Bile Transport Research Articles

Molecular Mechanism of Intrahepatic Cholestasis in Sickle Cell Disease

Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects over three million people worldwide. Sickle Cell Anemia, the most common form of SCD is caused by the substitution of hydrophilic glutamic acid with hydrophobic valine at the sixth position in β-globin chain. The mutated hemoglobin (HbS) polymerizes under hypoxic conditions to promote erythrocyte sickling. Sickling promotes vaso-occlusion (ischemia) and hemolysis, which are two major pathophysiological events contributing to end organ damage. SCD patients can develop Sickle Cell Intrahepatic Cholestasis (SCIC), which is a rare but lethal form of hepatopathy. Besides SCIC, cholethiasis (gallstone formation), viral hepatitis and choledocholithiasis (stones in bile duct) have also been reported in SCD patients. The morbidity associated with sickle hepatopathy is expected to rise with growing life expectancy of SCD patients. Rescue therapies to halt the progression of sickle hepatopathy will be highly beneficial, however, development of such therapies is stalled by the lack of understanding of the molecular mechanism underlying sickle hepatopathy.We assessed the liver pathophysiology in a humanized mouse model of SCD in Townes SS mice homozygous for Hbatm1(HBA)Tow and homozygous for Hbbtm2(HBG1,HBB*)Tow). Littermate sickle cell trait mice (Townes AS mice homozygous for Hbatm1(HBA)Tow and compound heterozygous for Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow) were used as control mice. Our findings reveal that SCD but not control mice developed progressive intrahepatic cholestasis with age characterized by increased level of serum bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cholesterol. Immunohistochemical analysis of SCD mice liver revealed increased ductular reaction, inflammation, and fibrosis, which is hallmark of cholestatic liver disease. Immunoelectron microscopy revealed damage to bile canalicular structure and loss of several bile transporters from hepatocyte membrane. Remarkably, biochemical analysis revealed that intrahepatic cholestasis in SCD mice was associated with loss of Wnt signaling in hepatocytes. Our preliminary findings suggest that further studies are needed to elucidate the molecular mechanism underlying progressive cholestasis in SCD mice. Profound understanding of the regulation of cholestasis and hepatobiliary injury in SCD could potentially benefit the development of new therapies to attenuate sickle hepatopathy in SCD patients. DisclosuresNo relevant conflicts of interest to declare.

Protective effect of the ethyl acetate fraction of Qinghai-Tibet Plateau medicinal plant Chrysosplenium axillare Maxim. against ANIT-induced cholestatic liver injury in mice

BackgroundChrysosplenium axillare Maxim. has been used as a traditional Tibetan herb medicine for hepatobiliary disorder in Qinghai-Tibet Plateau for more than one thousand years. Hypothesis/purposewe hypothesized that the ethyl acetate-soluble portion extracted from C. axillare (ECA) could exert hepatoprotection effect against α-naphthylisothiocyanate (ANIT)-induced hepatotoxity and acute intrahepatic cholestasis in mice and aimed to investigate its potential mechanism. Study designmale KM mice with acute intrahepatic cholestasis induced by ANIT were orally administered with the ECA (100 and 50 mg/kg B.W.). Mice receiving vehicle (0.5% CMC-Na) served as control. Methods48 h after ANIT administration, mice were sacrificed. Blood was collected for serum enzymes and components analysis using commercial kits. Livers were removed for histopathological examination and protein preparation. The protein expression levels of farnesoid X receptor (FXR), bile-salt export pump (BSEP), multidrug resistance associated protein2 (MRP2), Na+-taurocholate co-transporting polypeptide (NTCP), small heterodimer partner (SHP), and cholesterol 7α-hydroxylase (CYP7A1) were evaluated by a western blot analysis. A phytochemical analysis was conducted to characterize the chemical constituents of ECA. Resultsoral administration of ECA at both doses (P < 0.05 or P < 0.01) can prevent ANIT-induced increases in levels of serum enzymes and components, including AST, ALT, ALP, DBIL, TBA, and TBIL and improve the liver pathologic change induced by ANIT exposure. Western blot analysis showed that the expression levels of FXR, BSEP, MRP2, NTCP, and SHP in ECA (100 and 50 mg/kg) groups were up-regulated versus model group (P < 0.05 or P < 0.01), and those of CYP7A1 in both ECA treated groups were down-regulated versus model group (P < 0.01). One new highly O-methylated flavonoids, chrysosplenoside I (1), together with fourteen analogues (2–15), was isolated from ECA and structurally identified by spectra analysis. Among them, the two abundant ingredients, chrysosplenosides A and I (11 and 1), were quantitatively determined to have a total content account for 32.2% of ECA. ConclusionECA protected against mice liver damage with acute intrahepatic cholestasis induced by ANIT via regulation of the expression of hepatic proteins involved in bile acids synthesis, transportation and re-absorption. These results confirmed the hepatoprotective efficacy of traditional Tibet herb medicine C. axillare and provided the first insight into its related mechanism as well as the potential active ingredients.

