Hepatocyte estrogen sulfotransferase inhibition protects female mice from concanavalin A-induced T cell-mediated hepatitis independent of estrogens.

Autoimmune hepatitis (AIH) is a typical T cell-mediated chronic liver disease with a higher incidence in females. However, the molecular mechanism for the female predisposition is poorly understood. Estrogen sulfotransferase (Est) is a conjugating enzyme best known for its function in sulfonating and deactivating estrogens. The goal of this study is to investigate whether and how Est plays a role in the higher incidence of AIH in females. Concanavalin A (ConA) was used to induce T cell-mediated hepatitis in female mice. We first showed that Est was highly induced in the liver of ConA-treated mice. Systemic or hepatocyte-specific ablation of Est, or pharmacological inhibition of Est, protected female mice from ConA-induced hepatitis regardless of ovariectomy, suggesting the effect of Est inhibition was estrogen independent. In contrast, we found that hepatocyte-specific transgenic reconstitution of Est in the whole-body Est knockout (EstKO) mice abolished the protective phenotype. Upon the ConA challenge, EstKO mice exhibited a more robust inflammatory response with elevated production of proinflammatory cytokines and changed liver infiltration of immune cells. Mechanistically, we determined that ablation of Est led to the hepatic induction of lipocalin 2 (Lcn2), whereas ablation of Lcn2 abolished the protective phenotype of EstKO females. Our findings demonstrate that hepatocyte Est is required for the sensitivity of female mice to ConA-induced and T cell-mediated hepatitis in an estrogen-independent manner. Est ablation may have protected female mice from ConA-induced hepatitis by upregulating Lcn2. Pharmacological inhibition of Est might be a potential strategy for the treatment of AIH.

Inhibition of p53 Sulfoconjugation Prevents Oxidative Hepatotoxicity and Acute Liver Failure.

BACKGROUND & AIMS: Sulfoconjugation of small molecules or protein peptides is a key mechanism to ensure biochemical and functional homeostasis in mammals. The PAPS synthase 2 (PAPSS2) is the primary enzyme to synthesize the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), in which oxidative stress is a key pathogenic event, whereas sulfation of APAP contributes to its detoxification. The goal of this study was to determine whether and how PAPSS2 plays a role in APAP-induced ALF. METHODS: Gene expression was analyzed in APAP-induced ALF in patients and mice. Liver-specific Papss2-knockout mice using Alb-Cre (Papss2ΔHC) or AAV8-TBG-Cre (Papss2iΔHC) were created and subjected to APAP-induced ALF. Primary human and mouse hepatocytes were used for in vitro mechanistic analysis. RESULTS: The hepatic expression of PAPSS2 was decreased in APAP-induced ALF in patients and mice. Surprisingly, Papss2ΔHC mice were protected from APAP-induced hepatotoxicity despite having a decreased APAP sulfation, which was accompanied by increased hepatic antioxidative capacity through the activation of the p53-p2-Nrf2 axis. Treatment with a sulfation inhibitor also ameliorated APAP-induced hepatotoxicity. Gene knockdown experiments showed that the hepatoprotective effect of Papss2ΔHC was Nrf2, p53, and p21 dependent. Mechanistically, we identified p53 as a novel substrate of sulfation. Papss2 ablation led to p53 protein accumulation by preventing p53 sulfation, which disrupts p53-MDM2 interaction and p53 ubiquitination and increases p53 protein stability. CONCLUSIONS: We have uncovered a previously unrecognized and p53-mediated role of PAPSS2 in controlling oxidative response. Inhibition of p53 sulfation may be explored for the clinical management of APAP overdose.

Inhibition of Estrogen Sulfotransferase (SULT1E1/EST) Ameliorates Ischemic Acute Kidney Injury in Mice.

