Vascularized Hepatocellular Carcinoma on a Chip to Control Chemoresistance through Cirrhosis, Inflammation and Metabolic Activity.

Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient-specific treatment strategies. In this work, we created novel three-dimensional vascularized HCC-on-a-chips (HCCoC), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness. HCC inflammation was controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450-3A4 (CYP3A4) was investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allowed for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). We showed that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increase vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, we show that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy-associated endothelial porosity increase. The HCCoCs were shown to have utility in uncovering the impact of the tumor microenvironment (TME) during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies.

Leptin Enhances Hepatic Fibrosis and Inflammation in a Mouse Model of Cholestasis.

Leptin is an adipokine with roles in food intake and energy metabolism through its actions on neurons in the hypothalamus. The role of leptin in obesity and cardiovascular disorders is well documented. However, its influence on liver conditions such as cholestasis is poorly understood. The effects of exogenous leptin and leptin-neutralizing antibody on biliary hyperplasia, hepatic fibrosis, and inflammation in the multidrug resistance protein 2 knockout (Mdr2KO) mouse model of cholestasis were assessed by quantifying markers specific for cholangiocytes, activated hepatic stellate cells (HSCs), and cytokines. Serum and hepatic leptin were increased in Mdr2KO mice compared with FVB/NJ (FVBN) controls, and exogenous leptin enhanced biliary hyperplasia and liver fibrosis in Mdr2KO and FVBN mice. Leptin administration increased hepatic expression of C-C motif chemokine ligand 2 and IL-6 in Mdr2KO mice. In contrast, leptin-neutralizing antibody reduced intrahepatic bile duct mass and decreased HSC activation in Mdr2KO mice compared with FVBN controls. Sex-related differences were noted, with female Mdr2KO mice having more leptin than males. In cholangiocytes and LX2 cells in vitro, leptin increased phosphorylated Akt and stimulated cell proliferation. Leptin receptor siRNA and inhibitors of Akt phosphorylation impaired leptin-induced cell proliferation and proinflammatory cytokines. The current data suggest that leptin is abnormally increased in cholestatic mice, and excess leptin increases ductular reaction, hepatic fibrosis, and inflammation via leptin receptor-mediated phosphorylation of Akt in cholangiocytes and HSCs.

Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP3A4 Metabolic Activity.

Variations in tumor biology from patient to patient combined with the low overall survival rate of hepatocellular carcinoma (HCC) present significant clinical challenges. During the progression of chronic liver diseases from inflammation to the development of HCC, microenvironmental properties, including tissue stiffness and oxygen concentration, change over time. This can potentially impact drug metabolism and subsequent therapy response to commonly utilized therapeutics, such as doxorubicin, multi-kinase inhibitors (e.g., sorafenib), and other drugs, including immunotherapies. In this study, we utilized four common HCC cell lines embedded in 3D collagen type-I gels of varying stiffnesses to mimic normal and cirrhotic livers with environmental oxygen regulation to quantify the impact of these microenvironmental factors on HCC chemoresistance. In general, we found that HCC cells with higher baseline levels of cytochrome p450-3A4 (CYP3A4) enzyme expression, HepG2 and C3Asub28, exhibited a cirrhosis-dependent increase in doxorubicin chemoresistance. Under the same conditions, HCC cell lines with lower CYP3A4 expression, HuH-7 and Hep3B2, showed a decrease in doxorubicin chemoresistance in response to an increase in microenvironmental stiffness. This differential therapeutic response was correlated with the regulation of CYP3A4 expression levels under the influence of stiffness and oxygen variation. In all tested HCC cell lines, the addition of sorafenib lowered the required doxorubicin dose to induce significant levels of cell death, demonstrating its potential to help reduce systemic doxorubicin toxicity when used in combination. These results suggest that patient-specific tumor microenvironmental factors, including tissue stiffness, hypoxia, and CYP3A4 activity levels, may need to be considered for more effective use of chemotherapeutics in HCC patients.

