Experimental rat model for alcohol‐induced osteonecrosis of the femoral head

Alcohol‐induced osteonecrosis of the femoral head (ONFH) is observed in alcohol abusers and patients with alcoholic fatty liver disease. It has been reported that Toll‐like receptor 4 (TLR4) signalling plays a crucial role in the pathogenesis of alcoholic fatty liver disease. We previously reported a corticosteroid‐induced ONFH rat model, and suggested that TLR4 signalling contributes to the pathogenesis of ONFH. Thus, it is thought that the pathogenesis of alcohol‐induced ONFH is probably similar to that of corticosteroid‐induced ONFH. The aim of this study was to develop a new animal model for alcohol‐induced ONFH and to evaluate the relationship between the pro‐inflammatory response via TLRs and the development of ONFH in rats. Male Wistar rats were fed a Lieber–DeCarli liquid diet containing 5% ethanol (experimental group) or dextran (control group) for 1–24 weeks. Histopathological and biochemical analyses were performed. Feeding the ethanol‐containing liquid diet resulted in the development of ONFH with hepatic steatosis, hepatic dysfunction and hyperlipidaemia, whereas feeding the dextran‐containing diet did not cause ONFH. However, we could not recognize any relationship between the pro‐inflammatory response via TLR4 and the development of alcohol‐induced ONFH. Thus in this study we have developed a new rat model for alcohol‐induced ONFH based on the feeding of an ethanol liquid diet. ONFH was observed within seven days from the start of feeding with 5% ethanol‐containing liquid diet. Although this was linked to hepatic steatosis, a TLR4 association was not a feature of this model.     

紫草素对非酒精性脂肪性肝炎的影响及分子机制研究

目的 探究紫草素对非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)是否具有改善作用及其作用机制.方法 给予C57BL/6小鼠胆碱和蛋氨酸缺乏(methionine choline deficient,MCD)饮食诱导NASH模型,检测小鼠体质量、血清转氨酶、血脂以及肝脏中脂质含量...     

Deletion of Thioredoxin-interacting protein ameliorates high fat diet-induced non-alcoholic steatohepatitis through modulation of Toll-like receptor 2-NLRP3-inflammasome axis: Histological and immunohistochemical study

Endemic prevalence of obesity is associated with alarming increases in non-alcoholic steatohepatitis (NASH) with limited available therapeutics. Toll-like receptor2 (TLR2) and Nod-like receptor protein 3 (NLRP3) Inflammasome are implicated in hepatic steatosis, inflammation and fibrosis; the histological landmark stages of NASH. TXNIP, a member of α-arrestin family activates NLRP3 in response to various danger stimuli. The aim of current work was to investigate the effect of TXNIP genetic deletion on histological manifestations of high fat diet-induced steatohepatitis and activation of TLR2-NLRP3-inflammasome axis. Wild-type mice (WT) and TXNIP knock out (TKO) littermates were randomized to normal diet (WT-ND and TKO-ND) or high fat diet (HFD, 60% fat) (WT-HFD and TKO-HFD). After 8-weeks, liver samples from all groups were evaluated by histological, immunohistochemical and western blot analysis. HFD resulted in significant induction of micro and macrovesicular hepatic steatosis, that was associated with increased inflammatory immune cell infiltration in WT-HFD compared with WT-ND and TKO-ND controls, but not in TKO-HFD group. In parallel, WT-HFD group showed significant fibrosis and α-SMA expression; a marker of pro-fibrotic stellate-cell activation, in areas surrounding the central vein and portal circulation, versus all other groups. Western blot revealed increased activation of TLR2-NLRP3 inflammasome pathway and downstream IL-1β and TNFα in WT-HFD group, but not in TKO-HFD group. IL-1β expression coincided within the same areas of steatosis, inflammatory cell infiltration, collagen deposition and α-SMA expression in WT-HFD mice, that was significantly reduced in TKO-HFD mice. In conclusion, TXNIP deletion ameliorates the HFD-induced steatosis, inflammatory and fibrotic response via modulation of TLR2-NLRP3 inflammasome axis. Targeting TXNIP-TLR2-NLRP3 pathway may provide potential therapeutic modalities for NASH treatment.     