BACKGROUND: Studies have suggested that estrogens may protect mice from AKI. Estrogen sulfotransferase (SULT1E1, or EST) plays an important role in estrogen homeostasis by sulfonating and deactivating estrogens, but studies on the role of SULT1E1 in AKI are lacking. METHODS: We used the renal ischemia-reperfusion model to investigate the role of SULT1E1 in AKI. We subjected wild-type mice, Sult1e1 knockout mice, and Sult1e1 knockout mice with liver-specific reconstitution of SULT1E1 expression to bilateral renal ischemia-reperfusion or sham surgery, either in the absence or presence of gonadectomy. We assessed relevant biochemical, histologic, and gene expression markers of kidney injury. We also used wild-type mice treated with the SULT1E1 inhibitor triclosan to determine the effect of pharmacologic inhibition of SULT1E1 on AKI. RESULTS: AKI induced the expression of Sult1e1 in a tissue-specific and sex-specific manner. It induced expression of Sult1e1 in the liver in both male and female mice, but Sult1e1 induction in the kidney occurred only in male mice. Genetic knockout or pharmacologic inhibition of Sult1e1 protected mice of both sexes from AKI, independent of the presence of sex hormones. Instead, a gene profiling analysis indicated that the renoprotective effect was associated with increased vitamin D receptor signaling. Liver-specific transgenic reconstitution of SULT1E1 in Sult1e1 knockout mice abolished the protection in male mice but not in female mice, indicating that Sult1e1's effect on AKI was also tissue-specific and sex-specific. CONCLUSIONS: SULT1E1 appears to have a novel function in the pathogenesis of AKI. Our findings suggest that inhibitors of SULT1E1 might have therapeutic utility in the clinical management of AKI.

Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice.

BACKGROUND & AIMS: The role of aryl hydrocarbon receptor (AhR) in liver fibrosis is controversial because loss and gain of AhR activity both lead to liver fibrosis. The goal of this study was to investigate how the expression of AhR by different liver cell types, hepatic stellate cells (HSCs) in particular, affects liver fibrosis in mice. METHODS: We studied the effects of AhR on primary mouse and human HSCs, measuring their activation and stimulation of fibrogenesis using RNA-sequencing analysis. C57BL/6J mice were given the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE); were given carbon tetrachloride (CCl4); or underwent bile duct ligation. We also performed studies in mice with disruption of Ahr specifically in HSCs, hepatocytes, or Kupffer cells. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and immunoblotting. RESULTS: AhR was expressed at high levels in quiescent HSCs, but the expression decreased with HSC activation. Activation of HSCs from AhR-knockout mice was accelerated compared with HSCs from wild-type mice. In contrast, TCDD or ITE inhibited spontaneous and transforming growth factor ß-induced activation of HSCs. Mice with disruption of Ahr in HSCs, but not hepatocytes or Kupffer cells, developed more severe fibrosis after administration of CCl4 or bile duct ligation. C57BL/6J mice given ITE did not develop CCl4-induced liver fibrosis, whereas mice without HSC AhR given ITE did develop CCl4-induced liver fibrosis. In studies of mouse and human HSCs, we found that AhR prevents transforming growth factor ß-induced fibrogenesis by disrupting the interaction of Smad3 with ß-catenin, which prevents the expression of genes that mediate fibrogenesis. CONCLUSIONS: In studies of human and mouse HSCs, we found that AhR prevents HSC activation and expression of genes required for liver fibrogenesis. Development of nontoxic AhR agonists or strategies to activate AhR signaling in HSCs might be developed to prevent or treat liver fibrosis.

Cell Type-Specific Roles of CD38 in the Interactions of Isoniazid with NAD+ in the Liver.

