Cholestatic Patients Research Articles

Structure-guided discovery of bile acid derivatives for treating liver diseases without causing itch.

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

Serum matrix metalloproteinase-7 for discriminating biliary atresia: a diagnostic accuracy and validation study

BackgroundPrompt and precise differential diagnosis of biliary atresia (BA) among cholestatic patients is of great importance. Matrix metalloproteinase-7 (MMP-7) holds great promise as a diagnostic marker for BA. This study aimed to investigate the accuracy of age-specific serum MMP-7 for discriminating BA from other cholestatic pediatric patients.MethodsThis was a single center diagnostic accuracy and validation study including both retrospective and prospective cohorts. Serum MMP-7 concentrations were measured using an ELISA kit, the trajectory of which with age was investigated in a healthy infants cohort aged 0 to 365 days without hepatobiliary diseases (n = 284). Clinical BA diagnosis was based on intraoperative cholangiography and subsequent histological examinations. The diagnostic accuracy of age-specific cutoffs of serum MMP-7 were assessed in a retrospective cohort of cholestatic patients (n = 318, with 172 BA) and validated in a prospective cohort (n = 687, including 395 BA).ResultsThe MMP-7 concentration declines non-linearly with age, showing higher levels in healthy neonates as well as higher cutoff value in neonatal cholestasis. The area under the ROC curve (AUROC) was 0.967 (95% confidence interval [CI]: 0.946–0.988) for the retrospective cohort, and the cutoff of 18 ng/mL yielded 93.0% (95%CI: 88.1-96.3%), 93.8% (95%CI: 88.6-97.1%), 94.7% (95%CI: 90.1-97.5%), and 91.9% (95%CI: 86.4-95.8%) for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. The performance of MMP-7 was successfully validated in the larger prospective cohort, resulting in a diagnostic sensitivity of 95.9% (379/395; 95% CI: 93.5–97.7%), a specificity of 87.3% (255/292; 95% CI: 83.0–90.9%), a PPV of 91.1% (379/416; 95% CI: 87.9–93.7%), and a NPV of 94.1% (255/271; 95% CI: 90.6–96.6%), respectively. Besides, higher cutoff value of 28.1 ng/mL achieved the best sensitivity, specificity, PPV, and NPV for infants aged 0–30 days, which was 86.4% (95% CI: 75.0–94.0%), 95.5% (95% CI: 77.2–99.9%), 98.1% (95% CI: 89.7–100%), and 72.4% (95% CI: 52.8–87.3%), respectively.ConclusionsThe serum MMP-7 is accurate and reliable in differentiating BA from non-BA cholestasis, showing its potential application in the diagnostic algorithm for BA and significant role in the future research regarding pathogenesis of BA.

Drug disposition in cholestasis: An important concern.

Cholestasis, a chronic liver condition, disrupts bile acid homeostasis and complicates drug disposition, posing significant challenges in medicating cholestatic patients. Drug metabolism enzymes and transporters (DMETs) are pivotal in drug clearance. Research indicates that cholestasis leads to alterations in both hepatic and extrahepatic DMETs, with changes in expression and function documented in rodents and humans. This review synthesizes the modifications in key drug disposition components within cholestasis, focusing on cytochrome P450 (CYP450), drug transporters, and their substrates. Additionally, we briefly discuss certain drugs that have demonstrated efficacy in restoring DMET expression in cholestatic conditions. Ultimately, these insights necessitate a reevaluation of drug selection and dosing guidelines for patients with cholestasis.

Discrimination of Etiologically Different Cholestasis by Modeling Proteomics Datasets.

Cholestasis is characterized by disrupted bile flow from the liver to the small intestine. Although etiologically different cholestasis displays similar symptoms, diverse factors can contribute to the progression of the disease and determine the appropriate therapeutic option. Therefore, stratifying cholestatic patients is essential for the development of tailor-made treatment strategies. Here, we have analyzed the liver proteome from cholestatic patients of different etiology. In total, 7161 proteins were identified and quantified, of which 263 were differentially expressed between control and cholestasis groups. These differential proteins point to deregulated cellular processes that explain part of the molecular framework of cholestasis progression. However, the clustering of different cholestasis types was limited. Therefore, a machine learning pipeline was designed to identify a panel of 20 differential proteins that segregate different cholestasis groups with high accuracy and sensitivity. In summary, proteomics combined with machine learning algorithms provides valuable insights into the molecular mechanisms of cholestasis progression and a panel of proteins to discriminate across different types of cholestasis. This strategy may prove useful in developing precision medicine approaches for patient care.