动脉粥样硬化小鼠肝脏脂质代谢相关的PPAR-γ/LXR-α/ABCG1通路及炎症因子的变化和化瘀祛痰方在其中的作用

目的:观察动脉粥样硬化(AS)小鼠肝脏脂质代谢相关的过氧化物酶体增殖物激活受体γ(PPAR-γ)/肝X受体α(LXR-α)/ATP结合盒转运体G1(ABCG1)通路和炎症因子的变化,以及化瘀祛痰方在其中的作用,探讨化瘀祛痰方对肝脏脂质代谢及炎症反应的影响及作用机制。方法:将24只ApoE-/-小鼠随机分为模型组、化瘀祛痰方组和辛伐他汀组,8只C57BL/6J小鼠作为正常对照组。除正常对照组给予基础饲料外,其余各组给予高脂饲料。造模12周后,灌胃给药,化瘀祛痰方组与辛伐他汀组给予相应药物,正常对照组与模型组给予等体积的生理盐水。8周后用全自动生物化学分析仪检测血清甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)的含量;HE和油红O染色观察肝脏组织病理及脂质的变化情况;ELISA法检测肝脏游离脂肪酸(FFA)、TG、肿瘤坏死因子α(TNF-α)、Toll样受体4(TLR4)和白细胞介素1β(IL-1β)的含量;Western blot法检测PPAR-γ、LXR-α和ABCG1的蛋白表达。结果:与正常对照组比较,模型组小鼠血清TC、TG和LDL-C含量显著上升(P<0.01),HDL-C含量显著降低(P<0.01);肝脏脂肪变性,细胞变大,脂质沉积明显,FFA和TG含量显著上升(P<0.01),炎症因子TLR4、TNF-α和IL-1β的含量显著增加(P<0.01),脂质代谢通路相关因子PPAR-γ、LXR-α和ABCG1的蛋白表达显著降低(P<0.05或P<0.01)。与模型组比较,服用辛伐他汀和化瘀祛痰方可使小鼠血清TC、TG和LDL-C含量显著下降(P<0.05或P<0.01),HDL-C含量显著上升(P<0.01);肝脏脂肪变性减轻,脂质沉积不同程度降低,FFA和TG含量显著下降(P<0.05或P<0.01),TLR4、TNF-α和IL-1β含量显著降低(P<0.01),PPAR-γ、LXR-α和ABCG1的蛋白表达显著增高(P<0.05或P<0.01)。结论:化瘀祛痰方可能通过调控肝脏PPAR-γ/LXR-α/ABCG1通路及减弱肝脏TRL4介导的炎症反应来达到抗AS的作用。     

α,β-Amyrin prevents steatosis and insulin resistance in a high-fat diet-induced mouse model of NAFLD via the AMPK-mTORC1-SREBP1 signaling mechanism

Nonalcoholic fatty liver disease (NAFLD), characterized by hepatosteatosis and steatohepatitis, is intrinsically related to obesity. Our previous study reported on the anti-obese activity of α,β-amyrin (AMY), a pentacyclic triterpene isolated from Protium heptaphyllum. This study investigated its ability to prevent fatty liver and the underlying mechanism using the mouse model of NAFLD. NAFLD was induced in male Swiss mice fed a high fat diet (HFD) for 15 weeks. The controls were fed a normal chow diet (ND). The mice were simultaneously treated with AMY at 10 and 20 mg/kg or fenofibrate at 50 mg/kg. Lipid levels along with metabolic and inflammatory parameters were assessed in liver and serum. The liver sections were histologically examined using H&E staining. RT-qPCR and western blotting assays were performed to analyze signaling mechanisms. Mice fed HFD developed severe hepatic steatosis with elevated triglycerides and lipid droplets compared with ND controls. This was associated with a decrease in AMP-activated protein kinase (AMPK) activity, an increase of mechanistic target of rapamycin complex 1 (mTORC1) signaling, and enhanced sterol regulatory element binding protein 1 (SREBP1) expression, which have roles in lipogenesis, inhibition of lipolysis, and inflammatory response. AMY treatment reversed these signaling activities and decreased the severity of hepatic steatosis and inflammatory response, evidenced by serum and liver parameters as well as histological findings. AMY-induced reduction in hepatic steatosis seemed to involve AMPK-mTORC1-SREBP1 signaling pathways, which supported its beneficial role in the prevention and treatment of NAFLD.     

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.     

