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.     

Effect of chronic psychosocial stress on nonalcoholic steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which may progress towards inflammation (nonalcoholic steatohepatitis (NASH)). NAFLD is regarded as a consequence of a sedentary, food-abundant lifestyle which, in the modern world, often coincides with chronically high levels of perceived psychosocial stress. Here, we aimed to characterize the effect of chronic psychosocial stress on the development of NAFLD/NASH in male mice either fed with standard chow or NASH-inducing high fat diet. Chronic psychosocial stress was induced by chronic subordinate colony housing (CSC), a pre-clinically validated paradigm relevant for human affective and somatic disorders. Single housed (SHC) mice served as controls. Under standard chow conditions CSC mice revealed lower hepatic triglyceride levels but higher hepatic TNFα, MCP-1 and HMOX mRNA expression, while serum transaminase levels did not significantly differ from SHC mice. Under the NASH-inducing high-fat diet CSC and SHC mice showed similar body weight-gain and serum levels of glucose and adiponectin. Moreover, liver histology as well as TNFα, MCP-1 and HMOX expression were similar in CSC and SHC mice fed with HFD. Surprisingly, CSC showed even significantly lower transaminase levels than SHC mice fed with the same NASH-inducing diet. Together, these data indicate that under normal dietary conditions the CSC model induces noticeable hepatic oxidative stress and inflammation without causing manifest hepatocellular injury. In contrast, CSC exhibited a protective effect on hepatocellular injury in a dietary NASH-model. Identification of the underlying mechanisms of this phenomenon may lead to novel therapeutic strategies to prevent progression of NAFLD.     

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.     

A model of insulin resistance and nonalcoholic steatohepatitis in rats: role of peroxisome proliferator-activated receptor-alpha and n-3 polyunsaturated fatty acid treatment on liver injury

Insulin resistance induces nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH). We used a high-fat, high-calorie solid diet (HFD) to create a model of insulin resistance and NASH in nongenetically modified rats and to study the relationship between visceral adipose tissue and liver. Obesity and insulin resistance occurred in HFD rats, accompanied by a progressive increase in visceral adipose tissue tumor necrosis factor (TNF)-alpha mRNA and in circulating free fatty acids. HFD also decreased adiponectin mRNA and peroxisome proliferator-activated receptor (PPAR)-alpha expression in the visceral adipose tissue and the liver, respectively, and induced hepatic insulin resistance through TNF-alpha-mediated c-Jun N-terminal kinase (JNK)-dependent insulin receptor substrate-1Ser307 phosphorylation. These modifications lead to hepatic steatosis accompanied by oxidative stress phenomena, necroinflammation, and hepatocyte apoptosis at 4 weeks and by pericentral fibrosis at 6 months. Supplementation of n-3 polyunsaturated fatty acid, a PPARalpha ligand, to HFD-treated animals restored hepatic adiponectin and PPARalpha expression, reduced TNF-alpha hepatic levels, and ameliorated fatty liver and the degree of liver injury. Thus, our model mimics the most common features of NASH in humans and provides an ideal tool to study the role of individual pathogenetic events (as for PPARalpha down-regulation) and to define any future experimental therapy, such as n-3 polyunsaturated fatty acid, which ameliorated the degree of liver injury.     

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.     

Induction of macrophage scavenger receptor MARCO in nonalcoholic steatohepatitis indicates possible involvement of endotoxin in its pathogenic process

Nonalcoholic steatohepatitis (NASH) is one of the life-threatening hepatic diseases; however, its pathogenesis is still unknown. To evaluate the causative role of hyperlipidaemia and high-fat diet, we compared C57BL/6 mice with inherited hyperlipidaemic model mice (LDLR(-/-)mice and ApoE(-/-) mice) fed a normal or a high-fat diet. LDLR(-/-) and ApoE(-/-) mice fed the normal diet showed significantly higher serum cholesterol level than that of C57BL/6 mice fed the high-fat diet. These mice, however, have shown neither significant elevation of serum alanine transaminase (ALT) level nor histopathologic features of steatohepatitis. High-fat diet groups of all three strains showed histopathological characteristics of steatohepatitis with elevated serum ALT levels and high expression of macrophage scavenger receptor MARCO mRNA in the liver. Semiquantitative endotoxin analysis showed an elevated serum endotoxin level in the portal vein but not in the vena cava in ApoE(-/-) mice fed the high-fat diet. These results indicate that long-term feeding of a high-fat diet induces NASH, whereas hyperlipidaemia alone is not enough to induce NASH. Liver-restricted induction of MARCO in mice with high-fat diet and portal endotoxaemia in ApoE(-/-) mice fed the high-fat diet suggest the possible involvement of endotoxin in the pathogenesis of NASH.     

