Chalcone derivatives ameliorate lipopolysaccharide-induced acute lung injury and inflammation by targeting MD2.

Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are known as the common causes of respiratory failure in critically ill patients. Myeloid differentiation 2 (MD2), a co-receptor of toll like receptor 4 (TLR4), plays an important role in LPS-induced ALI in mice. Since MD2 inhibition by pharmacological inhibitors or gene knockout significantly attenuates ALI in animal models, MD2 has become an attractive target for the treatment of ALI. In this study we identified two chalcone-derived compounds, 7w and 7x, as new MD2 inhibitors, and investigated the therapeutic effects of 7x and 7w in LPS-induced ALI mouse model. In molecular docking analysis we found that 7w and 7x, formed pi-pi stacking interactions with Phe151 residue of the MD2 protein. The direct binding was confirmed by surface plasmon resonance analysis (with KD value of 96.2 and 31.2 µM, respectively) and by bis-ANS displacement assay. 7w and 7x (2.5, 10 µM) also dose-dependently inhibited the interaction between lipopolysaccharide (LPS) and rhMD2 and LPS-MD2-TLR4 complex formation. In mouse peritoneal macrophages, 7w and 7x (1.25-10 µM) dose-dependently inhibited LPS-induced inflammatory responses, MAPKs (JNK, ERK and P38) phosphorylation as well as NF-κB activation. Finally, oral administration of 7w or 7x (10 mg ·kg-1 per day, for 7 days prior LPS challenge) in ALI mouse model significantly alleviated LPS-induced lung injury, pulmonary edema, lung permeability, inflammatory cells infiltration, inflammatory cytokines expression and MD2/TLR4 complex formation. In summary, we identify 7w and 7x as new MD2 inhibitors to inhibit inflammatory response both in vitro and in vivo, proving the therapeutic potential of 7w and 7x for ALI and inflammatory diseases.

Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression.

AIMS: Wnt signaling plays a critical role in vascular calcification (VC). Wnt factors induce different physiological and pathological effects on cardiovascular functions. Wnt1, a ligand of Wnt/ß-catenin signaling, promotes pro-angiogenesis and reduces myocardial infarction. The role of Wnt1 on VC in chronic kidney disease (CKD) is not fully understood. METHODS AND RESULTS: We used human vascular smooth muscle cells (VSMCs) and a rat model of chronic renal failure (CRF), and observed a native protective mechanism by which VC is reduced via the activation of Wnt1 and its transcriptional target ANKH inorganic pyrophosphate transport regulator (ANKH) gene. ANKH is an essential calcification inhibitor that effluxes inorganic pyrophosphate (PPi) from VSMCs to play an inhibitory role in VC. Vascular ANKH and plasma PPi were significantly downregulated in the rat model of CRF. The knockdown or inhibition of ANKH reversed the effect of Wnt1 on VC in VSMCs. Clinical analysis revealed low plasma levels of Wnt1 and PPi were associated with CKD in patients. Applying a Wnt/ß-catenin signaling agonist can alleviate the progression of VC. CONCLUSION: This work reveals the ANKH regulation of Wnt1 in VSMCs is essential for blocking VC. Our findings may contribute to the development of medications that target Wnt signaling and/or ANKH to inhibit VC.

DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.

Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. CONCLUSION: The dysregulation of SIRT1-DEPTOR-mTORC1 signaling is a critical determinant of ALD pathology; targeting SIRT1 and DEPTOR and selectively inhibiting mTORC1-S6K1 signaling may have therapeutic potential for treating ALD in humans. (Hepatology 2018).

Heterozygous Ldlr-Deficient Hamster as a Model to Evaluate the Efficacy of PCSK9 Antibody in Hyperlipidemia and Atherosclerosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis and atherogenesis. However, there are only limited rodent models, with a functional low-density lipoprotein receptor (LDLR) pathway and cholesteryl ester transfer protein (CETP) to evaluate the drug candidates targeting the PCSK9/LDLR pathway, that are translatable to humans. Here, by using our recently generated LDLR heterozygote (Ldlr+/-) hamster model with functional LDLR pathway and CETP function, we seek to evaluate the effect of a PCSK9 antibody, evolocumab, on dyslipidemia and atherosclerosis compared with ezetimibe, an effective inhibitor of cholesterol absorption, as a positive therapeutic control. We show that the plasma levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) were significantly increased in Ldlr+/- hamsters fed a high-fat high-cholesterol (HFHC) diet; therefore, areas of atherosclerotic lesion in the aorta were obviously increased and positively correlated with plasma LDL-C and TC. Circulating free PCSK9 was downregulated by the HFHC diet and was undetectable in the evolocumab treated group, as expected. Most importantly, either evolocumab or ezetimibe treatment prevented HFHC diet-induced hyperlipidemia and subsequent atherosclerotic plaque formation. The results indicate that Ldlr+/- hamsters fed an HFHC diet represent an ideal rodent model to evaluate drug candidates that affect LDLR pathways.

