis in CLD varies according to etiology, but in cirrhosis and cholestatic liver disease it is driven primarily by decreased bone formation, which differs from the increased bone resorption seen in postmenopausal osteoporosis. Direct toxic effects from iron and alcohol play a role in hemochromatosis and alcoholic liver disease, respectively. Chronic inflammation also has been proposed to mediate bone disease in viral hepatitis and nonalcoholic fatty liver disease. Treatment trials specific to osteoporosis in CLD are small, confined to primary biliary cholangitis and post-transplant patients, and have not consistently demonstrated a benefit in this population. As it stands, prevention of osteoporosis in CLD relies on the mitigation of risk factors such as smoking and alcohol use, treatment of underlying hypogonadism, and encouraging a healthy diet and weight-bearing exercise. The primary medical intervention for the treatment of osteoporosis in CLD remains bisphosphonates though a benefit in terms of fracture reduction has never been shown. This review outlines what is known regarding the pathogenesis of bone disease in CLD and summarizes current and emerging therapies.

Cholestasis-associated reproductive toxicity in male and female rats: The fundamental role of mitochondrial impairment and oxidative stress.

Cholestasis is a significant decrease in bile flow. The liver is the primary organ affected by cholestasis. Chronic cholestasis could entail to tissue fibrotic changes and liver cirrhosis. Other organs, including heart, kidneys, nervous system, skeletal muscles, as well as the reproductive system, might also be affected during cholestasis. Although the cholestasis-associated pathological and biochemical alterations in organs such as liver have been widely investigated, there is little information about complications such as cholestasis-induced reproductive toxicity. The current study aimed to evaluate the pathologic effects of cholestasis on reproductive organs in both male and female animals. Rats underwent bile duct ligation (BDL) surgery. Markers of reproductive toxicity, including serum hormonal changes, tissue histopathological alterations, biomarkers of oxidative stress, and markers of mitochondrial impairment, were evaluated. Increased serum markers of liver injury and elevated level of cytotoxic molecules such as bile acids and bilirubin were evident in BDL animals. On the other hand, the serum level of hormones such as testosterone was suppressed in BDL rats. Significant histopathological alterations were also evident in the testis and ovary of BDL animals. A significant increase in oxidative stress markers, including ROS formation, lipid peroxidation, protein carbonylation, and depleted glutathione and antioxidant reservoirs were also detected in BDL rats. Moreover, mitochondrial depolarization decreased dehydrogenases activity, and depleted ATP content was detected in sperm isolated from the BDL group. These data indicate that cholestasis-associated reproductive toxicity in male and female rats is restrictedly coupled with severe oxidative stress and mitochondrial impairment.

Significance of biopsy with ERCP for diagnosis of bile duct invasion of DLBCL.

Obstructive jaundice is an initial symptom in 1-2% of diffuse large B cell lymphoma (DLBCL) cases. The major cause of bile duct obstruction in patients with DLBCL is extrinsic compression by enlarged lymph nodes. In such cases, the existence of bile duct invasion of lymphoma is rarely mentioned or observed pathologically, so the ratio of bile duct invasion to the total cases of obstructive jaundice, and its significance remains unknown. We report two cases of DLBCL presenting as an obstructive jaundice, in which we demonstrated bile duct invasion pathologically by biopsy from the wall of common bile duct with endoscopic retrograde cholangiopancreatography (ERCP). Endoscopic stent placement is a minimally invasive procedure to relieve cholestasis and is effective for diagnosing bile duct invasion. This procedure should thus be performed in all cases of obstructive jaundice caused by lymphoma to evaluate for bile duct invasion. Our cases suggest that ERCP may be useful as a diagnostic procedure for bile duct invasion.

Bile Duct Obstruction Leads to Increased Intestinal Expression of Breast Cancer Resistance Protein With Reduced Gastrointestinal Absorption of Imatinib.

Liver dysfunction reduces systemic clearance of drugs that are primarily eliminated by the liver. However, liver dysfunction can cause a reduction in the plasma concentration profiles of certain drugs, including several tyrosine kinase inhibitors, after oral administration. The aim of the present study was to clarify the reduction in oral absorption of a tyrosine kinase inhibitor, imatinib, and the mechanisms of action involved under conditions of hepatic dysfunction, focusing on intestinal transporters. The maximum plasma concentration of imatinib after oral administration in mice subjected to bile duct ligation (BDL) was lower than that in sham-operated mice, whereas the plasma concentration profile after intravenous administration was essentially unaffected by BDL. The change in maximum plasma concentration was compatible with a reduction in small intestinal permeability of imatinib observed in the in situ closed loop. Gene expression of abcg2 was increased in BDL mice compared with that in sham-operated mice. Expression of breast cancer resistance protein and P-glycoprotein in the small intestinal brush border membrane fraction from BDL mice was also increased compared with that in sham-operated mice. In summary, the intestinal absorption and permeability of imatinib was decreased in BDL mice, and this may be attributed to the up-regulation of the efflux transporter(s).

Cholestasis affects enteral tolerance and prospective weight gain in the NICU.