Bile duct matrix metalloproteinase-7 expression: a new modality for diagnosis of biliary atresia

BackgroundBiliary atresia (BA) is an obliterative cholangiopathy of infancy that results in cholestasis and liver fibrosis. This fibrosis is due to an imbalance in extracellular matrix (ECM) breakdown and deposition. The mechanism by which the progressive injury occurs is not fully elucidated. Matrix metalloproteinases (MMPs) are involved in ECM turnover but also have non-ECM-related functions. Matrix metalloproteinase 7 (MMP7) has been suggested as a promising biomarker in diagnosing BA.ObjectiveThe aim of this study was to assess the hepatic expression of MMP-7 in infants with BA.Patients and methodsThe study was a retrospective-prospective case–control study that included 50 patients who were categorized into two groups, BA group (25 patients) and non-BA cholestatic patients as a control group (25 patients). Liver biochemistry, liver biopsy, histopathology, and immunohistochemical staining for primary antibody MMP-7 were performed for all studied patients.ResultsBile duct MMP7 expression was significantly higher in infants with BA than in non-BA cholestasis (P = 0.003), While the hepatic MMP-7 intensity did not differ significantly between both groups (P > 0.05). Bile duct expression of MMP-7 had a significant positive correlation with the BA Score (P = 0.017), while hepatic MMP-7 intensity had a significant positive correlation with alanine transaminase levels (P = 0.007) and a significant negative correlation with γ glutamyl transferase in the BA group (P = 0. 038). There was no statistically significant difference among different stages of fibrosis as regards the median of the hepatic MMP-7 intensity score and MMP-7 bile duct expression in infants with BA. There was no statistically significant difference between infants with successful and failed Kasai as regard the hepatic MMP-7 intensity and its bile duct expression.ConclusionBile duct expression of MMP-7 measured by immunohistochemistry is useful for the diagnosis of BA, but it is limited in predicting the stage of liver fibrosis and the outcome of Kasai portoenterostomy (KPE).

Serum procalcitonin as a marker of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD)

BackgroundNeonatal Intrahepatic Cholestasis (NICCD), as the early-age stage of Citrin deficiency involving liver dysfunction, lacks efficient diagnostic markers. Procalcitonin (PCT) has been identified as a biomarker for infection as well as various organ damage. This study aimed to explore the potential of PCT as a biomarker for NICCD. MethodsIn a single-center retrospective case-control study. Serum PCT concentrations before and after treatment of 120 NICCD patients, as the study group, were compared to the same number of cholestatic hepatitis patients, as the control group. The potential value of PCT to discriminate NICCD from control disease was further explored using Receiver Operating Characteristic (ROC) curve analysis and compared to those of other inflammatory markers. ResultsThere was a significantly higher level of PCT in NICCD patients than in the control group. PCT concentrations were only weakly correlated with neutrophil counts and CRP levels (p ˂ 0.05). At a cut-off value of 0.495 ng/mL, PCT exhibited a significantly higher diagnostic value compared to other inflammatory markers for discriminating NICCD from the control, with a sensitivity of 90.8 % and specificity of 98.3 %. ConclusionPCT might be used as an initial biomarker to discriminate children with NICCD from another hepatitis disease.

The non-HDL-C to APOB ratio as a predictor of inaccurate LDL-C measurement in patients with chronic intrahepatic cholestasis and jaundice: a retrospective study.