Probiotic antigens stimulate hepatic natural killer T cells

Increasing evidence suggests that gut flora play an important role in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). Our previous studies show that hepatic natural killer T (NKT) cells play a significant role in the pathogenesis of NAFLD. In this study, we explore the mechanism by which modification of gut flora leads to the alteration of hepatic NKT cells and improvement of steatosis. Mice were fed a high‐fat (HF) diet to induce NAFLD. Some of them also received different doses of mixed‐strain probiotics (VSL#3); single‐strain probiotic (Bifidobacterium infantis) or antibiotics. Animal weight, glucose tolerance, liver steatosis and hepatic NKT cells were assessed. Lipid extracts from probiotics were tested for their ability to activate NKT cells. Toll‐like receptor 4 (TLR4) knockout mice were also evaluated for their responses to HF diet. High‐dose VSL#3 was more effective than low‐dose VSL#3 and B. infantis for the improvement of hepatic NKT cell depletion and steatosis. The lipids extracted from VSL#3 stimulated NKT cells both in vivo and in vitro. In contrast, lipids from B. infantis decreased α‐GalCer‐mediated NKT cell activation in vitro, but were able to stimulate NKT cells. TLR4 knockout mice have a similar response to HF‐diet‐induced NKT cell depletion and obesity. These results suggest that alterations in the gut flora have profound effects on hepatic NKT cells and steatosis, which are both strain‐specific and dose‐dependent, but not through TLR4 signalling. Furthermore, these data suggest that probiotics may contain bacterial glycolipid antigens that directly modulate the effector functions of hepatic NKT cells.     

Dysregulation of hepatic cAMP levels via altered Pde4b expression plays a critical role in alcohol‐induced steatosis

Alcohol‐induced hepatic steatosis is a significant risk factor for progressive liver disease. Cyclic adenosine monophosphate (cAMP) signalling has been shown to significantly regulate lipid metabolism; however, the role of altered cAMP homeostasis in alcohol‐mediated hepatic steatosis has never been studied. Our previous work demonstrated that increased expression of hepatic phosphodiesterase 4 (Pde4), which specifically hydrolyses and decreases cAMP levels, plays a pathogenic role in the development of liver inflammation/injury. The aim of this study was to examine the role of PDE4 in alcohol‐induced hepatic steatosis. C57BL/6 wild‐type and Pde4b knockout (Pde4b^?/?) mice were pair‐fed control or ethanol liquid diets. One group of wild‐type mice received rolipram, a PDE4‐specific inhibitor, during alcohol feeding. We demonstrate for the first time that an early increase in PDE4 enzyme expression and a resultant decrease in hepatic cAMP levels are associated with the significant reduction in carnitine palmitoyltransferase 1A (Cpt1a) expression. Notably, alcohol‐fed (AF) Pde4b^?/? mice and AF wild‐type mice treated with rolipram had significantly lower hepatic free fatty acid content compared with AF wild‐type mice. Importantly, PDE4 inhibition in alcohol‐fed mice prevented the decrease in hepatic Cpt1a expression via the Pparα/Sirt1/Pgc1α pathway. These results demonstrate that the alcohol‐ induced increase in hepatic Pde4, specifically Pde4b expression, and compromised cAMP signalling predispose the liver to impaired fatty acid oxidation and the development of steatosis. Moreover, these data also suggest that hepatic PDE4 may be a clinically relevant therapeutic target for the treatment of alcohol‐induced hepatic steatosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Hepatic steatosis causes induction of the chemokine RANTES in the absence of significant hepatic inflammation

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to cirrhosis. Hepatocellular lipid accumulation is a hallmark of both nonalcoholic steatosis and steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine CCL5/RANTES plays an important role in the progression of hepatic inflammation and fibrosis. We here aimed to investigate its expression in NAFLD. Incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation, and corresponding dose-dependent RANTES induction in vitro. Furthermore, we observed significantly elevated hepatic RANTES expression in a dietary model of NAFLD, in which mice were fed a high-fat diet for 12 weeks. This diet induced significant hepatic steatosis but only minimal inflammation. In contrast to the liver, RANTES expression was not induced in visceral adipose tissue of the group fed with high-fat diet. Finally, RANTES serum levels were elevated in patients with ultrasound-diagnosed NAFLD. In conclusion, our data indicate hepatocytes as cellular source of elevated hepatic as well as circulating RANTES levels in response to hepatic steatosis. Noteworthy, upregulation of RANTES in response to lipid accumulation occurs in the absence of relevant inflammation, which further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.     