Inflammation is independent of steatosis in a murine model of steatohepatitis.

Obesity and alcohol consumption synergistically promote steatohepatitis, and neutrophil infiltration is believed to be associated with steatosis. However, the underlying mechanisms remain obscure. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a complex role in lipid metabolism and inflammation; therefore, the purpose of this study was to dissect its role in regulating steatosis and neutrophil infiltration in a clinically relevant mouse steatohepatitis model of 3-month high-fat diet (HFD) feeding plus a binge of ethanol (HFD-plus-binge ethanol). Hepatocyte-specific Pparg disruption reduced liver steatosis but surprisingly increased hepatic neutrophil infiltration after HFD-plus-binge ethanol. Knockout or knockdown of the PPARγ target gene, fat-specific protein 27, reduced steatosis without affecting neutrophil infiltration in this model. Moreover, hepatocyte-specific deletion of the Pparg gene, but not the fat-specific protein 27 gene, markedly up-regulated hepatic levels of the gene for chemokine (C-X-C motif) ligand 1 (Cxcl1, a chemokine for neutrophil infiltration) in HFD-plus-binge ethanol-fed mice. In vitro, deletion of the Pparg gene also highly augmented palmitic acid or tumor necrosis factor alpha induction of Cxcl1 in mouse hepatocytes. In contrast, activation of PPARγ with a PPARγ agonist attenuated Cxcl1 expression in hepatocytes. Palmitic acid also up-regulated interleukin-8 (a key chemokine for human neutrophil recruitment) expression in human hepatocytes, which was attenuated and enhanced by cotreatment with a PPARγ agonist and antagonist, respectively. Finally, acute ethanol binge markedly attenuated HFD-induced hepatic PPARγ activation, which contributed to the up-regulation of hepatic Cxcl1 expression post-HFD-plus-binge ethanol. CONCLUSION: Hepatic PPARγ plays an opposing role in controlling steatosis and neutrophil infiltration, leading to dissociation between steatosis and inflammation; acute ethanol gavage attenuates hepatic PPARγ activation and subsequently up-regulates hepatic CXCL1/interleukin-8 expression, thereby exacerbating hepatic neutrophil infiltration. (Hepatology 2017;66:108-123).

Intermedin Restores Hyperhomocysteinemia-induced Macrophage Polarization and Improves Insulin Resistance in Mice.

Hyperhomocysteinemia (HHcy) is a condition characterized by an abnormally high level of homocysteine, an inflammatory factor. This condition has been suggested to promote insulin resistance. To date, the underlying molecular mechanism remains largely unknown, and identifying novel therapeutic targets for HHcy-induced insulin resistance is of high priority. It is well known that intermedin (IMD), a calcitonin family peptide, exerts potent anti-inflammatory effects. In this study, the effects of IMD on HHcy-induced insulin resistance were investigated. Glucose tolerance and insulin tolerance tests were performed on mice treated with IMD by minipump implantation (318 ng/kg/h for 4 weeks) or adipocyte-specific IMD overexpression mice (Adipo-IMD transgenic mice). The expression of genes and proteins related to M1/M2 macrophages and endoplasmic reticulum stress (ERS) was evaluated in adipose tissues or cells. The expression of IMD was identified to be lower in the plasma and adipose tissues of HHcy mice. In both IMD treatment by minipump implantation and Adipo-IMD transgenic mice, IMD reversed HHcy-induced insulin resistance, as revealed by glucose tolerance and insulin tolerance tests. Further mechanistic study revealed that IMD reversed the Hcy-elevated ratio of M1/M2 macrophages by inhibiting AMP-activated protein kinase activity. Adipo-IMD transgenic mice displayed reduced ERS and lower inflammation in adipose tissues with HHcy. Soluble factors from Hcy-treated macrophages induced adipocyte ERS, which was reversed by IMD treatment. These findings revealed that IMD treatment restores the M1/M2 balance, inhibits chronic inflammation in adipose tissues, and improves systemic insulin sensitivity of HHcy mice.