Cholestasis is characterized by the accumulation of bile in the liver or biliary system due to obstruction or impaired flow, necessitating lipid profiling to assess lipid metabolism abnormalities. Intrahepatic cholestasis, being the most significant type of cholestasis, further complicates the assessment of lipid abnormalities. However, the accuracy of low-density lipoprotein cholesterol (LDL-C) measurement in intrahepatic cholestasis patients remains uncertain. This study aimed to evaluate the consistency of the homogeneous assay and the Friedewald formula in detecting LDL-C levels and identify factors influencing LDL-C test results in intrahepatic patients with cholestasis. Retrospective analysis of laboratory data was conducted on intrahepatic cholestatic patients. Correlations between LDL-C values obtained using the homogeneous method (LDL-C(D)) and the Friedewald formula (LDL-C(F)), as well as associations between high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (ApoA1), LDL-C(D) and LDL-C(F), and apolipoprotein B (ApoB), were analyzed. Logistic regression analyses were employed to identify diagnostic indicators for inaccurate LDL-C measurements in intrahepatic cholestatic patients. Compared to patients with intrahepatic cholestasis without jaundice, the correlation between LDL-C(F) and LDL-C(D) was weaker in those with jaundice. Additionally, HDL-C exhibited a strong correlation with ApoA1 in both jaundice and non-jaundice cholestasis cases. Elevated non-HDL-C to APOB ratio (NH-C/B Ratio) levels (>4.5) were identified as a reliable predictor of inaccurate LDL-C measurements in patients with chronic intrahepatic cholestasis accompanied by jaundice. LDL-C measurement reliability is moderately weaker in patients with intrahepatic cholestasis accompanied by jaundice. Elevated levels of the NH-C/B ratio serve as a significant predictor of inaccurate LDL-C measurements in this chronic patient population, highlighting its clinical relevance for diagnostic assessments.

Factors associated with biliary atresia in children with cholestasis in Surabaya

Introduction. Biliary atresia (BA) represents a progressive and obstructive fibrotic cholangiopathy, serving as a leading cause of liver failure and pediatric liver transplantation worldwide. Identifying risk factors associated with BA incidence is crucial for expediting diagnosis; however, limited research has been conducted in Indonesia. This study aims to investigate the characteristics and analyze the factors associated with the occurrence of BA. Methods. A retrospective observational analysis of medical records encompassing 523 cholestatic patients at the Pediatric Hepatology Outpatient Department and Pediatric Ward of Dr. Soetomo General Academic Hospital, Surabaya, from 2011 to 2022 was conducted. The data was segregated into BA and non-BA groups, and diagnostic chi-square tests were employed to determine interrelationships among variables. Subsequently, logistic regression analysis was performed to identify the most impactful variable. Results. Notably, age at first time visiting specialist (p = 0.003), gestational age (p <0.001), birth weight (p = 0.039), and domicile (p = 0.028) exhibited substantial associations with BA. Moreover, gestational age emerged as the most influential factor (p = 0.004). Infants with normal birth weight (≥2,500 grams) exhibited strongest factor associated factor of BA cholestasis. Conclusion. Initial specialist consultations were predominantly observed within the 0-6 months age range, while term infant and normal birth weight were linked to a higher likelihood of BA cholestasis. Residence in rural areas exhibited a significant correlation with BA incidence.

Rhodanine Stain Improves Utility of Leipzig Score to Diagnose Hepatic Wilson’s Disease in Noncholestatic Patients with Fibrosis

Abstract Introduction: Cholestasis may increase the difficulty of diagnosing Wilson’s disease (WD). We aimed to compare Leipzig score including hepatic parenchymal copper concentration or rhodanine stain, to diagnose hepatic WD, in noncholestatic patients with fibrosis. Materials and Methods: We defined cholestasis by biochemical and/or histological criteria. Of noncholestatic patients who had liver fibrosis and liver copper estimation, those with isolated hepatic WD comprised WD study group and those with liver disease other than WD, with either low serum ceruloplasmin, high urinary copper, or Kayser–Fleischer ring, were controls. WD diagnosis by Leipzig score was labeled highly likely, probable, or unlikely. Results: Twenty WD study group patients (12 males; 21 [6–52] years; median [range]) and 18 controls (13 males; 32 [10–69] years, all had high urinary copper) were recruited. Rhodanine stain was positive in 60% WD patients and in 33% of controls. With rhodanine stain, Leipzig score had sensitivity of 100% (95% confidence interval: 83.2%–100%), specificity of 66.7% (41%–86.7%), and positive likelihood ratio of 3 (1.56–5.77) to diagnose probable/highly likely WD. In contrast, on adding hepatic parenchymal copper concentration, Leipzig score had sensitivity of 100% (83.2%–100%), but specificity was 38.9% (17.3%–64.2%). In controls, Leipzig score (including rhodanine stain) of ≤ 2 ruled out WD. Conclusion: On excluding cholestatic patients, rhodanine stain performs better than hepatic parenchymal copper concentration to diagnose hepatic WD using Leipzig score in patients with fibrosis. Widespread availability is an added advantage of rhodanine stain.