Protease-activated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western diet

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet-induced NAFLD has not been investigated. Using an established mouse model of Western diet-induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell-derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor-deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet.     

Effect of Monascus purpureus-fermented rice on lipidemia and fatty liver in quail

Monascus purpureus-fermented rice (MR), a preparation which has been shown to be hypolipidemic and antiatherogenic in rabbits and hamsters was fed to quail maintained on a high fat diet to determine if it could influence lipidemia and hepatic steatosis. MR was fed at two levels (0.8 or 1.6 g/kg/d), and compared with a lipoptropic preparation (dongbaogantai 0.6 g/kg/d) and an established hypolipidemic compound (lovastatin 6 mg/kg/d). All the test compounds lowered serum lipids and liver cholesterol levels. Dongbaogantai inhibited hepatic steatosis to the greatest extent (78%), lovastatin inhibited steatosis by 29% and the low and high doses of MR by 25 and 43%, respectively. These serum cholesterol lowering agents have been shown to reduce hepatic steatosis induced by dietary means.     

The differential effects of low birth weight and Western diet consumption upon early life hepatic fibrosis development in guinea pig

Key points

• Postnatal intake of a high saturated fat/high sugar diet, the Western diet (WD), is a risk factor for liver fibrosis. Recently, adverse in utero conditions resulting in low birth weight (LBW) have also been associated with postnatal fibrosis development.
• We demonstrate that suboptimal in utero conditions resulting in LBW are associated with changes in hepatic profibrotic genes in conjunction with minimal liver fibrosis in young non‐overweight adult guinea pigs.
• Our results also indicate that WD promotes liver steatosis, enhanced expression of hepatic genes and proteins of the proinflammatory, profibrotic, cell death and collagen deposition pathways in conjunction with mild hepatic fibrosis.
• Our data highlight that pathways responsible for the initiation of a profibrotic state and ultimately hepatic fibrosis appear different depending upon the insult, an in utero‐induced LBW outcome or a postnatal WD exposure.

Abstract

Postnatal intake of an energy dense diet, the Western diet (WD), is a strong risk factor for liver fibrosis. Recently, adverse in utero conditions resulting in low birth weight (LBW) have also been associated with postnatal fibrosis development. We assessed the independent and possible synergistic effects of placental insufficiency‐induced LBW and postnatal WD consumption on liver fibrosis in early adulthood, with a specific focus on changes in inflammation and apoptosis pathways in association with fibrogenesis. Male LBW (uterine artery ablation) and normal birth weight (NBW) guinea pig pups were fed either a control diet (CD) or WD from weaning to 150 days. Significant steatosis, mild lobular inflammation, apoptosis and mild stage 1 fibrosis (perisinusoidal or portal) were evident in WD‐fed offspring (NBW/WD and LBW/WD). In LBW/CD versus NBW/CD offspring, increased transforming growth factor‐beta 1 and matrix metallopeptidase mRNA and sma‐ and Mad‐related protein 4 (SMAD4) were present in conjunction with minimal stage 1 portal fibrosis. Further, connective tissue growth factor mRNA was increased and miR‐146a expression decreased in LBW offspring, irrespective of diet. Independent of birth weight, WD‐fed offspring exhibited increased expression of fibrotic genes as well as elevated inflammatory and apoptotic markers. Moreover, the augmented expression of collagen, type III, alpha 1 and tumor necrosis factor‐alpha was associated with increased recruitment of RNA polymerase II and enhanced histone acetylation (K9) to their respective promoters. These data support a role for both LBW and postnatal WD as factors contributing to hepatic fibrosis development in offspring through distinct pathways.     