Bioengineering of a CLiP-derived tubular biliary-duct-like structure for bile transport in vitro.

The integration of a bile drainage structure into engineered liver tissues is an important issue in the advancement of liver regenerative medicine. Primary biliary cells, which play a vital role in bile metabolite accumulation, are challenging to obtain in vitro because of their low density in the liver. In contrast, large amounts of purified hepatocytes can be easily acquired from rodents. The in vitro chemically induced liver progenitors (CLiPs) from primary mature hepatocytes offer a platform to produce biliary cells abundantly. Here, we generated a functional CLiP-derived tubular bile duct-like structure using the chemical conversion technology. We obtained an integrated tubule-hepatocyte tissue via the direct coculture of hepatocytes on the established tubular biliary-duct-like structure. This integrated tubule-hepatocyte tissue was able to transport the bile, as quantified by the cholyl-lysyl-fluoresceinassay, which was not observed in the un-cocultured structure or in the biliary cell monolayer. Furthermore, this in vitro integrated tubule-hepatocyte tissue exhibited an upregulation of hepatic marker genes. Together, these findings demonstrated the efficiency of the CLiP-derived tubular biliary-duct-like structures regarding the accumulation and transport of bile.

Secretin Treatment Promotes Hepatic Progenitor Cell Activation, Ductal‐Canalicular Junction Formation and Amelioration of Liver Damage in a Model of Late‐Stage Primary Biliary Cholangitis

Introduction PBC is a cholestatic disease characterized by bile duct loss, ductular reaction and liver fibrosis. The canals of Hering (CoH) are the site of ductulo-canalicular junction (DCJ) that maintain proper bile drainage from the liver, and contain the hepatic progenitor cell (HPC) population. CoH loss is noted in PBC and correlates with scoring. Secretin binds to its receptor (SR, expressed by large cholangiocytes) to promote biliary proliferation during cholestasis; however, during large duct damage, small cholangiocytes acquire large cholangiocyte phenotypes including SR expression. Secretin/SR expression is downregulated in late-stage PBC; therefore, we aimed to understand the role of the secretin/SR axis during PBC. We hypothesized that secretin treatment restores DCJ through HPC activation and ameliorates liver phenotypes associated with late-stage PBC. Methods Female 34 wk old wild-type (WT) and dominant-negative transforming growth factor-β receptor II (dnTGFβRII, model of late-stage PBC) mice were treated with control or secretin (2.5 nmol/kg BW) for 8 wk by IP implanted minipumps. Cholangitis activity, hepatitis activity and bile duct loss were determined by the Nakanuma scoring system. Ductular reaction was measured by semi-quantitative CK-19 immunostaining. Liver fibrosis was evaluated by Sirius Red staining, collagen-1a immunostaining and qPCR for α-SMA. Hepatic stellate cell (HSC) presence was determined by desmin immunostaining. Cholestasis was indicated by hepatic total bile acid (TBA) levels using EIA. DCJ presence was evaluated by co-immunostaining for ABC transporter bile salt export pump (BSEP)/CK-19 and semi-quantification. HPC activation was assessed by Sox9 staining and semi-quantification. Results dnTGFβRII mice had an increase in cholangitis and hepatitis activity; however, 8 wks of secretin treatment significantly reduces cholangitis, but not hepatitis, activity. dnTGFβRII mice present with ductular reaction and significant bile duct loss, which was reversed with 8 wks secretin treatment. Hepatic TBA levels, liver fibrosis and HSC presence are increased in dnTGFβRII mice, but significantly reduced following 8 wks of secretin treatment. In line with human studies, dnTGFβRII mice have significant loss of DCJ and reduced HPC number; however, 8 wks of secretin treatment restores these parameters. Conclusion Long-term secretin treatment (i) reduces ductular reaction, (ii) reverses bile duct loss, (iii) decreases liver fibrosis, (iv) promotes HPC activity and (v) restores DCJ presence in a model of late-stage PBC. Secretin maintenance of the DCJ may be due to (i) the restoration of bile flow and release of toxic bile acids or (ii) secretin binding to SR acquired on small cholangiocytes/HPCs. Treatment with secretin may be a therapeutic option for late-stage PBC patients.