Probiotics improve the neurometabolic profile of rats with chronic cholestatic liver disease.

Chronic liver disease leads to neuropsychiatric complications called hepatic encephalopathy (HE). Current treatments have some limitations in their efficacy and tolerability, emphasizing the need for alternative therapies. Modulation of gut bacterial flora using probiotics is emerging as a therapeutic alternative. However, knowledge about how probiotics influence brain metabolite changes during HE is missing. In the present study, we combined the advantages of ultra-high field in vivo 1H MRS with behavioural tests to analyse whether a long-term treatment with a multistrain probiotic mixture (VIVOMIXX) in a rat model of type C HE had a positive effect on behaviour and neurometabolic changes. We showed that the prophylactic administration of this probiotic formulation led to an increase in gut Bifidobacteria and attenuated changes in locomotor activity and neurometabolic profile in a rat model of type C HE. Both the performance in behavioural tests and the neurometabolic profile of BDL + probiotic rats were improved compared to the BDL group at week 8 post-BDL. They displayed a significantly lesser increase in brain Gln, a milder decrease in brain mIns and a smaller decrease in neurotransmitter Glu than untreated animals. The clinical implications of these findings are potentially far-reaching given that probiotics are generally safe and well-tolerated by patients.

Inhibition of thrombospondin-1 reduces glutathione activity and worsens acute liver injury during acetaminophen hepatotoxicity in mice.

Acetaminophen (N-Acetyl-p-Aminophenol or APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the United States and Western Europe. Previous studies have shown that TGFß1 is elevated during APAP-induced hepatotoxicity and promotes liver injury by reducing liver regeneration while inducing hepatocyte senescence. At this time, little is known about the role of proteins that activate latent TGFß1 and their effects during APAP-induced hepatotoxicity. Thrombospondin-1 (TSP1) is a homotrimeric protein that can not only activate latent TGFß1 but can also interact with other proteins including Nrf2 to induce antioxidant signaling. The aim of the current study was to assess the role of thrombospondin-1 (TSP1) in both TGFß1 activation and its contribution to APAP-induced liver injury. C57Bl/6 mice or TSP1 null mice (TSP1-/-) were administered 300 mg/kg or 600 mg/kg of APAP. TGFß1 signaling, TSP1 expression, measures of hepatic injury, Nrf2 expression, measures of oxidative/nitrosative stress and GSH metabolism were assessed. The expression of TGFß1, TSP1 and phosphorylation of SMAD proteins increased in APAP-treated mice compared to controls. TSP1-/- mice had reduced TGFß1 expression and phosphorylation of SMAD proteins but increased liver injury. Hepatocyte cell death was increased in TSP1-/- mice and this was associated with decreased Nrf2 activity, decreased GSH levels and increased oxidative stress in comparison to wild-type C57Bl/6 mice. Together, these data demonstrate that elimination of TSP1 protein in APAP-treated mice reduces TGFß1 signaling but leads to increased liver injury by reducing Nrf2 expression and GSH activity, ultimately resulting in increased cell death.

Ghrelin reverses ductular reaction and hepatic fibrosis in a rodent model of cholestasis.

The orexigenic peptide ghrelin (Ghr) stimulates hunger signals in the hypothalamus via growth hormone secretagogue receptor (GHS-R1a). Gastric Ghr is synthetized as a preprohormone which is proteolytically cleaved, and acylated by a membrane-bound acyl transferase (MBOAT). Circulating Ghr is reduced in cholestatic injuries, however Ghr's role in cholestasis is poorly understood. We investigated Ghr's effects on biliary hyperplasia and hepatic fibrosis in Mdr2-knockout (Mdr2KO) mice, a recognized model of cholestasis. Serum, stomach and liver were collected from Mdr2KO and FVBN control mice treated with Ghr, des-octanoyl-ghrelin (DG) or vehicle. Mdr2KO mice had lower expression of Ghr and MBOAT in the stomach, and lower levels of circulating Ghr compared to WT-controls. Treatment of Mdr2KO mice with Ghr improved plasma transaminases, reduced biliary and fibrosis markers. In the liver, GHS-R1a mRNA was expressed predominantly in cholangiocytes. Ghr but not DG, decreased cell proliferation via AMPK activation in cholangiocytes in vitro. AMPK inhibitors prevented Ghr-induced FOXO1 nuclear translocation and negative regulation of cell proliferation. Ghr treatment reduced ductular reaction and hepatic fibrosis in Mdr2KO mice, regulating cholangiocyte proliferation via GHS-R1a, a G-protein coupled receptor which causes increased intracellular Ca2+ and activation of AMPK and FOXO1, maintaining a low rate of cholangiocyte proliferation.

