SD大鼠酒精性脂肪肝模型的监测分析

目的：通过血清生化指标和病理学的监测分析来建立标准的SD大鼠酒精性脂肪肝动物模型。方法：选取40只SD大鼠,随机分为两组,模型组采用直接饮酒法,于第8、12和20周时检测大鼠血清生化指标：丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、甘油三酯（TG）,并于第8、12周时随机采集5只大鼠肝组织,20周时采集剩余所有大鼠肝组织并进行病理学分析。结果：模型组于第8、12和20周时体重增长量均低于对照组（P〈0．01）,血清ALT、AST均高于对照组（P〈0．01）,第8周和12周时TG高于对照组（P〈0．01）。病理学结果显示肝组织从8周至20周呈现出酒精性脂肪肝、重度酒精性脂肪肝伴肝炎和酒精性肝纤维化等演变过程。结论：直接饮酒法可成功地复制出酒精性脂肪肝动物模型,通过监测分析可了解酒精性脂肪肝病变的整个过程,为今后建立酒精性脂肪肝和肝纤维化动物模型提供了理论参考。     

彩超与GGT联合检测对酒精依赖患者酒精肝的诊断价值

目的:研究分析彩超和谷氨酰转肽酶(GGT)联合检测对酒精依赖患者酒精性脂肪肝诊断的临床意义。方法:对2013年5月-2014年4月于我院住院并诊断为酒精依赖的患者39例(研究组)行肝脏彩超及GGT检测,另选取同期来源于本院职工、进修医护人员40例为对照组,对其结果进行分析。结果:研究组血清GGT为(189.95±226.52)U/L,显著高于对照组的(26.85±18.94)U/L,差异有统计学意义(t=4.54,P0.001);研究组中彩超诊断为脂肪肝者的GGT水平与非脂肪肝者有明显差异(P0.05),且高于对照组中的脂肪肝者,差异有统计学意义(P0.05)。结论:对于酒精依赖患者,血清GGT是敏感性较高的检测指标,GGT的检测有利于酒精性疾病的早期发现。彩超与GGT联合检测能提高临床对酒精性脂肪肝的检出率。     

Metabonomic and microbiological analysis of the dynamic effect of vancomycin-induced gut microbiota modification in the mouse

The effects of the antibiotic vancomycin (2 x 100 mg/kg/day) on the gut microbiota of female mice (outbred NMRI strain) were studied, in order to assess the relative contribution of the gut microbiome to host metabolism. The host's metabolic phenotype was characterized using (1)H NMR spectroscopy of urine and fecal extract samples. Time-course changes in the gut microbiotal community after administration of vancomycin were monitored using 16S rRNA gene PCR and denaturing gradient gel electrophoresis (PCR-DGGE) analysis and showed a strong effect on several species, mostly within the Firmicutes. Vancomycin treatment was associated with fecal excretion of uracil, amino acids and short chain fatty acids (SCFAs), highlighting the contribution of the gut microbiota to the production and metabolism of these dietary compounds. Clear differences in gut microbial communities between control and antibiotic-treated mice were observed in the current study. Reduced urinary excretion of gut microbial co-metabolites phenylacetylglycine and hippurate was also observed. Regression of urinary hippurate and phenylacetylglycine concentrations against the fecal metabolite profile showed a strong association between these urinary metabolites and a wide range of fecal metabolites, including amino acids and SCFAs. Fecal choline was inversely correlated with urinary hippurate. Metabolic profiling, coupled with the metagenomic study of this antibiotic model, illustrates the close inter-relationship between the host and microbial "metabotypes", and will provide a basis for further experiments probing the understanding of the microbial-mammalian metabolic axis.     

Assessing the impact of thermal acclimation on physiological condition in the zebrafish model

The zebrafish has become a valuable vertebrate model organism in a wide range of scientific disciplines, but current information concerning the physiological temperature response of adult zebrafish is rather scarce. In this study, zebrafish were experimentally acclimated for 28 days to 18, 26 or 34 °C and a suite of non-invasive and invasive methods was applied to determine the thermal dependence of zebrafish physiological condition. With decreasing temperature, the metabolic rate of zebrafish decreased, as shown by the decreasing oxygen uptake and ammonia excretion rates, limiting the critical swimming speed, probably due to a decreased muscle fibre power output. In response to exercise, fuel stores were mobilized to the liver as shown by the increased hepatosomatic index, liver total absolute energetic value and liver carbohydrate concentration but due to the low metabolic rate they could not be adequately addressed to power swimming activity at 18 °C. Conversely, the increased metabolic performance at high temperature came with an increased metabolic cost resulting in decreased energy status reflected particularly well by the non-invasive condition factor and invasive measures of carcass protein concentration, carcass total absolute energetic value and liver carbohydrate concentration. We showed that the combined measurement of the relative condition factor and critical swimming speed is a powerful non-invasive tool for long-term follow-up studies. Invasive methods were redundant for measuring general energy status but they provided detailed information concerning metabolic reorganization. With this study we proved that the usefulness of the zebrafish as a model organism can easily be expanded to include physiological studies and we provided a reference dataset for the selection of measures of physiological responses for future studies using the zebrafish.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