Tectorigenin alleviates intrahepatic cholestasis by inhibiting hepatic inflammation and bile accumulation via activation of PPARγ.

Increasing evidence suggests that human cholestasis is closely associated with the accumulation and activation of hepatic macrophages. Research indicates that activation of PPARγ exerts liver protective effects in cholestatic liver disease (CLD), particularly by ameliorating inflammation and fibrosis, thus limiting disease progression. However, existing PPARγ agonists, such as troglitazone and rosiglitazone, have significant side effects that prevent their clinical application in the treatment of CLD. In this study, we found that tectorigenin alleviates intrahepatic cholestasis in mice by activating PPARγ. Wild-type mice were intragastrically administered α-naphthylisothiocyanate (ANIT) or fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to simultaneously establish an experimental model of intrahepatic cholestasis and tectorigenin intervention, followed by determination of intrahepatic cholestasis and the mechanisms involved. In addition, PPARγ-deficient mice were administered ANIT and/or tectorigenin to determine whether tectorigenin exerts its liver protective effect by activating PPARγ. Treatment with tectorigenin alleviated intrahepatic cholestasis by inhibiting the recruitment and activation of hepatic macrophages and by promoting the expression of bile transporters via activation of PPARγ. Furthermore, tectorigenin increased expression of the bile salt export pump (BSEP) through enhanced PPARγ binding to the BSEP promoter. In PPARγ-deficient mice, the hepatoprotective effect of tectorigenin during cholestasis was blocked. In conclusion, tectorigenin reduced the recruitment and activation of hepatic macrophages and enhanced the export of bile acids by activating PPARγ. Taken together, our results suggest that tectorigenin is a candidate compound for cholestasis treatment.

Spatial Organization of the Transport of Interstitial Fluid and Lymph in Rat Liver (Scanning Electron Microscopy of Injection Replicas).

Using the method of scanning electron microscopy of injection replicas, we studied the movement of a new injection mass between the blood microcirculation system, interstitial space, lymphatic system, and bile transport system in rat liver under normal conditions and 3 days after the occlusion of the common bile duct. The casts of the perisinusoidal spaces of Disse's after injection of the injection mass through the portal vein and common bile duct were obtained. Their direct transition not only in "leakages" structurally related to lymphatic capillaries in interlobular spaces, but also in perivascular spaces around the portal and hepatic veins. The flow of the injection mass through the perivascular spaces leads to the formation of peculiar "sheaths" around hepatic veins and components of the portal complex. The proposed approach allows effective visualization of the structural basis of interaction of various compartments of the fluid microcirculation in the liver under normal and pathological conditions.

Lactobacillus delbrueckii Interfere With Bile Acid Enterohepatic Circulation to Regulate Cholesterol Metabolism of Growing-Finishing Pigs via Its Bile Salt Hydrolase Activity.