Gut microbiota depletion aggravates bile acid-induced liver pathology in mice with a human-like bile acid composition.

Cyp2c70-deficient mice have a human-like bile acid (BA) composition due to their inability to convert chenodeoxycholic acid (CDCA) into rodent-specific muricholic acids (MCAs). However, the hydrophobic BA composition in these animals is associated with liver pathology. Although Cyp2c70-ablation has been shown to alter gut microbiome composition, the impact of gut bacteria on liver pathology in Cyp2c70-/- mice remains to be established. Therefore, we treated young-adult male and female wild-type (WT) and Cyp2c70-/- mice with antibiotics (AB) with broad specificity to deplete the gut microbiota and assessed the consequences on BA metabolism and liver pathology. Female Cyp2c70-/- mice did not tolerate AB treatment, necessitating premature termination of the experiment. Male Cyp2c70-/- mice did tolerate AB but showed markedly augmented liver pathology after 6 weeks of treatment. Dramatic downregulation of hepatic Cyp8b1 expression (-99%) caused a reduction in the proportions of 12α-hydroxylated BAs in the circulating BA pools of AB-treated male Cyp2c70-/- mice. Interestingly, the resulting increased BA hydrophobicity strongly correlated with various indicators of liver pathology. Moreover, genetic inactivation of Cyp8b1 in livers of male Cyp2c70-/- mice increased liver pathology, while addition of ursodeoxycholic acid to the diet prevented weight loss and liver pathology in AB-treated female Cyp2c70-/- mice. In conclusion, depletion of gut microbiota in Cyp2c70-/- mice aggravates liver pathology at least in part by increasing the hydrophobicity of the circulating BA pool. These findings highlight that the potential implications of AB administration to cholestatic patients should be evaluated in a systematic manner.

Evaluation of renal arterial resistive index in patients with extrahepatic cholestasis.

Biliary obstruction has been shown to cause acute renal failure. The Renal Resistive Index (RRI) has been recognized to be an important index for evaluating changes in renal plasma flow and renal damage in cholestatic patients. We aimed to investigate the effects of cholestasis on renal hemodynamics in patients with extrahepatic cholestasis by RRI. The prospective study included patients with extrahepatic cholestasis due to benign biliary stricture, choledocholithiasis, or periampullary tumor between January 1, 2022, and December 31, 2022. Renal and liver function tests, as well as renal doppler ultrasound for RRIs, were conducted before and after cholestasis treatment. Patients who experienced cholestasis resolution after treatment showed lower cholestasis enzymes and bilirubin values and higher glomerular filtration rates compared to pre-treatment values. RRI values significantly decreased in patients with resolved cholestasis compared to pre-treatment levels (p=0.009). Patients with malignant cholestasis had higher RRI values than those with benign cholestasis (p=0.006). Bilirubin levels were higher (p=0.001), and glomerular filtration rates were lower (p=0.046) in patients with malignant cholestasis compared to those with benign cholestasis. Acute renal injury in cholestatic patients can be demonstrated non-invasively by RRI and is reversible once cholestasis has resolved. Patients with benign cholestasis had lower RRI values than those with cholestasis due to periampullary tumors.

Co-Relation between MELD Scoring System prior Living Donor Liver Transplantation and Occurrence of Post-Transplant Biliary Complications