NAD+ is a critical molecule that is involved in multiple cellular functions. CD38 is a multifunctional enzyme with NAD+ nucleosidase activity. Our previous work revealed the CD38-dependent interactions of isoniazid (INH), an antituberculosis drug, with NAD+ to form INH-NAD adduct. In the current work, our metabolomic analysis discovered a novel NAD+ adduct with acetylisoniazid (AcINH), a primary INH metabolite mediated by N-acetyltransferase (NAT), and we named it AcINH-NAD. Using Nat1/2(-/-) and Cd38(-/-) mice, we determined that AcINH-NAD formation is dependent on both NAT and CD38. Because NAT is expressed in hepatocytes (HP), whereas CD38 is expressed in Kupffer cells (KC) and hepatic stellate cells (HSC), we explored cell type-specific roles of CD38 in the formation of AcINH-NAD as well as INH-NAD. We found that both INH-NAD and AcINH-NAD were produced in the incubation of INH or AcINH with KC and HSC but not in HP. These data suggest that hepatic nonparenchymal cells, such as KC and HSC, are the major cell types responsible for the CD38-dependent interactions of INH with NAD+ in the liver. SIGNIFICANCE STATEMENT: The current study identified AcINH-NAD as a novel metabolite of INH in the liver. Our work also revealed the essential roles of nonparenchymal cells, including Kupffer cells and hepatic stellate cells, in the CD38-dependent interactions of NAD+ with INH, leading to the formation of both INH-NAD and AcINH-NAD in the liver. These data can be used to guide the future studies on the mechanisms of INH and NAD+ interactions and their contributions to INH-induced liver injury.

An Unexpected Role of Cholesterol Sulfotransferase and its Regulation in Sensitizing Mice to Acetaminophen-Induced Liver Injury.

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. The sulfotransferase-mediated sulfation of APAP is widely believed to be a protective mechanism to attenuate the hepatotoxicity of APAP. The cholesterol sulfotransferase SULT2B1b is best known for its activity in catalyzing the sulfoconjugation of cholesterol to synthesize cholesterol sulfate. SULT2B1b can be transcriptionally and positively regulated by the hepatic nuclear factor 4α (HNF4α). In this study, we uncovered an unexpected role for SULT2B1b in APAP toxicity. Hepatic overexpression of SULT2B1b sensitized mice to APAP-induced liver injury, whereas ablation of the Sult2B1b gene in mice conferred resistance to the APAP hepatotoxicity. Consistent with the notion that Sult2B1b is a transcriptional target of HNF4α, overexpression of HNF4α sensitized mice or primary hepatocytes to APAP-induced hepatotoxicity in a Sult2B1b-dependent manner. We conclude that the HNF4α-SULT2B1b axis has a unique role in APAP-induced acute liver injury, and SULT2B1b induction might be a risk factor for APAP hepatotoxicity.

Homocysteine deteriorates intrahepatic derangement and portal-systemic collaterals in cirrhotic rats.

In liver cirrhosis, the altered levels of vasoactive substances, especially endothelin-1 (ET-1) and nitric oxide (NO) lead to elevated intrahepatic resistance, increased portal-systemic collaterals and abnormal intra- and extra-hepatic vascular responsiveness. These derangements aggravate portal hypertension-related complications such as gastro-oesophageal variceal bleeding. Homocysteine, a substance implicated in cardiovascular diseases, has been found with influences on vasoresponsiveness and angiogenesis. However, their relevant effects in liver cirrhosis have not been investigated. In the present study, liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. In acute study, the results showed that homocysteine enhanced hepatic vasoconstriction to ET-1 but decreased portal-systemic collateral vasocontractility to arginine vasopressin (AVP). Homocysteine down-regulated hepatic phosphorylated endothelial NO synthase (p-eNOS) and p-Akt protein expressions. Inducible NOS (iNOS) and cyclooxygenase (COX)-2 expressions were up-regulated by homocysteine in splenorenal shunt (SRS), the most prominent intra-abdominal collateral vessel. In chronic study, BDL or thioacetamide (TAA) rats received homocysteine or vehicle for 14 days. The results revealed that homocysteine increased hepatic collagen fibre deposition and fibrotic factors expressions in both BDL- and TAA-induced liver fibrotic rats. Portal-systemic shunting and expressions of mesenteric angiogenetic factors [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), PDGF receptor ß (PDGFRß) and p-eNOS] were also increased in BDL rats. In conclusion, homocysteine is harmful to vascular derangements and liver fibrosis in cirrhosis.

Inhibition of heme-thiolate monooxygenase CYP1B1 prevents hepatic stellate cell activation and liver fibrosis by accumulating trehalose.