BACKGROUND: Intestinal Failure-Associated Liver Disease is characterized by cholestasis and hepatic dysfunction due to parenteral nutrition (PN) therapy. We described key features of cholestatic infants receiving PN to assess overall outcomes in this population at our institution. METHODS: This is a retrospective single center study of 163 neonates grouped into cholestatic (n = 63) and non-cholestatic (n = 100) as defined by peak conjugated bilirubin of ≥2.0 mg/dL or < 0.8 mg/dL, respectively. Univariate and multiple regression models were used to study associations between variables and outcomes of interest. RESULTS: Lower Apgar scores (4 ± 3 vs. 6 ± 3, p-value = <0.005 at 1 min; 6 ± 2 vs. 7 ± 2, p < 0.005 at 5 min) and lower birth weight (adj ß [SE] = 0.62 [0.27], p-value = 0.024) were risk factors for developing cholestasis. Cholestatic infants were more likely to have had gastrointestinal surgery (31 [49%] vs. 15 [15%], p-value <0.005), received PN for a longer duration (40 ± 39 days vs. 11 ± 7 days, p-value <0.005), and started enteral feeds later in life (86 ± 23 days vs. 79 ± 20 days, p-value <0.005) when compared to non-cholestatic infants. Weight percentiles in cholestatic infants were lower both at hospital discharge (14 ± 19 vs. 24 ± 22, p-value <0.005) and at 6 months of age (24 ± 28 vs. 36 ± 31, p-value = 0.05). CONCLUSIONS: Cholestasis in the NICU is a multifactorial process, but it has a long lasting effect on prospective weight gain in infants who receive PN in the NICU. This finding highlights the importance of follow-up for adequate growth and the potential benefit from aggressive nutritional support.

DUCTULAR REACTION AT THE EARLY AND LATE STAGES OF BILIARY OBSTRUCTION: IS THE MECHANISM THE SAME?

Ductular reaction (DR) is one of the classical features of biliary obstruction in humans as well as in experimental models. It is the histological phenomenon expressed by the increased number of ductular profiles (DPs), which is especially intensively studied in rodents' model of common bile duct ligation (CBDL). However, some issues related with DR in long-term CBDL are not clear enough. The liver tissue of male Wistar rats (150-200 g) subjected to CBDL were studied histologically (H&E). The architectonics of the biliary tract was studied by investigation of the corrosion casts obtained by retrograde injection (trough CBD) the solidified mass prepared on the basis of "Protacryl M" widely implemented in orthopedic dentistry. The casts were investigated by electro-optic device ProScope-HR with magnification X10, X50 and X200. DR accompanying CBDO is featured by the development of new DPs, the number of which is permanently increased in association with the prolongation of the terms of Cholestasis. DPs continue to develop in all areas of liver lobules - from portal/periportal region up to the regions containing central veins (hepatic veins tributaries of small calibers). The part of DPs has wide lumens, while the lumens of another part of DPs are hardly notable. In spite of evident ductular metaplasia of hepatic tissue, the small groups of normal by form and structure hepatocytes forming the separate small islets could be revealed. Histological features of these hepatocytes support the proposition that these cells maintain the functional activity on the 4th-5th weeks after CBDO. The results of investigations of biliary corrosion casts on the 4th and 5th weeks of CBDO show that solidifying mass cannot reach the small ducts/ductules and especially bile canaliculi. This fact confirms the interruption of ductular-canalicular junctions (IDCJ). We propose that due to IDCJ the increased pressure in preserved bile canaliculi should stipulate the mitotic activity and biliary trans-differentiation of hepatocytes on the late terms of CBDO and support the generation of ductal-like structures and appearance of new DPs. These ductules generally are not drained into the entire biliary tree.

Fish oil-based lipid emulsion in the treatment of parenteral nutrition-associated cholestasis.

BACKGROUND: Parenteral nutrition-associated cholestasis (PNAC) is a serious complication in preterm infants receiving prolonged parenteral nutrition. Soybean lipid emulsion (SLE) seems to have a role in its pathogenesis, whereas fish oil-based emulsion (FOLE) seems to be able to reverse cholestasis. This study aimed to evaluate the effectiveness of a FOLE in reversing PNAC. METHODS: The effectiveness in reversing PNAC was evaluated in prospective cohort study of very preterm infants when compared to historical controls: twenty-six infants (27.0 ± 2.6 weeks GA; 724 ± 204 g) who developed cholestasis while receiving SLE were shifted to receive FOLE and were compared with 30 infants (27.3 ± 2.5 weeks GA¸ 838 ± 277 g) who continued to receive SLE at diagnosis of cholestasis. RESULTS: Time to reversal of cholestasis was the same in the two study groups (45 ± 21 vs 43 ± 32 days). CONCLUSIONS: FOLE does not seem to be superior to SLE in reversing cholestasis. Considering that definitive data on the actual efficacy of FOLE to reverse PNAC are lacking, larger randomized trials are required, mainly to asses if FOLE may have a role in PNAC prevention rather than PNAC treatment.

Cholestasis-induced bile acid elevates estrogen level via farnesoid X receptor-mediated suppression of the estrogen sulfotransferase SULT1E1.

The liver is the main site of estrogen metabolism, and liver disease is usually associated with an abnormal estrogen status. However, little is known about the mechanism underlying this connection. Here, we investigated the effects of bile acid (BA)-activated farnesoid X receptor (FXR) on the metabolism of 17ß-estradiol (E2) during blockage of bile flow (cholestasis). Correlations between BA levels and E2 concentrations were established in patients with cholestasis, and hepatic expression profiles of key genes involved in estrogen metabolism were investigated in both WT and FXR-/- mice. We found that the elevated E2 level positively correlated with BA concentrations in the patients with cholestasis. We further observed that bile duct ligation (BDL) increases E2 levels in mouse serum, and this elevation effect was alleviated by deleting the FXR gene. Of note, FXR down-regulated the expression of hepatic sulfotransferase SULT1E1, the primary enzyme responsible for metabolic estrogen inactivation. At the molecular level, we found that FXR competes with the protein acetylase CREB-binding protein (CBP) for binding to the transcription factor hepatocyte nuclear factor 4α (HNF4α). This competition decreased HNF4α acetylation and nuclear retention, which, in turn, repressed HNF4α-dependent SULT1E1 gene transcription. These findings suggest that cholestasis induces BA-activated FXR activity, leading to downstream inhibition of SULT1E1 and hence impeding hepatic degradation of estrogen.