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Abstract Gulf War Illness (GWI) describes a series of symptoms suffered by veterans of the Gulf war, consisting of cognitive, neurological and gastrointestinal dysfunctions. Two chemicals associated with GWI are the insecticide permethrin (PER) and the nerve gas prophylactic pyridostigmine-bromide (PB). In this study we assessed the effects of PER and PB exposure on the pathology and subsequent alcohol (EtOH)-induced liver injury, and the influence of a macrophage depletor, PLX3397, on EtOH-induced liver damage in PER/PB-treated mice. Male C57BL/6 mice were injected daily with vehicle or PER/PB for 10 days, followed by 4 months recovery, then treatment with PLX3397 and a chronic-plus-single-binge EtOH challenge for 10 days. PER/PB exposure resulted in the protracted increase in liver transaminases in the serum and induced chronic low-level microvesicular steatosis and inflammation in GWI vs Naïve mice up to 4 months after cessation of exposure. Furthermore, prior exposure to PER/PB also resulted in exacerbated response to EtOH-induced liver injury, with enhanced steatosis, ductular reaction and fibrosis. The enhanced EtOH-induced liver damage in GWI-mice was attenuated by strategies designed to deplete macrophages in the liver. Taken together, these data suggest that exposure to GWI-related chemicals may alter the liver’s response to subsequent ethanol exposure.

Abstract 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.

Abstract 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 (TGFβ1) is upregulated due to liver failure in mice and inhibiting circulating TGFβ reduced HE progression. However, the specific contributions of TGFβ1 on brain cell populations and neuroinflammation during HE are not known. Therefore, the aim of this study was to characterize hepatic and brain TGFβ1 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 (TGFβR2) null mice (TGFβR2ΔNeu) were treated with azoxymethane (AOM) to induce acute liver failure and HE. The activity of circulating TGFβ1 was inhibited in C57Bl/6 mice via injection of a neutralizing antibody against TGFβ1 (anti-TGFβ1) 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 TGFβ1 signaling in HT-22 and EOC-20 cells. Results TGFβ1 was expressed and upregulated in the liver following AOM injection. Pharmacological inhibition of TGFβ1 after AOM injection attenuated neurological decline, microglia activation, and neuroinflammation with no significant changes in liver damage. TGFβR2ΔNeu 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 TGFβ1 or in TGFβR2ΔNeu mice. TGFβ1 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 TGFβ1 following acute liver failure results in activation of neuronal TGFβR2 signaling, driving neuroinflammation and neurological decline during AOM-induced HE.

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-β-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-β-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.

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.

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 (TGFβ1) is elevated during liver injury and influences hepatocyte senescence during APAP-induced hepatotoxicity. This study investigated TGFβ1 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 TGFβ 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 TGFβ1 signaling plays a significant role in APAP-induced liver injury by influencing necrotic cell death, inflammation, oxidative stress, and hepatocyte regeneration. In conclusion, targeting TGFβ1 or downstream signaling may be a possible therapeutic target for the management of APAP-induced liver injury. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hepatic cholestasis is associated with a significant suppression of the hypothalamuspituitary- 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. [ABSTRACT FROM AUTHOR]