Microbiota-targeted therapies for hypercholesterolemia get more and more attention and are recognized as an effective strategy for preventing and treating cardiovascular disease. The experiment was conducted to investigate the cholesterol-lowering mechanism of Lactobacillus delbrueckii in a pig model. Twelve barrows (38.70 ± 5.33 kg) were randomly allocated to two groups and fed corn–soybean meal diets with either 0% (Con) or 0.1% Lactobacillus delbrueckii (Con + LD) for 28 days. L. delbrueckii–fed pigs had lower serum contents of total cholesterol (TC), total bile acids (TBAs), and triglyceride, but higher fecal TC and TBA excretion. L. delbrueckii treatment increased ileal Lactobacillus abundance and bile acid (BA) deconjugation and affected serum and hepatic BA composition. Dietary L. delbrueckii downregulated the gene expression of ileal apical sodium-dependent bile acid transporter (ASBT) and ileal bile acid binding protein (IBABP), and hepatic farnesoid X receptor (FXR), fibroblast growth factor (FGF19), and small heterodimer partner (SHP), but upregulated hepatic high-density lipoprotein receptor (HDLR), low-density lipoprotein receptor (LDLR), sterol regulatory element binding protein-2 (SREBP-2), and cholesterol-7α hydroxylase (CYP7A1) expression. Our results provided in vivo evidence that L. delbrueckii promote ileal BA deconjugation with subsequent fecal TC and TBA extraction by modifying ileal microbiota composition and induce hepatic BA neosynthesis via regulating gut–liver FXR–FGF19 axis.

Impaired Bile Secretion Promotes Hepatobiliary Injury in Sickle Cell Disease.

Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

Loss of FXR Signaling Promotes Chronic Liver Injury in Sickle Cell Disease

Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary crisis remains poorly understood. We have used intravital imaging to show that sinusoidal ischemia occurs in the liver of transgenic-humanized SCD mice under basal condition. SCD mice manifested progressive hepatomegaly, liver injury and hyperbilirubinemia. RNA-sequence analysis identified dysregulation of genes encoding proteins responsible for fibrosis, bile acid synthesis, and bile transport in the SCD mice liver. Live injury was the result of NF-κB-dependent inhibition of FXR signaling and its downstream targets in hepatocytes, leading to loss of canalicular bile transport and bile metabolism. Blocking NF-κB activation rescued FXR-signaling, ameliorated liver injury and resolved sinusoidal ischemia in SCD mice. Moreover, administration of FXR agonist (GW4064) also ameliorated liver injury seen in SCD mice. These findings are the first to identify that FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these pathways could potentially benefit the development of new therapies to treat sickle cell hepatic crisis. Disclosures No relevant conflicts of interest to declare.

High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell–Derived Organoids

Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution. We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform. HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis. Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.

Picroside II alleviates liver injury induced by alpha-naphthylisothiocyanate through AMPK-FXR pathway

Alpha-naphthylisothiocyanate (ANIT) is a typical hepatotoxicant that causes cholestasis, which causes toxic bile acid accumulation in the liver and leads to liver injury. Picroside II (PIC), one of the dominant effective components extracted from Picrorhiza scrophulariiflora Pennell, exhibits many pharmacological effects. However, the role of AMP-activated protein kinase (AMPK)-Farnesoid X receptor (FXR) pathway in the hepatoprotective effect of PIC against ANIT-induced cholestasis remains largely unknown. This study aimed to investigate the mechanisms of PIC on ANIT-induced cholestasis in vivo and in vitro. Our results showed that PIC protected against ANIT-induced liver injury in primary mouse hepatocytes, and decreased serum biochemical markers and lessened histological injuries in mice. ANIT inhibited FXR and its target genes of bile acid synthesis enzymes sterol-12α-hydroxylase (CYP8B1), and increase bile acid uptake transporter Na + −dependent taurocholate transporter (NTCP), efflux transporter bile salt export pump (BSEP) and bile acid metabolizing enzymes UDP-glucuronosyltransferase 1a1 (UGT1A1) expressions. PIC prevented its downregulation of FXR, NTCP, BSEP and UGT1A1, and further reduced CYP8B1 by ANIT. Furthermore, ANIT activated AMPK via ERK1/2-LKB1 pathway. PIC inhibited ERK1/2, LKB1 and AMPK phosphorylation in ANIT-induced cholestasis in vivo and in vitro. AICAR, an AMPK agonist, blocked PIC-mediated changes in FXR, CYP8B1 and BSEP expression in vitro. Meanwhile, U0126, an ERK1/2 inhibitor, further repressed ERK1/2-LKB1-AMPK pathway phosphorylation. In conclusion, PIC regulated bile acid-related transporters and enzymes to protect against ANIT-induced liver injury, which related to ERK1/2-LKB1-AMPK pathway. Thus, this study extends the understanding of the anti-cholestasis effect of PIC and provides new therapeutic targets for cholestasis treatment.