Development and functional consequences of LPS tolerance in sinusoidal endothelial cells of the liver

Kupffer cells and liver sinusoidal endothelial cells (LSEC) clear portal venous blood from gut-derived bacterial degradation products such as lipopolysaccharide (LPS) without inducing a local inflammatory reaction. LPS tolerance was reported for Kupffer cells, but little is known whether sensitivity of LSEC toward LPS is dynamically regulated. Here, we demonstrate that LSEC react to LPS directly as a function of constitutive Toll-like receptor 4 (TLR4)/CD14 expression but gain a LPS-refractory state upon repetitive stimulation without loss of scavenger activity. LPS tolerance in LSEC is characterized by reduced nuclear localization of nuclear factor-kappaB upon LPS rechallenge. In contrast to monocytes, however, TLR4 surface expression of LSEC is not altered by LPS stimulation and thus does not account for LPS tolerance. Mechanistically, LPS tolerance in LSEC is linked to prostanoid production and may account for cross-tolerance of LPS-treated LSEC to interferon-gamma stimulation. Functionally, LPS tolerance in LSEC results in reduced leukocyte adhesion following LPS rechallenge as a consequence of decreased CD54 surface expression. Furthermore, LPS tolerance is operative in vivo, as we observed by intravital microscopy-reduced leukocyte adhesion to LSEC and improved sinusoidal microcirculation in the liver after repetitive LPS challenges. Our results support the notion that LPS tolerance in organ-resident scavenger LSEC contributes to local hepatic control of inflammation.     

Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease

Mechanisms associated with the progression of simple steatosis to non-alcoholic fatty liver disease (NAFLD) remain undefined. Regulatory T cells (T(regs)) play a critical role in regulating inflammatory processes in non-alcoholic steatohepatitis (NASH) and because T helper type 17 (Th17) functionally oppose T(reg)-mediated responses, this study focused on characterizing the role of Th17 cells using a NAFLD mouse model. C57BL/6 mice were fed either a normal diet (ND) or high fat (HF) diet for 8 weeks. Mice in the HF group had a significantly higher frequency of liver Th17 cells compared to ND-fed mice. Neutralization of interleukin (IL)-17 in HF mice ameliorated lipopolysaccharide (LPS)-induced liver injury reflected by decreased serum alanine aminotransferase (ALT) levels and reduced inflammatory cell infiltrates in the liver. In vitro, HepG2 cells cultured in the presence of free fatty acids (FFA; oleic acid and palmitic acid) for 24 h and IL-17 developed steatosis via insulin-signalling pathway interference. IL-17 and FFAs synergized to induce IL-6 production by HepG2 cells and murine primary hepatocytes which, in combination with transforming growth factor (TGF-β), expanded Th17 cells. It is likely that a similar process occurs in NASH patients, as there were significant levels of IL-17(+) cell infiltrates in NASH patient livers. The hepatic expression of Th17 cell-related genes [retinoid-related orphan receptor gamma (ROR)γt, IL-17, IL-21 and IL-23] was also increased significantly in NASH patients compared to healthy controls. Th17 cells and IL-17 were associated with hepatic steatosis and proinflammatory response in NAFLD and facilitated the transition from simple steatosis to steatohepatitis. Strategies designed to alter the balance between Th17 cells and T(regs) should be explored as a means of preventing progression to NASH and advanced liver diseases in NAFLD patients.     

Initiation of alcoholic fatty liver and hepatic inflammation with a specific recall immune response in alcohol-consuming C57Bl/6 mice

Whether immunological responses are involved in initiation and progression of alcoholic liver disease is unclear. We describe a mouse model of alcoholic liver injury characterized by steatosis and hepatic inflammation initiated by a recall immune response. Mice immune to Listeria monocytogenes fed a liquid diet containing ethanol and challenged with viable bacteria developed steatosis within 24 h and, at a later time, elevated serum alanine aminotransferase levels, indicating more liver damage in this group. Listeria antigen also induced steatosis and increased serum alanine aminotransferase levels in immune ethanol-consuming mice. The production of tumour necrosis factor by a recall immune response in this model is a major, but not the only, component in initiation of alcoholic liver disease.     