甘氨酸对小鼠体重及脂质代谢的影响

目的： 观察甘氨酸（Gly）对正常饮食和高脂饮食小鼠体重及血脂代谢的影响。 方法： 健康成年昆明种小鼠80只,雌雄各半，随机分4组：对照组、高脂饮食组（HF）、Gly组、Gly＋HF组，按各组要求分别给予正常饲料和高脂饲料喂养。以Gly 等药物定量腹腔注射处理。连续12 d，观察体重变化，实验末取血测定血脂4项（TC、TG、HDL-C、LDL），摘取雄性小鼠附睾周围脂肪垫和肾周脂肪垫，摘取雌性鼠腹部脂肪垫和肾周脂肪垫，称重并记录，留取肝脏组织并观察病理变化。 结果： HF组体重明显低于对照组，体脂含量无明显差异；Gly组的体重和体脂含量均明显低于对照组； Gly＋HF组体重和体脂含量均明显低于HF组； Gly组血清TC和TG的含量显著低于对照组； Gly＋HF组血脂4项与HF组相比没有显著差异；光镜下HF组肝组织可见弥漫性脂肪变性，Gly＋HF组肝组织脂肪变性少，肝细胞内脂滴小且少。 结论： Gly对正常饮食和高脂饮食的小鼠都有降低体重和体脂含量的作用，并能降低血脂，有预防肝脂肪变的作用。     

Manganese superoxide dismutase is reduced in the liver of male but not female humans and rodents with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is among the most common liver diseases. Oxidative stress is one of the pathogenic mechanisms contributing to the progression of simple fatty liver to non-alcoholic steatohepatitis (NASH). Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidative enzyme and here its expression in rodent and human NAFLD has been analyzed. MnSOD is found reduced in the liver of male mice fed a high fat diet and male ob/ob mice. Female mice fed an atherogenic diet to induce NASH have MnSOD protein levels comparable to controls. In a cohort of 30 controls, 41 patients with fatty liver and 39 NASH patients, MnSOD mRNA is significantly lower in the steatotic and NASH liver. When analyzed in both genders separately reduction of MnSOD expression is only found in males. Here, MnSOD mRNA negatively correlates with steatosis grade but not with extent of fibrosis or inflammation. MnSOD is, however, not reduced in primary human hepatocytes (PHH) treated with palmitate or oleate to increase cellular triglycerides. Lipopolysaccharide, TNF, IL-6, TGFβ and leptin which are all raised in NAFLD do not affect MnSOD in PHH. Adiponectin which attenuates oxidative stress partly by increasing MnSOD in macrophages does not induce MnSOD in PHH. In summary, current data show that hepatic MnSOD is reduced in male but not female humans and rodents with NAFLD.     

Role of AMP-activated protein kinase and adiponectin during development of hepatic steatosis in high-fat diet-induced obesity in rats

Obesity, an abnormal condition of adipose tissue, has recently been recognized as a major cause of metabolic syndromes, especially non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to examine the possible involvement of adipokines in the development of fatty liver. Sprague-Dawley (SD) rats fed a high-fat (HF) diet for 15 weeks developed increased hepatocellular vacuolation, hepatic triglyceride (TG) content and serum TG, total cholesterol and free fatty acid levels, with increases in adipose tissue mass. The serum concentration of adiponectin decreased slightly in these animals. Western blotting analysis demonstrated a decrease in the levels of AMP-activated protein kinase (AMPK) and phosphorylated-AMPK in the livers of these rats. These results indicate similarities between the diet-induced obesity rat model of NAFLD and human NAFLD, thus making the rat a useful model for the further study of NAFLD, including the interactions between adipokines and hepatic fat metabolism.     

Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis

Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the "two hit" hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor-deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies.     