The Influence of Chronic Liver Diseases on Hepatic Vasculature: A Liver-on-a-chip Review.

In chronic liver diseases and hepatocellular carcinoma, the cells and extracellular matrix of the liver undergo significant alteration in response to chronic injury. Recent literature has highlighted the critical, but less studied, role of the liver vasculature in the progression of chronic liver diseases. Recent advancements in liver-on-a-chip systems has allowed in depth investigation of the role that the hepatic vasculature plays both in response to, and progression of, chronic liver disease. In this review, we first introduce the structure, gradients, mechanical properties, and cellular composition of the liver and describe how these factors influence the vasculature. We summarize state-of-the-art vascularized liver-on-a-chip platforms for investigating biological models of chronic liver disease and their influence on the liver sinusoidal endothelial cells of the hepatic vasculature. We conclude with a discussion of how future developments in the field may affect the study of chronic liver diseases, and drug development and testing.

Coordinated Targeting of Galanin Receptors on Cholangiocytes and Hepatic Stellate Cells Ameliorates Liver Fibrosis in Multidrug Resistance Protein 2 Knockout Mice.

Galanin (Gal) is a peptide with a role in neuroendocrine regulation of the liver. In this study, we assessed the role of Gal and its receptors, Gal receptor 1 (GalR1) and Gal receptor 2 (GalR2), in cholangiocyte proliferation and liver fibrosis in multidrug resistance protein 2 knockout (Mdr2KO) mice as a model of chronic hepatic cholestasis. The distribution of Gal, GalR1, and GalR2 in specific liver cell types was assessed by laser-capture microdissection and confocal microscopy. Galanin immunoreactivity was detected in cholangiocytes, hepatic stellate cells (HSCs), and hepatocytes. Cholangiocytes expressed GalR1, whereas HSCs and hepatocytes expressed GalR2. Strategies were used to either stimulate or block GalR1 and GalR2 in FVB/N (wild-type) and Mdr2KO mice and measure biliary hyperplasia and hepatic fibrosis by quantitative PCR and immunostaining of specific markers. Galanin treatment increased cholangiocyte proliferation and fibrogenesis in both FVB/N and Mdr2KO mice. Suppression of GalR1, GalR2, or both receptors in Mdr2KO mice resulted in reduced bile duct mass and hepatic fibrosis. In vitro knockdown of GalR1 in cholangiocytes reduced α-smooth muscle actin expression in LX-2 cells treated with cholangiocyte-conditioned media. A GalR2 antagonist inhibited HSC activation when Gal was administered directly to LX-2 cells, but not via cholangiocyte-conditioned media. These data demonstrate that Gal contributes not only to cholangiocyte proliferation but also to liver fibrogenesis via the coordinate activation of GalR1 in cholangiocytes and GalR2 in HSCs.

Thrombospondin-1 Exacerbates Acute Liver Failure and Hepatic Encephalopathy Pathology in Mice by Activating Transforming Growth Factor ß1.