Adrenomedullin 2 Enhances Beiging in White Adipose Tissue Directly in an Adipocyte-autonomous Manner and Indirectly through Activation of M2 Macrophages.

Adrenomedullin 2 (ADM2) is an endogenous bioactive peptide belonging to the calcitonin gene-related peptide family. Our previous studies showed that overexpression of ADM2 in mice reduced obesity and insulin resistance by increasing thermogenesis in brown adipose tissue. However, the effects of ADM2 in another type of thermogenic adipocyte, beige adipocytes, remain to be understood. The plasma ADM2 levels were inversely correlated with obesity in humans, and adipo-ADM2-transgenic (tg) mice displayed resistance to high-fat diet-induced obesity with increased energy expenditure. Beiging of subcutaneous white adipose tissues (WAT) was more noticeably induced in high-fat diet-fed transgenic mice with adipocyte-ADM2 overexpression (adipo-ADM2-tg mice) than in WT animals. ADM2 treatment in primary rat subcutaneous adipocytes induced beiging with up-regulation of UCP1 and beiging-related marker genes and increased mitochondrial uncoupling respiration, which was mainly mediated by activation of the calcitonin receptor-like receptor (CRLR)·receptor activity-modifying protein 1 (RAMP1) complex and PKA and p38 MAPK signaling pathways. Importantly, this adipocyte-autonomous beiging effect by ADM2 was translatable to human primary adipocytes. In addition, M2 macrophage activation also contributed to the beiging effects of ADM2 through catecholamine secretion. Therefore, our study reveals that ADM2 enhances subcutaneous WAT beiging via a direct effect by activating the CRLR·RAMP1-cAMP/PKA and p38 MAPK pathways in white adipocytes and via an indirect effect by stimulating alternative M2 polarization in macrophages. Through both mechanisms, beiging of WAT by ADM2 results in increased energy expenditure and reduced obesity, suggesting ADM2 as a novel anti-obesity target.

Aging aggravates alcoholic liver injury and fibrosis in mice by downregulating sirtuin 1 expression.

BACKGROUND & AIMS: Aging is known to exacerbate the progression of alcoholic liver disease (ALD), but the underlying mechanisms remain obscure. The aim of this study was to use a chronic plus binge ethanol feeding model in mice to evaluate the effects of aging on alcohol-induced liver injury. METHODS: C57BL/6 mice were subjected to short-term (10days) ethanol plus one binge or long-term (8weeks) ethanol plus multiple binges of ethanol. Liver injury and fibrosis were determined. Hepatic stellate cells (HSCs) were isolated and used in in vitro studies. RESULTS: Middle-aged (12-14months) and old-aged (>16months) mice were more susceptible to liver injury, inflammation, and oxidative stress induced by short-term plus one binge or long-term plus multiple binges of ethanol feeding when compared to young (8-12weeks) mice. Long-term plus multiple binges of ethanol feeding induced greater liver fibrosis in middle-aged mice than that in young mice. Hepatic expression of sirtuin 1 (SIRT1) protein was downregulated in the middle-aged mice compared to young mice. Restoration of SIRT1 expression via the administration of adenovirus-SIRT1 vector ameliorated short-term plus binge ethanol-induced liver injury and fibrosis in middle-aged mice. HSCs isolated from middle-aged mice expressed lower levels of SIRT1 protein and were more susceptible to spontaneous activation in in vitro culture than those from young mice. Overexpression of SIRT1 reduced activation of HSCs from middle-aged mice in vitro with downregulation of PDGFR-α and c-Myc, while deletion of SIRT1 activated HSCs isolated from young mice in vitro. Finally, HSC-specific SIRT1 knockout mice were more susceptible to long-term chronic-plus-multiple binges of ethanol-induced liver fibrosis with upregulation of PDGFR-α expression. CONCLUSIONS: Aging exacerbates ALD in mice through the downregulation of SIRT1 in hepatocytes and HSCs. Activation of SIRT1 may serve as a novel target for the treatment of ALD. LAY SUMMARY: Aged mice are more susceptible to alcohol-induced liver injury and fibrosis, which is, at least in part, due to lower levels of sirtuin 1 protein in hepatocytes and hepatic stellate cells. Our findings suggest that sirtuin 1 activators may have beneficial effects for the treatment of alcoholic liver disease in aged patients.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

The Detrimental Role Played by Lipocalin-2 in Alcoholic Fatty Liver in Mice.