Activation of extracellular matrix-producing hepatic stellate cells (HSCs) is a key event in liver fibrogenesis. We showed that the expression of the heme-thiolate monooxygenase cytochrome P450 1B1 (CYP1B1) was elevated in human and mouse fibrotic livers and activated HSCs. Systemic or HSC-specific ablation and pharmacological inhibition of CYP1B1 attenuated HSC activation and protected male but not female mice from thioacetamide (TAA)-, carbon tetrachloride (CCl4)-, or bile duct ligation (BDL)-induced liver fibrosis. Metabolomic analysis revealed an increase in the disaccharide trehalose in CYP1B1-deficient HSCs resulting from intestinal suppression of the trehalose-metabolizing enzyme trehalase, whose gene we found to be a target of RARα. Trehalose or its hydrolysis-resistant derivative lactotrehalose exhibited potent antifibrotic activity in vitro and in vivo by functioning as an HSC-specific autophagy inhibitor, which may account for the antifibrotic effect of CYP1B1 inhibition. Our study thus reveals an endobiotic function of CYP1B1 in liver fibrosis in males, mediated by liver-intestine cross-talk and trehalose. At the translational level, pharmacological inhibition of CYP1B1 or the use of trehalose/lactotrehalose may represent therapeutic strategies for liver fibrosis.

The anti-fibrotic drug pirfenidone inhibits liver fibrosis by targeting the small oxidoreductase glutaredoxin-1.

Activation of the hepatic stellate cells (HSCs) is a key pathogenic event in liver fibrosis. Protein S-glutathionylation (PSSG) of cysteine residues is a distinct form of oxidative response that modifies protein structures and functions. Glutaredoxin-1 (GLRX) reverses PSSG by liberating glutathione (GSH). In this study, we showed that pirfenidone (PFD), an anti-lung fibrosis drug, inhibited HSC activation and liver fibrosis in a GLRX-dependent manner. Glrx depletion exacerbated liver fibrosis, and decreased GLRX and increased PSSG were observed in fibrotic mouse and human livers. In contrast, overexpression of GLRX inhibited PSSG and liver fibrosis. Mechanistically, the inhibition of HSC activation by GLRX may have been accounted for by deglutathionylation of Smad3, which inhibits Smad3 phosphorylation, leading to the suppression of fibrogenic gene expression. Our results have established GLRX as the therapeutic target of PFD and uncovered an important role of PSSG in liver fibrosis. GLRX/PSSG can be both a biomarker and a therapeutic target for liver fibrosis.

Activation of Liver X Receptor α Sensitizes Mice to T-Cell Mediated Hepatitis.

Autoimmune hepatitis (AIH) is an inflammatory disease of the liver. Liver X receptors (LXRs), including the α and ß isoforms, are previously known for their anti-inflammatory activities. The goal of this study is to determine whether and how LXR plays a role in AIH. LXRα gain-of-function and loss-of-function mouse models were used, in conjunction with the concanavalin A (ConA) model of T-cell mediated hepatitis. We first showed that the hepatic expression of LXRα was decreased in the ConA model of hepatitis and in human patients with AIH. In the ConA model, we were surprised to find that activation of LXRα in the constitutively activated VP-LXRα whole-body knock-in (LXRα-KI) mice exacerbated ConA-induced AIH, whereas the LXRα-/- mice showed attenuated ConA-induced AIH. Interestingly, hepatocyte-specific activation of LXRα in the fatty acid binding protein-VP-LXRα transgenic mice did not exacerbate ConA-induced hepatitis. Mechanistically, the sensitizing effect of the LXRα-KI allele was invariant natural killer T (iNKT)-cell dependent, because the sensitizing effect was abolished when the LXRα-KI allele was bred into the NKT-deficient CD1d-/- background. In addition, LXRα-enhanced ConA-induced hepatitis was dependent on interferon gamma. In contrast, adoptive transfer of hepatic iNKT cells isolated from LXRα-KI mice was sufficient to sensitize CD1d-/- mice to ConA-induced AIH. Conclusion: Activation of LXRα sensitizes mice to ConA-induced AIH in iNKT and interferon gamma-dependent manner. Our results suggest that LXRα plays an important role in the development of AIH.