Immunohistological analysis on distribution of smooth muscle tissues in livers of various vertebrates with attention to different liver architectures

BackgroundThe liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having non-periportal bile ducts). The former is detectable from the hagfish, which is the most ancestral vertebrate, to tetrapod livers whereas many actinopterygian livers have the latter. The aim of the present study is to reveal the distribution of smooth muscle tissue in livers of various vertebrates with attention to their architectures. MethodsSmooth muscle was immunohistochemically compared in hepatic blood vessels and bile ducts of various vertebrates, using an anti-alpha-smooth muscle actin (ASMA) antibody. ResultsSmooth muscle was noted in the gallbladder and hepatic artery in all vertebrates, including the hagfish. Bile ducts having ASMA-positive smooth muscles were absent in the hagfish, but detected in the Chondrichthyes and conserved in actinopterygians with or without portal triads during the evolution of vertebrates. In tetrapods having portal triads, reptiles had a tendency to have strongly ASMA-positive biliary smooth muscle tissues whereas other tetrapods had bile ducts with poor smooth muscle tissues. Although the hagfish livers never had ASMA-positive smooth muscle tissue in the walls of portal and central veins, it was observed in discontinuous distributions or not observed in portal veins and central veins of chondrichthyans and actinopterygians. By contrast, in most tetrapods, ASMA-positive smooth muscle tissue was detectable in portal veins, which supported the adjacent endothelial cells as a circular layer. Central veins did not consistently have smooth muscle tissue in these groups. Discussion and conclusionThe hagfish liver may retain more ancestral characteristics than other vertebrates in terms of smooth muscle distribution in the vascular and biliary systems. Actinopterygians might have a different mechanism of bile transport from tetrapods from their smooth muscle distribution in intrahepatic bile ducts. The circular smooth muscle distribution in portal veins might be a characteristic acquired by tetrapods.

Serum Cholesterol-Lowering Activity of β-Sitosterol Laurate Is Attributed to the Reduction of Both Cholesterol Absorption and Bile Acids Reabsorption in Hamsters.

The research was performed to delineate how β-sitosterol laurate (β-SLE) consumption influenced serum and hepatic lipids. The results showed that 220 mg/5 mL oil/kg body weight of β-SLE robustly reduced serum total triglyceride and cholesterol levels and the epididymal adipocyte size, and efficiently protected hepatic polyunsaturated fatty acids against lipid peroxidation through superoxide dismutase and glutathione transferase activity enhancement and malondialdehyde level reduction. Based on the changes of fecal cholesterol contents, fecal and hepatic bile acid (BAs) levels, and related protein expression, it was concluded that the mechanisms for lowering serum cholesterol by β-SLE involved (i) the enhanced excretion of fecal cholesterol via down-regulation of intestinal Niemann-Pick C1-like 1 protein; (ii) the increased conversion from cholesterol to primary BAs via up-regulation of cholesterol-7α-hydroxylase and sterol 27-hydroxylase, which was induced by the reduced BAs reabsorption through up-regulating ileal apical sodium-dependent bile acid transporter and ileal bile acid-binding protein.