Background Liver transplantation is considered the treatment of choice for patients with end stage liver disease who have failed standard medical and surgical therapy. The development of liver transplantation has revolutionized the field of hepatology and greatly improved the outlook of patients suffering from various liver diseases. The Model for EndStage Liver Disease, or MELD, is a scoring system for assessing the severity of chronic liver disease. It was initially developed to predict mortality within three months of surgery in patients who had undergone a transjugular intrahepatic portosystemic shunt (TIPS) procedure and was subsequently found to be useful in determining prognosis and prioritizing for receipt of a liver transplant. This score is now used by the United Network for Organ Sharing (UNOS) and Eurotransplant for prioritizing allocation of liver transplants instead of the older Child-Pugh score. Biliary tract complications are abundantly occurring post-operative, and they may include a variety of scenarios, as biliary leakage, ischemic bile duct injuries, and biliary stenosis. Aim of the Work Aim of the work is to study the relation between the Model of End Stage Liver Disease (MELD) scoring system and the occurrence of post liver transplantation biliary complications. Patients and Methods A combined retrospective and prospective cohort study was carried out. We retrospectively analyzed the available database of all patients that underwent LDLT, at Ain Shams Center of Organ Transplantation (ASCOT), during the period from January 2013 till May 2019, and suffered from post-operative biliary complications during the first 3 months Post-operative and correlated it with their MELD score before the operation. To do so, we studied 50 patients that are aged more than or equal 18 years old, any gender, that underwent living donor liver transplantation. We excluded patients with cholestatic liver disease and patients that underwent preoperative biliary intervention. All patients were subjected to complete history taking and thorough clinical examination, full laboratory workup, full radiological workup, post-operative cholangiography and Endoscopic retrograde colangio-pancreatography if needed.

Internal versus external biliary drainage in malignant biliary obstructions: is there a difference in the rate of infection?

Percutaneous biliary drainage is a frequently used method to provide biliary decompression in patients with biliary obstruction. To investigate the between drainage type and infection risk in patients treated with internal-external and external biliary drainage catheterization for malignant biliary obstruction. A total of 410 patients with malignant biliary obstruction who underwent internal-external or external biliary drainage catheterization between January 2012 and October 2016 were retrospectively evaluated. We investigated the correlation between percutaneous biliary drainage technique and infection frequency by evaluating patients with clinical findings, bile and blood cultures, complete blood counts, and blood biochemistry. There was no statistically significant difference between the selected patient groups (internal-external or external biliary drainage catheter placed) in terms of age, sex, primary diagnosis, receiving chemotherapy, catheter sizes, and outpatient-patient status. After catheterization, catheter-related infection was observed in 49 of 216 (22.7%) patients with internal-external and 18 of 127 (14.2%) patients with external biliary drainage catheters, according to the defined criteria. There was no difference in infection rate after the biliary drainage in the two groups (P > 0.05). There was also no difference concerning frequently proliferating microorganisms in bile cultures. Internal-external biliary drainage catheter placement does not bring an additional infection risk for uninfected cholestatic patients whose obstruction could be passed easily in the initial drainage.

Biliary atresia and liver transplantation in the United States: A contemporary analysis.

Biliary atresia (BA) remains the number one indication for paediatric liver transplantation (LT) worldwide but is an uncommon indication for older LT recipients. The impact of recent donor allocation changes, pervasive organ shortage and evolving LT practices on the BA LT population is unknown. We identified patients who underwent LT between January 2010 and December 2021 using the UNOS database. We compared clinical outcomes between patients with BA and those with non-BA cholestatic liver disease. Groups were stratified by age, <12 years (allocated via PELD system) and ≥12 years (allocated via MELD system). Waitlist outcomes were compared using competing-risk regression analysis, graft survival rates were compared using Kaplan-Meier time-to-event analysis and Cox proportional hazards modelling provided adjusted estimates. There were 2754 BA LT waitlist additions and 2206 BA LTs (1937 <12 years [younger], 269 ≥12 years [older]). There were no differences in waitlist mortality between BA and non-BA cholestatic patients. Among BA LT recipients, there were 441 (20.0%) living-donor liver transplantations (LDLT) and 611 (27.7%) split deceased-donor LTs. Five-year graft survival was significantly higher among BA versus non-BA cholestatic patients in the older group (88.3% vs. 79.5%, p < .01) but not younger group (89.3% vs. 89.5%). Among BA LT recipients, improved graft outcomes were associated with LDLT (vs. split LT: HR: 2, 95% CI: 1.03-3.91) and higher transplant volume (volume >100 vs. <40 BA LTs: HR: 3.41, 95% CI: 1.87-6.2). Liver transplant outcomes among BA patients are excellent, with LDLT and higher transplant centre volume associated with optimal graft outcomes.