High-Carbohydrate Diet Selectively Induces Tumor Necrosis Factor-α Production in Mice Liver

Obesity may represent a state of chronic low-grade inflammation associated with infiltration of adipose tissue by inflammatory cells. Tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1/JE), two important inflammatory cytokines, have been shown to be regulated according to changes in body adiposity. In this study on Swiss mice, we compared the influences of long-term high-carbohydrate (HC) or high-fat (HF) diet on adiposity, glucose tolerance, and secretion of TNF-α and MCP-1/JE by adipose tissue and liver. For 8 weeks, male Swiss mice (7–8 weeks) were fed either standard laboratory rodent diet (control group), HC diet (64% carbohydrate, 19% protein, and 11% fat), or HF diet (45% carbohydrate, 17% protein, and 38% fat), with the latter two diets having no fiber. Oral glucose tolerance test, triacylglycerol (TAG) plasma concentration, and systemic or tissue levels of the two proinflammatory cytokines were determined. Body weight increased by approximately 20% in mice fed the experimental diets compared with mice fed the control diet. Systemically, the hypercaloric diets induced hyperglycemia with impairment in glucose tolerance, elevated circulating TAG levels, and increased plasma concentrations of TNF-α and MCP-1/JE. In the target organs (adipose tissue and liver), both diets increased MCP-1/JE levels. However, the HC diet, but not the HF diet, was able to increase TNF-α concentration in the liver. These results have shown that the nature of nutrients influences the type of proinflammatory cytokines in target organs and may contribute to the comorbidities of obesity.     

Serum lipocalin-2 is a potential biomarker for the clinical diagnosis of nonalcoholic steatohepatitis

Background/AimsNonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis. We aimed to investigate the usefulness of a key biomarker, lipocalin-2 (LCN2), for the detection of NASH progression.MethodsA mouse NASH model was established using a high-fat diet and a high-sugar drinking water. Gene expression profile of the NASH model was analyzed using RNA sequencing. Moreover, 360 NAFLD patients (steatosis, 83; NASH, 277), 40 healthy individuals, and 87 patients with alcoholic fatty liver disease were recruited.ResultsInflammatory infiltration, focal necrosis in the leaflets, steatosis, and fibrosis were documented in the mouse liver. In total, 504 genes were differentially expressed in the livers of NASH mice, and showed significant functional enrichment in the inflammation-related category. Upregulated liver LCN2 was found to be significantly interactive with various interleukins and toll-like receptors. Serum LCN2 levels were significantly increased in NAFLD patients. Serum LCN2 levels were correlated with steatosis, intralobular inflammation, semiquantitative fibrosis score, and nonalcoholic fatty liver disease activity score. The area under the curve of serum LCN2 was 0.987 with a specificity of 100% and a sensitivity of 93.5% for NASH diagnosis, and 0.977 with almost the same specificity and sensitivity for steatosis.ConclusionsLCN2 might be involved in the transition from NAFL to NASH by mediating inflammation. Serum LCN2 levels might be a novel biomarker for the diagnosis of NASH.     

紫薯花色苷提取物对ApoE~(-/-)小鼠动脉粥样硬化和高脂血症的作用

目的:研究紫薯花色苷提取物对Apo E-/-小鼠动脉粥样硬化和高脂血症的作用。方法:将8周龄雄性ApoE~(-/-)小鼠随机分为对照组、高脂饮食组、花色苷低剂量组、花色苷高剂量组和立普妥组,共5组(n=10),除对照组小鼠饲喂普通饲料外,其它4组在饲喂高脂饲料8周后,再给予蒸馏水、紫薯花色苷提取物(1. 67 mg·kg~(-1)·d~(-1)和8. 35 mg·kg~(-1)·d~(-1))或立普妥(阿托伐他汀钙,2 mg·kg~(-1)·d~(-1))灌胃8周。心脏取血检测小鼠血清的总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C)水平;油红O和HE染色评估主动脉根部斑块面积和肝脏的脂肪变性程度; RT-q PCR方法检测主动脉和肝脏中炎症因子的表达。结果:与对照组比较,高脂饮食组小鼠血清的TC、LDL-C和HDL-C水平明显升高(P 0. 01),动脉的斑块面积明显增大(P 0. 01),肝脏的脂肪变性明显,白细胞介素1β(IL~(-1)β)、IL-6和单核细胞趋化蛋白1(MCP~(-1))的表达增加(P 0. 01),而过氧化物酶体增殖物激活受体α(PPAR-α)的表达降低(P 0. 05);高剂量紫薯花色苷提取物可以明显降低TC、TG和LDL-C水平(P 0. 01),减少动脉的斑块面积(P 0. 01),减轻肝脏的脂肪变性程度,同时还可以降低IL~(-1)β、IL-6和MCP~(-1)的表达(P 0. 05),提高PPAR-α的表达(P 0. 05)。结论:紫薯花色苷提取物可调节血脂,降低炎症细胞因子表达,从而抑制动脉粥样硬化的发展和高脂血症引起的肝组织脂肪变性。     