We have previously shown that the ethanol-mediated elevation of lipocaline-2 (LCN2) is closely associated with the development of alcoholic fatty liver disease (AFLD) in mice. Herein, we aimed to understand the functional significance of LCN2 induction by ethanol and to explore its underlying mechanisms. We evaluated the effects of LCN2 in an in vitro cellular alcoholic steatosis model and in an animal study using wild-type and LCN2 knockout mice fed for 4 weeks with an ethanol-supplemented Lieber-DeCarli diet. In the cellular model of alcoholic steatosis, recombinant LCN2 or overexpression of LCN2 exacerbated ethanol-induced fat accumulation, whereas knocking down LCN2 prevented steatosis in hepatocytes exposed to ethanol. Consistently, removal of LCN2 partially but significantly alleviated alcoholic fatty liver injury in mice. Mechanistically, LCN2 mediates detrimental effects of ethanol in the liver via disrupted multiple signaling pathways, including aberrant nicotinamide phosphoribosyltransferase-sirtuin 1 axis, perturbed endocrine metabolic regulatory fibroblast growth factor 15/19 signaling, and impaired chaperone-mediated autophagy. Finally, compared with healthy human livers, liver samples from patients with AFLD had lower gene expression of several LCN2-regualted molecules. Our study demonstrated a pivotal and causal role of LCN2 in the development of AFLD and suggested that targeting the LCN2 could be of great value for the treatment of human AFLD.

Animal Models of Alcoholic Liver Disease: Pathogenesis and Clinical Relevance.

Alcoholic liver disease (ALD), a leading cause of chronic liver injury worldwide, comprises a range of disorders including simple steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Over the last five decades, many animal models for the study of ALD pathogenesis have been developed. Recently, a chronic-plus-binge ethanol feeding model was reported. This model induces significant steatosis, hepatic neutrophil infiltration, and liver injury. A clinically relevant model of high-fat diet feeding plus binge ethanol was also developed, which highlights the risk of excessive binge drinking in obese/overweight individuals. All of these models recapitulate some features of the different stages of ALD and have been widely used by many investigators to study the pathogenesis of ALD and to test for therapeutic drugs/components. However, these models are somewhat variable, depending on mouse genetic background, ethanol dose, and animal facility environment. This review focuses on these models and discusses these variations and some methods to improve the feeding protocol. The pathogenesis, clinical relevance, and translational studies of these models are also discussed.

Intermedin1-53 attenuates vascular calcification in rats with chronic kidney disease by upregulation of α-Klotho.

Deficiency in α-Klotho is involved in the pathogenesis of vascular calcification. Since intermedin (IMD)1-53 (a calcitonin/calcitonin gene-related peptide) protects against vascular calcification, we studied whether IMD1-53 inhibits vascular calcification by upregulating α-Klotho. A rat model of chronic kidney disease (CKD) with vascular calcification induced by the 5/6 nephrectomy plus vitamin D3 was used for study. The aortas of rats with CKD showed reduced IMD content but an increase of its receptor, calcitonin receptor-like receptor, and its receptor modifier, receptor activity-modifying protein 3. IMD1-53 treatment reduced vascular calcification. The expression of α-Klotho was greatly decreased in the aortas of rats with CKD but increased in the aortas of IMD1-53-treated rats with CKD. In vitro, IMD1-53 increased α-Klotho protein level in calcified vascular smooth muscle cells. α-Klotho knockdown blocked the inhibitory effect of IMD1-53 on vascular smooth muscle cell calcification and their transformation into osteoblast-like cells. The effect of IMD1-53 to upregulate α-Klotho and inhibit vascular smooth muscle cell calcification was abolished by knockdown of its receptor or its modifier protein, or treatment with the protein kinase A inhibitor H89. Thus, IMD1-53 may attenuate vascular calcification by upregulating α-Klotho via the calcitonin receptor/modifying protein complex and protein kinase A signaling.

Fat-Specific Protein 27/CIDEC Promotes Development of Alcoholic Steatohepatitis in Mice and Humans.

BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is the progressive form of alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma. We studied mouse models and human tissues to identify molecules associated with ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in white adipose tissues and promotes formation of fat droplets. METHODS: C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27 (Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5% ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5 or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor (GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral vectors were used to express transgenes or small hairpin (sh) RNAs in cultured hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH and analyzed histologically and immunohistochemically and by transcriptome, immunoblotting, and real-time PCR analyses. RESULTS: Chronic-plus-binge ethanol feeding of mice, which mimics the drinking pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray analyses revealed similar alterations in expression of many hepatic genes in ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given injections of adenovirus-Fsp27shRNA had markedly reduced ASH following chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic AMP-responsive element binding protein H (CREBH) prevented the increases in Fsp27α and FSP27ß mRNAs, respectively, and reduced liver injury in this chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol exposure had synergistic effects in inducing production of mitochondrial reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA expression was increased in patients with AH and correlated with the degree of hepatic steatosis and disease severity including mortality. CONCLUSIONS: In mice, chronic-plus-binge ethanol feeding induces ASH that mimics some histological and molecular features observed in patients with AH. Hepatic expression of FSP27/CIDEC is highly up-regulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation contributes to alcohol-induced liver damage.

Short- or long-term high-fat diet feeding plus acute ethanol binge synergistically induce acute liver injury in mice: an important role for CXCL1.

UNLABELLED: Obesity and alcohol consumption often coexist and work synergistically to promote steatohepatitis; however, the underlying mechanisms remain obscure. Here, we demonstrate that feeding mice a high-fat diet (HFD) for as little as 3 days markedly exacerbated acute ethanol binge-induced liver neutrophil infiltration and injury. Feeding mice with an HFD for 3 months plus a single binge of ethanol induced much more severe steatohepatitis. Moreover, 3-day or 3-month HFD-plus-ethanol binge (3d-HFD+ethanol or 3m-HFD+ethanol) treatment markedly up-regulated the hepatic expression of several chemokines, including chemokine (C-X-C motif) ligand 1 (Cxcl1), which showed the highest fold (approximately 20-fold and 35-fold, respectively) induction. Serum CXCL1 protein levels were also markedly elevated after the HFD+ethanol treatment. Blockade of CXCL1 with a CXCL1 neutralizing antibody or genetic deletion of the Cxcl1 gene reduced the HFD+ethanol-induced hepatic neutrophil infiltration and injury, whereas overexpression of Cxcl1 exacerbated steatohepatitis in HFD-fed mice. Furthermore, expression of Cxcl1 messenger RNA was up-regulated in hepatocytes, hepatic stellate cells, and endothelial cells isolated from HFD+ethanol-fed mice compared to mice that were only given the HFD, with the highest fold induction observed in hepatocytes. In vitro stimulation of hepatocytes with palmitic acid up-regulated the expression of Cxcl1 messenger RNA, and this up-regulation was attenuated after treatment with an inhibitor of extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, or nuclear factor κB. In addition, hepatic or serum levels of free fatty acids were higher in HFD+ethanol-fed mice than in the control groups. CONCLUSION: An HFD combined with acute ethanol consumption synergistically induces acute liver inflammation and injury through the elevation of hepatic or serum free fatty acids and subsequent up-regulation of hepatic CXCL1 expression and promotion of hepatic neutrophil infiltration.

Liver is the major source of elevated serum lipocalin-2 levels after bacterial infection or partial hepatectomy: a critical role for IL-6/STAT3.

UNLABELLED: Lipocalin-2 (LCN2) was originally isolated from human neutrophils and termed neutrophil gelatinase-associated lipocalin (NGAL). However, the functions of LCN2 and the cell types that are primarily responsible for LCN2 production remain unclear. To address these issues, hepatocyte-specific Lcn2 knockout (Lcn2(Hep-/-)) mice were generated and subjected to bacterial infection (with Klesbsiella pneumoniae or Escherichia coli) or partial hepatectomy (PHx). Studies of Lcn2(Hep-/-) mice revealed that hepatocytes contributed to 25% of the low basal serum level of LCN2 protein (∼ 62 ng/mL) but were responsible for more than 90% of the highly elevated serum LCN2 protein level (∼ 6,000 ng/mL) postinfection and more than 60% post-PHx (∼ 700 ng/mL). Interestingly, both Lcn2(Hep-/-) and global Lcn2 knockout (Lcn2(-/-)) mice demonstrated comparable increases in susceptibility to infection with K. pneumoniae or E. coli. These mice also had increased enteric bacterial translocation from the gut to the mesenteric lymph nodes and exhibited reduced liver regeneration after PHx. Treatment with interleukin (IL)-6 stimulated hepatocytes to produce LCN2 in vitro and in vivo. Hepatocyte-specific ablation of the IL-6 receptor or Stat3, a major downstream effector of IL-6, markedly abrogated LCN2 elevation in vivo. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that STAT3 was recruited to the promoter region of the Lcn2 gene upon STAT3 activation by IL-6. CONCLUSION: Hepatocytes are the major cell type responsible for LCN2 production after bacterial infection or PHx, and this response is dependent on IL-6 activation of the STAT3 signaling pathway. Thus, hepatocyte-derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration.

Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice.

AIM: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice. METHODS: ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses. RESULTS: Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1. CONCLUSION: Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation.

[Secondary hyperuricemia in chronic renal failure promotes vascular calcification in rats].

The present study was aimed to explore the effects of hyperuricemia on vascular calcification in chronic renal failure (CRF) and the mechanisms. Adenine diet-induced CRF rat model was used. Twenty-three male 8-week-old Wistar rats were randomly divided into control group (Ctr, n = 5), CRF group (n = 8) and CRF plus allopurinol group (CRF + ALL, n = 10), and the rats were given standard diet plus standard drinking water, adenine diet plus standard drinking water and adenine diet plus allopurinol drinking for 6 weeks, respectively. Vascular calcification of abdominal aorta was identified by o-cresolphthalein complexone copper assay and Von Kossa staining. The mRNA expression levels of osteogenic/chondrogenic regulatory factors (Cbfα1, Msx2, Osx, and Sox9), vascular smooth muscle cell (VSMC) lineage markers (SM22a and Acta2) and calcification inhibitors (Mgp and Opn) were detected by real-time PCR. The results showed that the levels of serum phosphorus (Pi), urea nitrogen, creatinine and uric acid were significantly increased in the CRF rats, whereas allopurinol reversed the levels of serum urea nitrogen, creatinine and uric acid, except for serum Pi. The calcium content of rat abdominal aorta in the CRF group was significantly higher than that of the Ctr group (P < 0.05), but it was partially rescued in the CRF + ALL group (P < 0.05); Compared with the Ctr group, Cbfα1, Msx2, Osx and Sox9 mRNA levels of abdominal aorta in the CRF group were significantly up-regulated, while SM22a, Acta2, Mgp and Opn mRNA levels were down-regulated. In the CRF + ALL group, the changes of Msx2, Osx, SM22a and Opn mRNA levels were reversed (P < 0.05). Allopurinol had no effect on high Pi-induced VSMC calcification, and uric acid (6 and 7 mg/dL) significantly increased high Pi-induced VSMC calcification in vitro (P < 0.05). These results suggest that hyperuricemia in CRF may promote the osteoblast/chondrocyte-like cells differentiation of VSMC and further exacerbate vascular calcification.

Research Advances in the Mutation of TET2 Gene in Myeloid Maligancies.

TET2 gene is a member of TET oncogene family. It has been reported as a tumor suppressor gene with important roles in myelopiesis. Recent studies have shown that TET2 protein takes part in demethylation by converting 5-methylcytosine (5-mc) into 5-hydroxymethylcytosine (5-hmc). Somatic TET2 inactivation leads to abnormal myelopiesis and myeloid malignancies. In this review,the structure and function of TET2 and the relationship between TET gene mutation and myeloid malignancies are summarized.

Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via Klotho upregulation.

Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo.

STAT4 knockout mice are more susceptible to concanavalin A-induced T-cell hepatitis.

STAT4, which is activated mainly by IL-12, promotes inflammatory responses by inducing Th1 and Th2 cytokines. Recent genome-wide association studies indicate that STAT4 gene variants are associated with risk of various types of liver diseases, but how STAT4 contributes to liver disease pathogenesis remains obscure. In this study, STAT4 activation was detected in liver immune cells from patients with viral hepatitis and autoimmune hepatitis, as well as in a mouse model of concanavalin A (Con A)-induced hepatitis. Such STAT4 activation was detected mainly in T cells, natural killer T cells, and macrophages and Kupffer cells, and was diminished in Il12a(-/-) and Il12b(-/-) mice. As expected, disruption of the Stat4 gene reduced production of Th1 and Th2 cytokines, but surprisingly exacerbated Con A-induced liver injury. Similarly, disruption of Il12a or Il12b also augmented Con A-induced hepatocellular damage. Further studies showed that hepatic natural killer T (NKT) cells from Con A-treated Stat4(-/-) mice had higher levels of FasL expression and increased cytotoxicity against hepatocytes than those from Con A-treated WT mice. In vitro, blocking FasL attenuated Stat4(-/-) NKT cytotoxicity against hepatocytes. In conclusion, despite up-regulation of proinflammatory cytokines, STAT4 protects against acute T-cell hepatitis, which is mediated by direct or indirect down-regulation of FasL expression on NKT cells.