Severe hepatic insults can lead to acute liver failure and hepatic encephalopathy (HE). Transforming growth factor ß1 (TGFß1) has been shown to contribute to HE during acute liver failure; however, TGFß1 must be activated to bind its receptor and generate downstream effects. One protein that can activate TGFß1 is thrombospondin-1 (TSP-1). Therefore, the aim of this study was to assess TSP-1 during acute liver failure and HE pathogenesis. C57Bl/6 or TSP-1 knockout (TSP-1-/-) mice were injected with azoxymethane (AOM) to induce acute liver failure and HE. Liver damage, neurologic decline, and molecular analyses of TSP-1 and TGFß1 signaling were performed. AOM-treated mice had increased TSP-1 and TGFß1 mRNA and protein expression in the liver. TSP-1-/- mice administered AOM had reduced liver injury as assessed by histology and serum transaminase levels compared with C57Bl/6 AOM-treated mice. TSP-1-/- mice treated with AOM had reduced TGFß1 signaling that was associated with less hepatic cell death as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cleaved caspase 3 expression. TSP-1-/- AOM-treated mice had a reduced rate of neurologic decline, less cerebral edema, and a decrease in microglia activation in comparison with C57Bl/6 mice treated with AOM. Taken together, TSP-1 is an activator of TGFß1 signaling during AOM-induced acute liver failure and contributes to both liver pathology and HE progression.

The TGFß1 Receptor Antagonist GW788388 Reduces JNK Activation and Protects Against Acetaminophen Hepatotoxicity in Mice.

Acute liver failure is a serious consequence of acetaminophen (APAP)-induced hepatotoxic liver injury with high rates of morbidity and mortality. Transforming growth factor beta 1 (TGFß1) is elevated during liver injury and influences hepatocyte senescence during APAP-induced hepatotoxicity. This study investigated TGFß1 signaling in the context of inflammation, necrotic cell death, and oxidative stress during APAP-induced liver injury. Male C57Bl/6 mice were injected with 600 mg/kg APAP to generate liver injury in the presence or absence of the TGFß receptor 1 inhibitor, GW788388, 1 h prior to APAP administration. Acetaminophen-induced liver injury was characterized using histological and biochemical measures. Transforming growth factor beta 1 expression and signal transduction were assessed using immunohistochemistry, Western blotting and ELISA assays. Hepatic necrosis, liver injury, cell proliferation, hepatic inflammation, and oxidative stress were assessed in all mice. Acetaminophen administration significantly induced necrosis and elevated serum transaminases compared with control mice. Transforming growth factor beta 1 staining was observed in and around areas of necrosis with phosphorylation of SMAD3 observed in hepatocytes neighboring necrotic areas in APAP-treated mice. Pretreatment with GW788388 prior to APAP administration in mice reduced hepatocyte cell death and stimulated regeneration. Phosphorylation of SMAD3 was reduced in APAP mice pretreated with GW788388 and this correlated with reduced hepatic cytokine production and oxidative stress. These results support that TGFß1 signaling plays a significant role in APAP-induced liver injury by influencing necrotic cell death, inflammation, oxidative stress, and hepatocyte regeneration. In conclusion, targeting TGFß1 or downstream signaling may be a possible therapeutic target for the management of APAP-induced liver injury.

Elevated circulating TGFß1 during acute liver failure activates TGFßR2 on cortical neurons and exacerbates neuroinflammation and hepatic encephalopathy in mice.