斑马鱼elovl8缺失对抗冷胁迫下存活率及脂肪代谢的影响

研究利用CRISPR/Cas9技术构建了斑马鱼elovl8a-/-、elovl8b-/-和DKO(elovl8a和elovl8b)敲除模型,通过组织学观察、实时荧光定量PCR和脂肪酸组成分析等实验方法,探究了脂肪酸延长酶8(elovl8)缺失对斑马鱼抗冷胁迫能力的影响.结果表明elovl8缺失导致斑马鱼在低温环境下的存...     

制首乌醇提物及其主要成分大黄素对斑马鱼非酒精性脂肪肝的治疗作用

研究制首乌醇提物及其主要成分大黄素对斑马鱼非酒精性脂肪肝疾病(non-alcoholic fatty liver disease, NAFLD)的治疗作用。选取野生型斑马鱼若干,随机均分为对照组、模型组、制首乌醇提物(RPMP)组和大黄素(emodin)组,于胚胎受精后5天,对照组给于普通饲料AP100,模型组给予2 mg/mL蛋黄粉(EY powder),制首乌醇提物组分别给予2 mg/mL蛋黄粉与低剂量组(1 mg/mL)、中剂量组(1.5 mg/mL)、高剂量组(2 mg/mL),大黄素组分别给予2 mg/mL蛋黄粉与低剂量组(0.25μg/mL)、中剂量组(0.5μg/mL)、高剂量组(1μg/mL)。72 h后观察斑马鱼存活率和体重、体长、BMI的变化■,用整体油红染色评判脂肪肝发生率,石蜡切片观察肝组织病理形态,甘油三酯(TG)、总胆固醇(TC)试剂盒判断斑马鱼体脂含量。制首乌醇提物组与大黄素组能显著降低非酒精性脂肪肝斑马鱼的死亡率、BMI、TG含量,并显著改善斑马鱼幼鱼肝脏脂质沉积情况(P<0.05)。由此可见制首乌醇提物与大黄素可有效治疗斑马鱼NAFLD,且大黄素可能为制首乌治疗NAFLD的主要有效成分。     

Establishment of the Tree Shrew as an Alcohol-Induced Fatty Liver Model for the Study of Alcoholic Liver Diseases

Currently, the pathogenesis of alcoholic liver diseases (ALDs) is not clear. As a result, there is no effective treatment for ALDs. One limitation is the lack of a suitable animal model for use in studying ALDs. The tree shrew is a lower primate animal, characterized by a high-alcohol diet. This work aimed to establish a fatty liver model using tree shrews and to assess the animals’ suitability for the study of ALDs. Tree shrews were treated with alcohol solutions (10% and 20%) for two weeks. Hemophysiology, blood alcohol concentrations (BACs), oxidative stress factors, alcohol metabolic enzymes and hepatic pathology were checked and assayed with an automatic biochemical analyzer, enzyme-linked immunosorbent assay (ELISA), western blot, hematoxylin-eosin (HE) staining and oil red O staining, and magnetic resonance imaging (MRI). Compared with the normal group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), total cholesterol (TC), triglyceride (TG), reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly enhanced in alcohol-treated tree shrews. However, the activity of reduced glutathione hormone (GSH) and superoxide dismutase (SOD) declined. Notable changes in alcohol dehydrogenase(ADH1), aldehyde dehydrogenase(ALDH2), CYP2E1, UDP-glucuronosyl transferase 1A1 (UGT1A1) and nuclear factor erythroid-related factor 2 (Nrf2) were observed. HE and oil red O staining showed that hepatocyte swelling, hydropic degeneration, and adipohepatic syndrome occurred in the tree shrews. Alcohol can induce fatty liver-like pathological changes and result in alterations in liver function, oxidative stress factors, alcohol metabolism enzymes and Nrf2. Therefore, the established fatty liver model of tree shrews induced by alcohol should be a promising tool for the study of ALDs.     

Discovery of serum biomarkers of alcoholic fatty liver in a rodent model: C-reactive protein

Background  

Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model.     