Chronické cholestatické jaterní choroby, primární biliární cholangitida a primární sklerozující cholangitida

Cholestasis is defined as hepatocyte and cholangiocyte bile excretion failure or failure of bile transport to the duodenum. Primary biliary cholangitis (PBC) and primary sclerosing cholangitis as chronic progressive cholestatic diseases are the common reasons of chronic cholestasis. Altogether with cholestatic laboratory picture the pruritus, liver osteodystrophy and fatigue are associated symptoms in both diseases. All associated symptoms and complications are needed to be diagnosed and treated early. In case of liver cirrhosis complicatons of accompanied portal hypertension should be treated and liver transplantation must be considered in all those patients. Diagnosis of PBC is based on cholestatic laboratory features, animitochondrial antibody positivity or typical histological patern. Most patients are asymptomatic in time of diagnosis. First line therapy is ursodeoxycholic acid. In case of first line therapy failure, the prognosis is unfavourable. In this case, second line therapy must be considered. In case of PSC the diagnosis is based on MRCP finding mainly, laboratory test and liver biopsy in some cases. Progressive inflamatory and fibrosing impairment affecting intrahepatic and extrahepatict biliary ducts and strong association with inflamatory bowel disease, especially ulcerative colitis is typical for PSC. Endoscopic therapy with dilatation of dominant structure is crucial. The effect of pharmacotherapy is still being discussed and ursodeoxycholic acid could be used. During follow up patients are in the risk of bacterial cholangitis and malignant tumor development (cholangiogenic and colorectal carcinoma mainly). In PSC patients the severe pruritus and reccurent bacterial cholangitis could be an indication for the liver transplantation.

Mechanism of Hydrophobic Bile Acid-Induced Hepatocyte Injury and Drug Discovery.

Cholestatic liver disease is caused by the obstruction of bile synthesis, transport, and excretion in or outside the liver by a variety of reasons. Long-term persistent cholestasis in the liver can trigger inflammation, necrosis, or apoptosis of hepatocytes. Bile acid nuclear receptors have received the most attention for the treatment of cholestasis, while the drug development for bile acid nuclear receptors has made considerable progress. However, the targets regulated by bile acid receptor drugs are limited. Thus, as anticipated, intervention in the expression of bile acid nuclear receptors alone will not yield satisfactory clinical results. Therefore, this review comprehensively summarized the literature related to cholestasis, analyzed the molecular mechanism that bile acid damages cells, and status of drug development. It is hoped that this review will provide some reference for the research and development of drugs for cholestasis treatment in the future.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease

There are few invitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

NFkB mediated sustained inflammation promotes hepatobiliary injury in Sickle Cell Disease

Abstract Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepatopathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, progressive inflammation, hyperbilirubinemia, and increased ductular reaction under basal condition. NFkB activation in the SCD mice liver inhibited FXR signaling and its downstream targets, leading to loss of canallicular bile transport, sustained inflammation and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canallicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mice liver. These findings are the first to identify that NFkB-FXR dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury and liver inflammation in SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis.

Impaired bile secretion promotes hepatobiliary injury in Sickle Cell Disease.

Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary crisis remains poorly understood. Here, we use intravital imaging to show that sinusoidal ischemia occurs in the liver of transgenic‐humanized SCD mice under basal condition. SCD mice manifested progressive hepatomegaly, liver injury and hyperbilirubinemia. RNA‐sequence analysis identified dysregulation of genes encoding proteins responsible for fibrosis, bile acid synthesis, bile transport and cholesterol metabolism in the SCD mice liver. Immunohistochemical analysis confirmed inflammation, fibrosis and increased ductular reaction in SCD mice. Intravital imaging also revealed impaired bile secretion into the bile canaliculi, which was associated with loss of apical bile transporters and bile acid biosynthetic enzymes, hepatic bile accumulation, and activation of Farnesoid X receptor (FXR) and small heterodimer partner (Shp) in SCD mice liver. These findings are the first to identify that impaired bile acid synthesis and misexpression of bile transporters promote intrahepatic bile accumulation and impaired bile secretion, leading to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis.Support or Funding InformationTP‐S is funded by PLRC pilot and feasibility award and Community Liver Alliance Pilot Grant award.