BACKGROUND: Acute liver failure resulting from drug-induced liver injury can lead to the development of neurological complications called hepatic encephalopathy (HE). Hepatic transforming growth factor beta 1 (TGFß1) is upregulated due to liver failure in mice and inhibiting circulating TGFß reduced HE progression. However, the specific contributions of TGFß1 on brain cell populations and neuroinflammation during HE are not known. Therefore, the aim of this study was to characterize hepatic and brain TGFß1 signaling during acute liver failure and its contribution to HE progression using a combination of pharmacological and genetic approaches. METHODS: C57Bl/6 or neuron-specific transforming growth factor beta receptor 2 (TGFßR2) null mice (TGFßR2ΔNeu) were treated with azoxymethane (AOM) to induce acute liver failure and HE. The activity of circulating TGFß1 was inhibited in C57Bl/6 mice via injection of a neutralizing antibody against TGFß1 (anti-TGFß1) prior to AOM injection. In all mouse treatment groups, liver damage, neuroinflammation, and neurological deficits were assessed. Inflammatory signaling between neurons and microglia were investigated in in vitro studies through the use of pharmacological inhibitors of TGFß1 signaling in HT-22 and EOC-20 cells. RESULTS: TGFß1 was expressed and upregulated in the liver following AOM injection. Pharmacological inhibition of TGFß1 after AOM injection attenuated neurological decline, microglia activation, and neuroinflammation with no significant changes in liver damage. TGFßR2ΔNeu mice administered AOM showed no effect on liver pathology but significantly reduced neurological decline compared to control mice. Microglia activation and neuroinflammation were attenuated in mice with pharmacological inhibition of TGFß1 or in TGFßR2ΔNeu mice. TGFß1 increased chemokine ligand 2 (CCL2) and decreased C-X3-C motif ligand 1 (CX3CL1) expression in HT-22 cells and reduced interleukin-1 beta (IL-1ß) expression, tumor necrosis factor alpha (TNFα) expression, and phagocytosis activity in EOC-20 cells. CONCLUSION: Increased circulating TGFß1 following acute liver failure results in activation of neuronal TGFßR2 signaling, driving neuroinflammation and neurological decline during AOM-induced HE.

The Direct Contribution of Astrocytes and Microglia to the Pathogenesis of Hepatic Encephalopathy.

Hepatic encephalopathy is a neurological complication resulting from loss of hepatic function and is associated with poor clinical outcomes. During acute liver failure over 20% of mortality can be associated with the development of hepatic encephalopathy. In patients with liver cirrhosis, 1-year survival for those that develop overt hepatic encephalopathy is under 50%. The pathogenesis of hepatic encephalopathy is complicated due to the multiple disruptions in homeostasis that occur following a reduction in liver function. Of these, elevations of ammonia and neuroinflammation have been shown to play a significant contributing role to the development of hepatic encephalopathy. Disruption of the urea cycle following liver dysfunction leads to elevations of circulating ammonia, which enter the brain and disrupt the functioning of astrocytes. This results in dysregulation of metabolic pathways in astrocytes, oxidative stress and cerebral edema. Besides ammonia, circulating chemokines and cytokines are increased following liver injury, leading to activation of microglia and a subsequent neuroinflammatory response. The combination of astrocyte dysfunction and microglia activation are significant contributing factors to the pathogenesis of hepatic encephalopathy.

Gulf war illness-related chemicals increase CD11b/c+ monocyte infiltration into the liver and aggravate hepatic cholestasis in a rodent model.

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b+F4/80- macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

FXR-Mediated Cortical Cholesterol Accumulation Contributes to the Pathogenesis of Type A Hepatic Encephalopathy.

BACKGROUND & AIMS: Hepatic encephalopathy is a serious neurologic complication of acute and chronic liver diseases. We previously showed that aberrant bile acid signaling contributes to the development of hepatic encephalopathy via farnesoid X receptor (FXR)-mediated mechanisms in neurons. In the brain, a novel alternative bile acid synthesis pathway, catalyzed by cytochrome p450 46A1 (Cyp46A1), is the primary mechanism by which the brain regulates cholesterol homeostasis. The aim of this study was to determine if FXR activation in the brain altered cholesterol homeostasis during hepatic encephalopathy. METHODS: Cyp7A1-/- mice or C57Bl/6 mice pretreated with central infusion of FXR vivo morpholino, 2-hydroxypropyl-ß-cyclodextrin, or fed a cholestyramine-supplemented diet were injected with azoxymethane (AOM). Cognitive and neuromuscular impairment as well as liver damage and expression of Cyp46A1 were assessed using standard techniques. The subsequent cholesterol content in the frontal cortex was measured using commercially available kits and by Filipin III and Nile Red staining. RESULTS: There was an increase in membrane-bound and intracellular cholesterol in the cortex of mice treated with AOM that was associated with decreased Cyp46A1 expression. Strategies to inhibit FXR signaling prevented the down-regulation of Cyp46A1 and the accumulation of cholesterol. Treatment of mice with 2-hydroxypropyl-ß-cyclodextrin attenuated the AOM-induced cholesterol accumulation in the brain and the cognitive and neuromuscular deficits without altering the underlying liver pathology. CONCLUSIONS: During hepatic encephalopathy, FXR signaling increases brain cholesterol and contributes to neurologic decline. Targeting cholesterol accumulation in the brain may be a possible therapeutic target for the management of hepatic encephalopathy.