The Beneficial Effects of P2X7 Antagonism in Rats with Bile Duct Ligation-induced Cirrhosis.

Splanchnic angiogenesis in liver cirrhosis often leads to complications as gastroesophageal variceal hemorrhage and the treatment efficacy is adversely affected by poor portal-systemic collateral vasoresponsiveness related to nitric oxide (NO). Purinergic receptor subtype P2X7 participates in the modulation of inflammation, angiogenesis, fibrogenesis and vasoresponsiveness, but the relevant influence in cirrhosis is unknown. Common bile duct-ligated (CBDL) or sham-operated Spraque-Dawley rats received brilliant blue G (BBG, a P2X7 antagonist and food additive) or vehicle from the 15th to 28th day after operations, then hemodynamics, mesenteric angiogenesis, portal-systemic shunting, liver fibrosis, and protein expressions of angiogenic and fibrogenic factors were evaluated. The influence of oxidized ATP (oATP, another P2X7 receptor antagonist) on the collateral vasoresponsiveness to arginine vasopressin (AVP) was also surveyed. BBG decreased superior mesenteric artery (SMA) flow, portal-systemic shunting, mesenteric vascular density, and mesenteric protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), phospho (p)-VEGFR2, platelet-derived growth factor (PDGF), PDGF receptor beta (PDGFRß), cyclooxygenase (COX)-1, COX-2, and endothelial NO synthase (eNOS) in CBDL rats. BBG also ameliorated liver fibrosis and down-regulated hepatic interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), PDGF, IL-1ß, transforming growth factor-beta (TGF-ß), p-extracellular-signal-regulated kinases (ERK), and alpha-smooth muscle actin (α-SMA) expressions in CBDL rats. The collateral vasocontractility to AVP was enhanced by oATP. oATP down-regulated eNOS, inducible NOS (iNOS), VEGF, Akt, p-Akt, and nuclear factor-kappa B (NF-κB) expressions in splenorenal shunt, the most prominent intra-abdominal collateral vessel in rodents. P2X7 antagonism alleviates splanchnic hyperemia, severity of portal-systemic shunting, mesenteric angiogenesis, liver fibrosis, and enhances portal-systemic collateral vasoresponsiveness in cirrhotic rats. P2X7 blockade may be a feasible strategy to control cirrhosis and complications.

Resveratrol exhibits differential protective effects on fast- and slow-twitch muscles in streptozotocin-induced diabetic rats.

BACKGROUND: This study aimed to investigate the differential protective effect of resveratrol (RSV) on oxidative stress and metabolic signaling pathways in fast- and slow-twitch skeletal muscles of rats with diabetes. METHODS: Diabetic rats were induced by streptozotocin (STZ) for 2 weeks and then administered with RSV (1, 10 and 100 µg/kg per day) for 1 week. We determined oxidative stress and protein expression by lucigenin-mediated chemiluminescence and Western immunoblot. RESULTS: The superoxide anion production and copper-zinc superoxide dismutase (CuZnSOD) protein level were increased in fast-twitch muscle than in slow-twitch muscle of diabetes. The Akt and glycogen synthase kinase 3 (GSK-3) phosphorylations were reduced in both fast- and slow-twitch muscles of diabetes. Oxidative stress and GSK-3 dephosphorylation were corrected by RSV treatment in both fast- and slow-twitch muscles of diabetes. Furthermore, RSV treatment downregulated CuZnSOD protein level in diabetic fast-twitch muscle. In diabetic slow-twitch muscle, RSV treatment elevated manganese SOD (MnSOD) and phosphorylated Akt protein levels and reduced acetyl-CoA carboxylase (ACC) phosphorylation. CONCLUSIONS: Our results suggested that fast-twitch muscle incurred more oxidative stress, whereas slow-twitch muscle altered metabolic signaling molecules activities under diabetic status. The antidiabetic effect of RSV on fast- and slow-twitch skeletal muscles was mediated by different antioxidative and metabolic signals.