超声在评价大鼠酒精性脂肪肝模型中的应用

目的利用超声技术来评价大鼠酒精性脂肪肝动物模型。方法选取40只SD大鼠随机分为两组（n=20只）。模型组按每周测定的体重早晚各1次乙醇灌胃（10 g/kg）,第1周浓度为40%,第2、3周分别为45%和50%,第4周为55%灌胃直至12周;对照组给予等体积的生理盐水灌胃。造模于第4、8和12周时对两组大鼠进行超声监测,并从两组中各随机抽取3只大鼠进行肝脏病理学分析,与超声监测结果进行对比分析。结果超声与病理检查结果均提示酒精性脂肪肝造模成功,超声可以监测模型组大鼠肝脏脂肪病变从轻到重的渐变过程以及对照组大鼠无脂肪病变过程。这与肝组织的病理学诊断结果具有一致性。结论超声检测技术可以较好地进行活体评价大鼠酒精性脂肪肝动物模型。     

Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line

Metabolic profiling or metabolomics is the analysis of a larger number of small metabolic compounds within cells. While this technique has been utilized to study microbial and yeast strains under different physiochemical conditions, very little has been reported regarding its application in mammalian cell culture. Here, the physiological and metabolic changes observed during the proliferation arrest of an antibody producing GS-NS0 mouse myeloma cell line were studied using conventional biochemical analysis and one-dimensional nuclear magnetic resonance (NMR)-based metabolic profiling. Proliferation-arrested cells had increased antibody productivity, enhanced normalized mitochondrial membrane potential, and showed changes in the consumption of several amino acids. Further investigation into these physiological changes was carried out by ¹H NMR profiling followed by principle component analysis (PCA). The resulting data showed a clear separation of the arrested and control spectra that related to the altered metabolic state of the arrested culture. Metabolites associated with phosphatidylcholine homeostasis, lipid and fatty acid metabolism, and ascorbate formation were found to be present in significant amount in these cultures. Taken together, the results suggested that there was a link between the metabolic alterations and the hyper-productive state, possibly relating to vesicle recycling and secretory functions, and mechanism to counteract against the generation of reactive oxygen species. While the use of metabolic profiling is still in its infancy, its potential to enhance the understanding of physiological processes in mammalian cell lines used for antibody production is certain.     

(1)H NMR-based metabolomic profiling in mice infected with Mycobacterium tuberculosis

Tuberculosis (TB) is one of three major infectious diseases, and the control of TB is becoming more difficult because of the emergence of multidrug-resistant and extensively drug-resistant strains. In this study, we explored the (1)H NMR-based metabolomics of TB using an aerobic TB infection model. Global profiling was applied to characterize the responses of C57Bl/6 mice to an aerobic infection with virulent Mycobacterium tuberculosis (MTB). The metabolic changes in organs (i.e., the lung, the target organ of TB, and the spleen and liver, remote systemic organs) and in serum from control and MTB-infected rats were investigated to clarify the host-pathogen interactions in MTB-infected host systems. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed distinct separation between control and MTB-infected rats for all tissue and serum samples. Several tissue and serum metabolites were changed in MTB-infected rats, as compared to control rats. The precursors of membrane phospholipids, phosphocholine, and phosphoethanolamine, as well as glycolysis, amino acid metabolism, nucleotide metabolism, and the antioxidative stress response were altered based on the presence of MTB infection. This study suggests that NMR-based global metabolite profiling of organ tissues and serum could provide insight into the metabolic changes in host infected aerobically with virulent Mycobacterium tuberculosis.     

A diet-independent zebrafish model for NAFLD recapitulates patient lipid profiles and offers a system for small molecule screening

Non-alcoholic Fatty Liver Disease (NAFLD) or pathological hepatic lipid overload, is considered to affect obese individuals. However, NAFLD in lean individuals is prevalent, especially in South Asian population. The pathophysiology of lean NAFLD is not well understood and most animal models of NAFLD use the high-fat diet paradigm. To bridge this gap, we have developed a diet-independent model of NAFLD in zebrafish. We have previously shown that chronic systemic inflammation causes metabolic changes in the liver leading to hepatic fat accumulation in an IL6 overexpressing (IL6-OE) zebrafish model. In the present study, we compared the hepatic lipid composition of adult IL6-OE zebrafish to the controls and found an accumulation of saturated triacylglycerols and a reduction in the unsaturated triacylglycerol species reminiscent of NAFLD patients. Zebrafish is an ideal system for chemical genetic screens. We tested whether the hepatic lipid accumulation in the IL6-OE is responsive to chemical treatment. We found that PPAR-gamma agonist Rosiglitazone, known to reduce lipid overload in the high-fat diet models of NAFLD, could ameliorate the fatty liver phenotype of the IL6-OE fish. Rosiglitazone treatment reduced the accumulation of saturated lipids and showed a concomitant increase in unsaturated TAG species in our inflammation-induced NAFLD model. Our observations suggest that the IL6-OE model can be effective for small molecule screening to identify compounds that can reverse hepatic lipid accumulation, especially relevant to lean NAFLD.     