Direct Comparison of the Thioacetamide and Azoxymethane Models of Type A Hepatic Encephalopathy in Mice.

Acute liver failure is a devastating consequence of hepatotoxic liver injury that can lead to the development of hepatic encephalopathy. There is no consensus on the best model to represent these syndromes in mice, and therefore the aim of this study was to classify hepatic and neurological consequences of azoxymethane- and thioacetamide-induced liver injury. Azoxymethane-treated mice were euthanized at time points representing absence of minor and significant stages of neurological decline. Thioacetamide-treated mice had tissue collected at up to 3 days following daily injections. Liver histology, serum chemistry, bile acids, and cytokine levels were measured. Reflexes, grip strength measurement, and ataxia were calculated for all groups. Brain ammonia, bile acid levels, cerebral edema, and neuroinflammation were measured. Finally, in vitro and in vivo assessments of blood-brain barrier function were performed. Serum transaminases and liver histology demonstrate that both models generated hepatotoxic liver injury. Serum proinflammatory cytokine levels were significantly elevated in both models. Azoxymethane-treated mice had progressive neurological deficits, while thioacetamide-treated mice had inconsistent neurological deficits. Bile acids and cerebral edema were increased to a higher degree in azoxymethane-treated mice, while cerebral ammonia and neuroinflammation were greater in thioacetamide-treated mice. Blood-brain barrier permeability exists in both models but was likely not due to direct toxicity of azoxymethane or thioacetamide on brain endothelial cells. In conclusion, both models generate acute liver injury and hepatic encephalopathy, but the requirement of a single injection and the more consistent neurological decline make azoxymethane treatment a better model for acute liver failure with hepatic encephalopathy.

Glucocorticoids Cause Gender-Dependent Reversal of Hepatic Fibrosis in the MDR2-Knockout Mouse Model.

Hepatic cholestasis is associated with a significant suppression of the hypothalamus-pituitary-adrenal axis (HPA). In the present study, we tested the hypothesis that activation of the HPA axis by corticosterone treatment can reverse liver inflammation and fibrosis in a multidrug resistance protein 2 knockout (MDR2KO) transgenic mouse model of hepatic cholestasis. Friend Virus B NIH-Jackson (FVBN) control and MDR2KO male and female mice were treated with vehicle or corticosterone for two weeks, then serum and liver analyses of hepatic cholestasis markers were performed. Indicators of inflammation, such as increased numbers of macrophages, were determined. MDR2KO mice had lower corticotropin releasing hormone and corticosterone levels than FVBN controls in the serum. There was a large accumulation of CD68 and F4/80 macrophages in MDR2KO mice livers, which indicated greater inflammation compared to FVBNs, an effect reversed by corticosterone treatment. Intrahepatic biliary duct mass, collagen deposition and alpha smooth muscle actin (αSMA) were found to be much higher in livers of MDR2KO mice than in controls; corticosterone treatment significantly decreased these fibrosis markers. When looking at the gender-specific response to corticosterone treatment, male MDR2KO mice tended to have a more pronounced reversal of liver fibrosis than females treated with corticosterone.

Melatonin inhibits hypothalamic gonadotropin-releasing hormone release and reduces biliary hyperplasia and fibrosis in cholestatic rats.

Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestatic liver injury. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin-releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats have not been investigated. To test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin were assessed. Cholangiocyte proliferation and fibrosis were measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor antagonist cetrorelix acetate, and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation, and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation, and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs, and this was reversed by cetrorelix acetate cotreatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.NEW & NOTEWORTHY We have previously demonstrated that GnRH is expressed in cholangiocytes and promotes their proliferation during cholestasis. In addition, dark therapy, which increases melatonin, reduced cholangiocyte proliferation and fibrosis during cholestasis. This study expands these findings by investigating neural GnRH regulation by melatonin during BDL-induced cholestasis by infusing melatonin into the brain. Melatonin infusion reduced cholangiocyte proliferation and fibrosis, and these effects are due to GNRH receptor 1-dependent paracrine signaling between cholangiocytes and hepatic stellate cells.

Bile Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Promotes Neuroinflammation during Hepatic Encephalopathy in Mice.

Hepatic encephalopathy (HE) is a neuropsychiatric complication that occurs due to deteriorating hepatic function and this syndrome influences patient quality of life, clinical management strategies and survival. During acute liver failure, circulating bile acids increase due to a disruption of the enterohepatic circulation. We previously identified that bile acid-mediated signaling occurs in the brain during HE and contributes to cognitive impairment. However, the influences of bile acids and their downstream signaling pathways on HE-induced neuroinflammation have not been assessed. Conjugated bile acids, such as taurocholic acid (TCA), can activate sphingosine-1-phosphate receptor 2 (S1PR2), which has been shown to promote immune cell infiltration and inflammation in other models. The current study aimed to assess the role of bile-acid mediated S1PR2 signaling in neuroinflammation and disease progression during azoxymethane (AOM)-induced HE in mice. Our findings demonstrate a temporal increase of bile acids in the cortex during AOM-induced HE and identified that cortical bile acids were elevated as an early event in this model. In order to classify the specific bile acids that were elevated during HE, a metabolic screen was performed and this assay identified that TCA was increased in the serum and cortex during AOM-induced HE. To reduce bile acid concentrations in the brain, mice were fed a diet supplemented with cholestyramine, which alleviated neuroinflammation by reducing proinflammatory cytokine expression in the cortex compared to the control diet-fed AOM-treated mice. S1PR2 was expressed primarily in neurons and TCA treatment increased chemokine ligand 2 mRNA expression in these cells. The infusion of JTE-013, a S1PR2 antagonist, into the lateral ventricle prior to AOM injection protected against neurological decline and reduced neuroinflammation compared to DMSO-infused AOM-treated mice. Together, this identifies that reducing bile acid levels or S1PR2 signaling are potential therapeutic strategies for the management of HE.

The Neuropeptide Galanin Is Up-Regulated during Cholestasis and Contributes to Cholangiocyte Proliferation.

During the course of cholestatic liver diseases, mitotically dormant cholangiocytes proliferate and subsequently acquire a neuroendocrine phenotype. Galanin is a neuroendocrine factor responsible for regulation of physiological responses, such as feeding behavior and mood, and has been implicated in the development of fatty liver disease, although its role in biliary hyperplasia is unknown. Biliary hyperplasia was induced in rats via bile duct ligation (BDL) surgery, and galanin was increased in serum and liver homogenates from BDL rats. Treatment of sham and BDL rats with recombinant galanin increased cholangiocyte proliferation and intrahepatic biliary mass, liver damage, and inflammation, whereas blocking galanin expression with specific vivo-morpholino sequences inhibited hyperplastic cholangiocyte proliferation, liver damage, inflammation, and subsequent fibrosis. The proliferative effects of galanin were via activation of galanin receptor 1 expressed specifically on cholangiocytes and were associated with an activation of extracellular signal-regulated kinase 1/2, and ribosomal S6 kinase 1 signal transduction pathways and subsequent increase in cAMP responsive element binding protein DNA-binding activity and induction of Yes-associated protein expression. Strategies to inhibit extracellular signal-regulated kinase 1/2, ribosomal S6 kinase 1, or cAMP responsive element binding protein DNA-binding activity prevented the proliferative effects of galanin. Taken together, these data suggest that targeting galanin signaling may be effective for the maintenance of biliary mass during cholestatic liver diseases.