Intrahepatic insulin resistance in a murine model of steatohepatitis: effect of PPARgamma agonist pioglitazone

Hepatic insulin resistance is associated with hepatic steatosis and is thought to play an important role in the pathogenesis of steatohepatitis. Using a murine model of steatohepatitis (mice fed a diet deficient in methionine and choline-MCD diet), we tested the effects of the insulin-sensitising, PPARgamma agonist drug pioglitazone (PGZ) on systemic and intrahepatic insulin sensitivity and on liver pathology. Compared to controls, mice fed the MCD diet develop a significant steatohepatitis, have enhanced glucose tolerance and enhanced systemic response to insulin. PGZ did not affect the systemic insulin sensitivity in control or MCD-fed mice as assessed in vivo by intraperitoneal glucose or insulin dynamic tests. However, PGZ prevented hepatic fat accumulation and steatohepatitis induced by the MCD diet. This effect was associated with an increased mass of adipose tissue and increased expression and release of adiponectin, while hepatic acyl co-enzyme A oxidase and acyl-co-enzyme A carboxylase, regulating hepatic beta-oxidation of fatty acid, remained unchanged. Steatohepatitis in MCD-diet-fed mice was associated with intrahepatic insulin resistance as shown by a reduced phosphorylation of hepatic insulin receptor, and Akt in response to an insulin stimulus. PGZ to MCD-fed mice restored the activation of the insulin receptor and of the Akt pathway in response to insulin. In conclusion, PGZ alleviates steatosis and steatohepatitis induced by the MCD diet, an effect associated with correction of intrahepatic insulin resistance.     

Local coordination verses systemic disregulation: complexities in leukocyte recruitment revealed by local and systemic activation of TLR4 in vivo

The recruitment of leukocytes to a tissue is a critical step in the inflammatory response. Toll-like receptor 4 (TLR4) is an important receptor involved in the initiation of inflammatory responses. Administration of the ligand for TLR4, lipopolysaccharide, is often used to model inflammation--local responses to stimuli within a specific tissue and systemic responses such as those observed during endotoxic or septic shock. Here, we review work, which demonstrates that in response to local activation of TLR4, highly coordinated and multistep processes are initiated, ultimately resulting in the leukocyte's arrival at the inflamed tissue. In contrast, systemic activation of TLR4 results in nonspecific accumulation of leukocytes within the lung capillaries and liver sinusoids through mechanisms profoundly different than those involved in local tissue recruitment. Contrary to current dogma, leukocyte accumulation in the lung is dependent on endothelial rather than leukocyte activation. Finally, we discuss recent evidence suggesting that activation of leukocytes through TLR4, although still in the circulation, effectively paralyzes inflammatory cells, rendering them incapable of appropriate trafficking to inflamed tissues.     

利胆汤对大鼠急性重症胆管炎所致的肝损伤的作用机制

目的:研究利胆汤对急性重症胆管炎的大鼠所致肝损伤的作用机制.方法:选择健康的Wistar大鼠60只,3月龄,雄性,随机分组为对照组、模型组、胆宁片组、利胆汤低剂量组、中剂量组和高剂量组6组,每组10只.分别于术后立刻、术后6h、术后12h、术后24h4个时间点收集大鼠胆汁,以及检测大鼠胆总管压力;对照组和模型组灌胃给予0.9％的氯化钠;胆宁片组灌胃给予胆宁片0.5 g/(kg.d);各利胆汤剂量组:6,12和24 g/(kg.d),灌胃给药3天后处死大鼠.HE染色观察大鼠肝病理形态;ELISA检测的方法大鼠血清中的细胞因子IL-6,TNF-α水平;Q-PCR法检测大鼠肝脏巨噬细胞移动抑制因子(migration inhibitory factor,MIF)和Toll样受体4(Toll-like receptor 4,TLR4)基因的表达.结果:利胆汤能明显的降低血清细胞因子IL-6(P＜0.05)、TNF-α的表达(P＜0.0l),并且明显抑制MIF和TLR4的表达(P＜0.01).结论:利胆汤能够改善大鼠急性重症胆管炎所致的肝损伤,作用机制可能通过抑制MIF和TLR4的表达和下调IL-6,TNF-α的表达水平实现的.     

Therapeutic Administration of the Direct Thrombin Inhibitor Argatroban Reduces Hepatic Inflammation in Mice with Established Fatty Liver Disease

Thrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor–deficient LDLr^−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet.More than 70% of patients with abdominal obesity develop concurrent nonalcoholic fatty liver disease (NAFLD).¹ NAFLD, the hepatic manifestation of metabolic syndrome, is characterized by excess accumulation of lipids in the liver (ie, hepatic steatosis)^2,3 and affects approximately 25% of the Western population.⁴ Steatosis accompanied by marked histological inflammation is termed nonalcoholic steatohepatitis (NASH), which is the most severe form of NAFLD and a major cause of liver fibrosis and cirrhosis.^5,6 Progression from simple steatosis to NASH is indicative of a poor clinical outcome and currently has no effective pharmacological treatment options. In addition, both obesity and NAFLD are associated with an increased risk of developing type 2 diabetes mellitus⁷ and cardiovascular disease.^8,9 Therefore, there is an immediate need to identify novel pharmacological approaches to treat NAFLD.A significant commonality among obesity-related diseases is inflammation. Obesity and hepatic steatosis are associated with increased expression of many inflammatory mediators in the liver.¹⁰ The expression of several of these mediators, particularly those involved in leukocyte recruitment, is further increased in patients with NASH.¹⁰ Several compelling studies have demonstrated that inflammatory chemokines such as monocyte chemoattractant protein-1 (MCP-1) and the subsequent recruitment and activation of hepatic macrophages (ie, Kupffer cells) are essential components of NAFLD pathogenesis.^11–14 A systemic proinflammatory state, driven in part by hepatic inflammation, is associated with an increased risk of type 2 diabetes^15,16 and adverse cardiovascular outcomes.¹⁷ In particular, systemic levels of high sensitivity C-reactive protein (hs-CRP), a biomarker of risk for acute cardiovascular events,¹⁸ are primarily dictated by the proinflammatory environment in the liver. Indeed, hs-CRP levels are independently associated with hepatic steatosis in patients with metabolic syndrome.⁸ These studies indicate that increased hepatic inflammation is a focal point of multiple diseases stemming from the metabolic syndrome. Of importance, the molecular triggers of hepatic inflammation in metabolic diseases such as obesity are not completely understood. To this end, understanding the cellular and molecular pathways coordinating hepatic inflammation in metabolic disease could lead to the development of clinical therapies that target inflammation as an underlying cause of multiple interrelated diseases.Because the liver is the primary site of coagulation factor synthesis, liver diseases are often accompanied by a rebalancing of the hemostatic profile.¹⁹ Indeed, abdominal obesity, metabolic syndrome, and NAFLD are each associated with activation of the blood coagulation cascade, including increased generation of the serine protease thrombin.^20–23 Moreover, thrombin generation is increased in mouse models of diet-induced obesity and hypercholesterolemia.^24,25 Previous studies have shown that the induction of tissue factor on monocytes is essential for thrombin generation in mice fed a Western diet.²⁶ Various hepatic manifestations of diet-induced obesity, including hepatic steatosis, are reduced in tissue factor–deficient mice.²⁴ Moreover, we found previously that mice lacking a thrombin receptor, protease activated receptor-1 (PAR-1), did not develop hepatic steatosis when fed a Western diet.²⁴ Although compelling, these genetic approaches do not directly address the question of whether intervention with pharmacological agents, perhaps anticoagulants, can reduce established liver disease. Indeed, it is currently unclear whether pharmacological inhibition of thrombin alters the course of established diet-induced fatty liver disease in mice.To this end, we tested the hypothesis that pharmacological inhibition of thrombin could therapeutically reverse diet-induced hepatic inflammation and steatosis in hypercholesterolemic low density lipoprotein receptor–deficient (LDLr^−/−) mice.     

Hepatic expression of toll-like receptor 4 in primary biliary cirrhosis

Toll-like receptor 4 (TLR4) is a receptor for bacterial lipopolysaccharide, which is suggested to be involved in the pathogenesis of disease of hepatobiliary tracts. To explore a possible role for this receptor in primary biliary cirrhosis (PBC), we investigated the expression of TLR4 in liver tissues from PBC patients. We studied liver biopsy sections from 62 PBC patients and 41 patients with chronic hepatitis C (CHC). Expression of TLR4 in paraffin-embedded sections was analyzed by immunohistochemistry. The bile duct epithelial cells (BECs) of PBC liver tissues markedly expressed TLR4, whereas BECs of CHC liver tissues barely expressed TLR4. The TLR4 expression was also observed in periportal hepatocytes of PBC liver tissues and its expression was extended to interlobular hepatocytes in advanced stage PBC. Although periportal hepatocytes of CHC liver tissues expressed TLR4, its expression levels were not correlated with the fibrosis stage. Our data demonstrated that TLR4 was expressed in BECs and periportal hepatocytes in PBC livers, suggesting the possible involvement of bacterial pathogens and TLR4 in the inflammatory processes of PBC.