活血降脂方对小鼠脂肪肝的防治作用

目的:探讨活血降脂方对小鼠脂肪肝的防治作用及机制。方法:高脂饲料喂养小鼠,分别用不同剂量的活血降脂方(由人参、三七、天麻组成,命名为GST)给小鼠灌胃2周,检测血脂、肝组织甘油三酯(TG)含量,并观察肝指数和肝脏病理变化,筛选出药物的最佳用药剂量。此外,小鼠分为正常对照(NC)组,喂基础饲料;模型组喂高脂饲料。12周后将模型小鼠随机分为高脂(HF)组,正常饮食(ND)组和GST组。除HF组饲高脂饲料外,其余各组饲基础饲料;GST组给予GST灌胃2周,其余各组以同等容积蒸馏水灌胃。检测血清总胆固醇(TC)、TG、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)及肝组织TC、TG含量,观察肝指数、肝组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,肝组织病理变化及过氧化物酶体增殖物激活受体α(PPARα)、细胞色素P450 2E1(CYP2E1)的mRNA表达。结果:GST可显著降低血脂、肝脂和MDA水平,增加SOD活性,明显降低肝指数并改善肝组织脂肪变性,增加肝组织PPARαmRNA表达、抑制CYP2E1 mRNA的表达。结论:GST具有有效防治脂肪肝的作用,其机制可能与上调肝组织PPARαmRNA的表达、降低血清和肝组织TG含量、下调CYP2E1 mRNA的表达以及抗脂质过氧化反应有关。     

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.     

A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver pathologies and is associated with obesity and the metabolic syndrome. Here, we investigated the molecular mechanisms by which a novel cystine based glutathione precursor with added selenomethionine (F1) prevents hepatic steatosis in a moderate high fat dietary model of NAFLD. Adult (8 weeks old), male apolipoprotein E (ApoE)−/− mice were fed with a normal diet (ND) or high fat diet (HFD), consisting of 21% fat and 0.21% cholesterol, with or without dietary supplementation of F1 (3 g/kg food) for 16 weeks. Compared with ApoE−/− mice fed with ND with or without F1, ApoE−/− mice fed with HFD exhibited significant weight gain, hepatomegaly, and increased serum cholesterol and triglycerides levels with no change in serum albumin levels. High resolution light and electron microscopy revealed micro-and macro-vesicular steatosis in ApoE−/− mice fed on a HFD. HFD-induced obesity also led to increased lipogenesis, oxidative stress, activation of c-Jun-NH₂-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), perturbation of the BAX/BCL-2 rheostat, hepatocyte apoptosis, and activation of caspases 9 and 3. F1 fully prevented the adverse effects of HFD on serum triglyceride levels, body and liver weights, and hepatic steatosis and substantially attenuated HFD-induced increase in lipogenesis, oxidative stress, kinase activation, apoptotic signaling, and hepatocyte ultrastructural abnormalities. These results demonstrate that administration of F1, a glutathione precursor, ameliorates HFD-induced hepatic steatosis in ApoE−/− mice and emphasizes the role of oxidative stress in diet-induced obesity and hepatic steatosis.     

FAT/CD36在高脂喂养小鼠脂肪组织炎症中的作用

目的:探讨脂肪酸转运酶/白细胞分化抗原36(fatty acid translocase/CD36,FAT/CD36)在高脂饮食诱导的小鼠脂肪组织炎症中的作用。方法:将6周龄雄性C57BL/6J小鼠分别随机分为普通饮食组和高脂饮食组,喂养14周后,ELISA测定血清游离脂肪酸(FFA)含量,应用荧光实时定量PCR和Western blotting检测脂肪组织中FAT/CD36及炎症/趋化因子(IL-1β、IL-6、TNF-α、MCP-1、MIP-1)mRNA和蛋白的表达,免疫组织化学染色检测脂肪组织巨噬细胞浸润,比较高脂喂养14周的野生型小鼠和CD36基因敲除小鼠的脂肪组织炎症反应情况。结果:与普通饮食组相比,高脂饮食能增强C57BL/6J小鼠脂肪组织的FAT/CD36及炎症/趋化因子的表达,促进巨噬细胞在脂肪组织的浸润。与高脂饮食喂养的野生型小鼠相比,CD36基因敲除小鼠的脂肪组织炎症因子、趋化因子表达明显降低,脂肪组织巨噬细胞浸润减少。结论:高脂饮食通过上调脂肪组织FAT/CD36的表达激活了脂肪组织炎症。     

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.