γ-Mangostin abrogates AINT-induced cholestatic liver injury: Impact on Nrf2/NF-κB/NLRP3/Caspase-1/IL-1β/GSDMD signalling

γ-Mangostin (γ-MN) is one of the abundant xanthones separated from Garcinia mangostana (Clusiaceae) pericarps that has been reported to have varied bioactivities such as neuroprotective, cytotoxic, antihyperglycemic, antioxidant, and anti-inflammation. Yet, its effect on cholestatic liver damage (CLI) has not been investigated. This study explored the protective activity of γ-MN against alpha-naphthyl isothiocyanate (ANIT)-induced CLI in mice. The results showed that γ-MN protected against ANIT-induced CLI as indicated by reduced serum levels of hepatic injury parameters (e.g., ALT, AST, γ-GT, ALP, LDH, bilirubin, and total bile acids). ANIT-induced pathological lesions were improved in γ-MN pre-treated groups. γ-MN exerted potent antioxidant effects as it lowered the parameters of lipid peroxidation (4-HNE, PC, and MDA) and intensified the content and activity of antioxidants (TAC, GSH, GSH-Px, GST, and SOD) in the hepatic tissue. Furthermore, γ-MN enhanced the signalling of Nrf2/HO-1 as it augmented the mRNA expression of Nrf2/downstream genes (HO-1/GCLc/NQO1/SOD). The binding capacity and the immuno-expression of Nrf2 were also increased. γ-MN showed anti-inflammatory capacity as it suppressed the activation of NF-κB signalling, it decreased mRNA expression and levels of NF-κB/TNF-α/IL-6 and the immuno-expression of NF-κB/TNF-α. In addition, γ-MN inhibited the activation of NLRP3 inflammasome as it lowered the mRNA expression of NLRP3/caspase-1/IL-1β along with their levels as well as the immuno-expression of caspase-1/IL-1β. γ-MN also reduced the level of the pyroptotic parameter GSDMD. Collectively, this study demonstrated the potent hepatoprotective potential of γ-MN against CLI which was linked to its ability to potentiate Nrf2/HO-1 and to offset NF-κB/NLRP3/Caspase-1/IL-1β/GSDMD. Hence, γ-MN may be suggested as a new candidate for cholestatic patients.

Hepatic interleukin 32 attenuates liver injury through repression of necroptosis in cholestasis.

We aimed to evaluate the association between interleukin (IL)-32 and necroptosis in cholestatic liver injury. Levels of necroptosis-related markers in cholestatic and control patients, including the receptor-interacting serine-threonine kinase 3 (RIPK3), receptor-interacting serine-threonine kinase 1 (RIPK1), and mixed lineage kinase domain-like (MLKL) were measured. Animal experiments in C57BL/6J and transgenic mice withIL32β/γ overexpression were also conducted to confirm the effect of IL-32 on necroptosis in cholestasis, which was induced by α-naphthylisothiocyanate (ANIT) and 1% lithocholic acid (LCA). PLC/PRF/5-ASBT and primary mouse hepatocytes were utilized for the investigation of the regulation and mechanism of IL-32 in cholestasis. In the liver tissues of cholestatic patients, the mRNA and protein expressions of RIPK1, RIPK3, and MLKL were increased and associated with IL-32 expression. In addition, expressions of these indicatorsin the liver of 1% LCA- and ANIT-induced mouse models were significantly increased, while they were markedly decreased in hIL32βLTg and hIL32γLTg mice. After bile acid stimulation, IL-32 and phosphorylated Akt (p-Akt) expressions significantly elevated in a dose-dependent manner. After treated with tumor necrosis factor (TNF)-α, IL-32 inhibited MLKL expression in primary mouse hepatocytes. IL-32 is negatively associated with necroptosis in cholestatic patients. Moreover, IL-32 is induced by p-Akt and effectively attenuates necroptosis in ANIT- or 1% LCA-induced cholestasis.

Melatonin attenuates cholestatic liver injury via inhibition of the inflammatory response.