The role of Caspase‐1 in diet induced chronic liver injury and blood bile barrier regulation

End‐stage chronic liver disease is a major cause of mortality worldwide. Irrespective of the underlying cause, most chronic liver diseases are characterized by fibrosis, hepatocellular necrosis, inflammation, ductular reactions and proliferation of the liver progenitor cells. Vast differences exist between experimental models that mimic these processes, and their identification is fundamental for translational research. However, the underlying differences in the molecular mechanism driving liver injury in different models remains largely unknown due to our inability to visualize the initiation and progression of liver injury in vivo in mice. Recently we have introduced quantitative Liver Intravital Microscopy (qLIM) that enables real‐time assessment of bile transport and blood‐bile barrier (BBlB) integrity in the intact liver of live mice (Pradhan‐Sundd et al., Gastroenterology, 2018) to visualize injury progression in two widely used experimental models of chronic liver injury ‐the choline‐deficient ethionine‐supplemented (CDE) or 3, 5‐diethoxycarbonyl 1, 4‐dihydrocollidine (DDC) diet, respectively. qLIM analysis revealed that chronic liver injury involves physical breach of BBlB, mixing of blood with bile and loss of bile transport across hepatocytes in both CDE and DDC diet fed mice. Mechanistically we showed an association of disruption of the BBlB and loss of various tight junctional proteins with sustained inflammation and apoptotic cell death (Pradhan‐Sundd et al., Gastroenterology, 2018). Thus, we hypothesized that blocking Caspase ‐1 induced inflammatory response can inhibit the loss of BBlB and thus postpone the chronic liver injury initiation in these diet fed mice. Indeed, mice with deletion of caspase‐1 displayed reduced liver injury, ductular reaction, fibrosis, inflammation, and apoptosis after two weeks of CDE and DDC diet. Remarkably, qLIM analysis showed intact blood biliary barrier at day 4 and 6 of CDE and DDC fed mice suggestive of delay in injury initiation. Furthermore, we demonstrate that the recovery from CDE and DDC diet induced liver injury is significantly accelerated in caspase‐1 KO mouse. We also found upregulation several tight junctional claudins in the liver upon caspase ‐1 knockdown. Current investigation is underway to analyse the regulation of tight junctional claudins by Caspase‐1. These findings may lead insight to disease progression and possible treatment of diet induced chronic liver injury.

Canal of Hering loss is an initiating step for primary biliary cholangitis (PBC): A hypothesis

The origin and initiating features of PBC remain obscure despite decades of study. However, recent papers have demonstrated loss of canals of Hering (CoH) to be the earliest histologic change in liver biopsy specimens from patients with primary biliary cholangitis (PBC). We posit that CoH loss prior to significant inflammation or evidence of bile duct injury might be a very early, perhaps even an initiating lesion of PBC. As a potential target of inflammatory or toxic injury, CoH loss may initiate rather than follow the cascade of events leading to duct injury and loss and their sequelae. Toxins may be exogenous in origin, such as environmental toxins or drug exposures, or endogenous, resulting from genetic or epigenetic alterations in canalicular bile transporters upstream from the CoH. In turn, this hypothesis suggests that loss of CoH would lead to altered bile flow and composition injurious to downstream bile ducts, because bile composition has not been modulated by normal CoH physiologic functions or because, in the absence of CoH, canalicular fluid flow into the biliary tree is disrupted interfering with soluble trophic factors important for bile duct integrity. Regardless of the pathogenic mechanism causing CoH loss, only following such loss would the characteristic diagnostic findings of PBC become evident: damage to downstream interlobular and sub-lobular bile ducts. To the extent that the causal mechanisms for CoH loss can be identified, clinical identification (as through early identification of CoH loss) and intervention (depending on the inciting cause) may offer promise for treatment of this enigmatic disease.