Melatonin, an indole neurohormone secreted mainly by the pineal gland, has been found to be involved in a variety of liver diseases. However, the underlying mechanism by which melatonin ameliorates cholestatic liver injury is not fully understood. In this study, we investigated the mechanism by which melatonin attenuates cholestatic liver injury via inhibition of the inflammatory response. We measured the levels of serum melatonin in patients with obstructive cholestasis (n = 9), patients with primary biliary cholangitis (PBC) (n = 11), and control patients (n = 7). We performed experiments with C57BL/6J mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and melatonin to verify the role of melatonin in the mouse model of cholestasis. Primary mouse hepatocytes were used for in vitro studies to study the mechanisms of action of melatonin in cholestasis. The levels of serum melatonin were markedly increased and negatively correlated with serum markers of liver injury in cholestatic patients. As expected, oral administration of melatonin significantly attenuated cholestasis-induced liver inflammation and fibrosis in 0.1% DDC diet-fed mice. Further mechanistic studies in cholestatic mice and primary hepatocytes revealed that melatonin reduced the conjugate BA-stimulated expression of cytokines (e.g. Ccl2, Tnfα, and Il6) through the ERK/Egr1 signalling pathway in these models. The levels of serum melatonin are significantly elevated in cholestatic patients. Melatonin treatment ameliorates cholestatic liver injury by suppressing the inflammatory response in vivo and in vitro. Therefore, melatonin is a promising novel therapeutic strategy for cholestasis.

P4HA2 induces hepatic ductular reaction and biliary fibrosis in chronic cholestatic liver diseases.

Prolyl-4-hydroxylases (P4Hs) are key enzymes in collagen synthesis. The P4HA subunit (P4HA1, P4HA2, and P4HA3) contains a substrate binding and catalyzation domain. We postulated that P4HA2 would play a key role in the cholangiocyte pathology of cholestatic liver diseases. We studied humans with primary biliary cholangitis (PBC) and Primary sclerosing cholangitis (PSC), P4HA2 -/- mice injured by DDC, and P4HA2 -/- /MDR2 -/- double knockout mice. A parallel study was performed in patients with PBC, PSC, and controls using immunohistochemistry and immunofluorescence. In the murine model, the level of ductular reaction and biliary fibrosis were monitored by histology, qPCR, immunohistochemistry, and Western blotting. Expression of Yes1 Associated Transcriptional Regulator (YAP) phosphorylation was measured in isolated mouse cholangiocytes. The mechanism of P4HA2 was explored in RBE and 293T cell lines by using qPCR, Western blot, immunofluorescence, and co-immunoprecipitation. The hepatic expression level of P4HA2 was highly elevated in patients with PBC or PSC. Ductular reactive cholangiocytes predominantly expressed P4HA2. Cholestatic patients with more severe liver injury correlated with levels of P4HA2 in the liver. In P4HA2 -/- mice, there was a significantly reduced level of ductular reaction and fibrosis compared with controls in the DDC-induced chronic cholestasis. Decreased liver fibrosis and ductular reaction were observed in P4HA2 -/- /MDR2 -/- mice compared with MDR2 -/- mice. Cholangiocytes isolated from P4HA2 -/- /MDR2 -/- mice displayed a higher level of YAP phosphorylation, resulting in cholangiocytes proliferation inhibition. In vitro studies showed that P4HA2 promotes RBE cell proliferation by inducing SAV1 degradation, eventually resulting in the activation of YAP. P4HA2 promotes hepatic ductular reaction and biliary fibrosis by regulating the SAV1-mediated Hippo signaling pathway. P4HA2 is a potential therapeutic target for PBC and PSC.

Gamma-Glutamyl Transferase: A Friend against Cholestatic Itch? A Retrospective Observational Data Analysis in Patients with Extrahepatic Cholestasis.

We included 235 patients with chronic extrahepatic cholestasis due to pancreatic cancer, cholangiocarcinoma, or papillary carcinoma. GGT was significantly higher in patients without pruritus (median 967, IQR 587-1571) compared to patients with pruritus (median 561 IQR 266-1084 IU/l) (p < 0.01). In contrast, median alkaline phosphatase (AP) was 491 U/L (IQR; 353-684) in patients with pruritus and was not significantly different from 518 U/L (IQR; 353-726) in patients without pruritus (p = 0.524). Direct bilirubin was significantly higher in patients with pruritus compared to patients without pruritus (168 μmol/L (IQR; 95-256) vs. 120 μmol/L (IQR; 56.75-185.5)) (p < 0.01). After correcting for the extent of cholestasis via direct bilirubin, the negative association between GGT and pruritus remained significant and became stronger (p < 0.001). Serum GGT activity is inversely associated with the presence of cholestatic itch in patients with chronic extrahepatic cholestasis.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.