Ethanol consumption impairs regulation of fatty acid metabolism by decreasing the activity of AMP-activated protein kinase in rat liver

The mechanisms by which ethanol consumption causes accumulation of hepatic triacylglycerols are complex. AMP-activated protein kinase (AMPK) plays a central role in the regulation of lipid metabolism. Therefore, in the present study we investigated whether AMPK may have a role in the development of ethanol-induced fatty liver. Hepatocytes isolated from rats fed with an ethanol-containing liquid diet showed higher rates of fatty acid and triacylglycerol syntheses, but a decreased rate of fatty acid oxidation, concomitant to a lower activity of carnitine palmitoyltransferase I. Hepatocytes from both ethanol-fed and pair-fed control rats were incubated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator in intact cells. In both hepatocyte preparations AICAR strongly inhibited the activity of acetyl-CoA carboxylase in parallel to fatty acid synthesis, but cells from ethanol-fed rats showed significantly lower sensitivity to inhibition by AICAR. Moreover, AICAR strongly decreased triacylglycerol synthesis and increased fatty acid oxidation in control hepatocytes, but these effects were markedly attenuated in hepatocytes from ethanol-fed rats. In parallel, AMPK in liver of ethanol-fed rats showed a decreased specific activity and a lower sensitivity to changes in the AMP/ATP ratio, compared to the enzyme of control rats. These effects are consistent with the impairment of AMPK-mediated regulation of fatty acid metabolism after ethanol consumption, that will facilitate triacylglycerol accumulation. Taken together, these findings suggest that a decreased AMPK activity may have an important role in the development of alcoholic fatty liver.     

Gas Chromatography/Mass Spectrometry-Based Metabolomic Profiling Reveals Alterations in Mouse Plasma and Liver in Response to Fava Beans

Favism is a life-threatening hemolytic anemia resulting from the intake of fava beans by susceptible individuals with low erythrocytic glucose 6-phosphate dehydrogenase (G6PD) activity. However, little is known about the metabolomic changes in plasma and liver after the intake of fava beans in G6PD normal and deficient states. In this study, gas chromatography/mass spectrometry was used to analyze the plasma and liver metabolic alterations underlying the effects of fava beans in C3H- and G6PD-deficient (G6PDx) mice, and to find potential biomarkers and metabolic changes associated with favism. Our results showed that fava beans induced oxidative stress in both C3H and G6PDx mice. Significantly, metabolomic differences were observed in plasma and liver between the control and fava bean treated groups of both C3H and G6PDx mice. The levels of 7 and 21 metabolites in plasma showed significant differences between C3H-control (C3H-C)- and C3H fava beans-treated (C3H-FB) mice, and G6PDx-control (G6PDx-C)- and G6PDx fava beans-treated (G6PDx-FB) mice, respectively. Similarly, the levels of 7 and 25 metabolites in the liver showed significant differences between C3H and C3H-FB, and G6PDx and G6PDx-FB, respectively. The levels of oleic acid, linoleic acid, and creatinine were significantly increased in the plasma of both C3H-FB and G6PDx-FB mice. In the liver, more metabolic alterations were observed in G6PDx-FB mice than in C3H-FB mice, and were involved in a sugar, fatty acids, amino acids, cholesterol biosynthesis, the urea cycle, and the nucleotide metabolic pathway. These findings suggest that oleic acid, linoleic acid, and creatinine may be potential biomarkers of the response to fava beans in C3H and G6PDx mice and therefore that oleic acid and linoleic acid may be involved in oxidative stress induced by fava beans. This study demonstrates that G6PD activity in mice can affect their metabolic pathways in response to fava beans.     

Role of adipose tissue in methionine–choline-deficient model of non-alcoholic steatohepatitis (NASH)

Methionine–choline-deficient (MCD) diet is a widely used dietary model of non-alcoholic steatohepatitis (NASH) in rodents. However, the contribution of adipose tissue to MCD-induced steatosis, and inflammation as features of NASH are not fully understood. The goal of this study was to elucidate the role of adipose tissue fatty acid (FA) metabolism, adipogenesis, lipolysis, inflammation and subsequent changes in FA profiles in serum and liver in the pathogenesis of steatohepatitis. We therefore fed ob/ob mice with control or MCD diet for 5 weeks. MCD-feeding increased adipose triglyceride lipase and hormone sensitive lipase activities in all adipose depots which may be attributed to increased systemic FGF21 levels. The highest lipase enzyme activity was exhibited by visceral WAT. Non-esterified fatty acid (NEFA)-18:2n6 was the predominantly elevated FA species in serum and liver of MCD-fed ob/ob mice, while overall serum total fatty acid (TFA) composition was reduced. In contrast, an overall increase of all FA species from TFA pool was found in liver, reflecting the combined effects of increased FA flux to liver, decreased FA oxidation and decrease in lipase activity in liver. NAFLD activity score was increased in liver, while WAT showed no changes and BAT showed even reduced inflammation. Conclusion: This study demonstrates a key role for adipose tissue lipases in the pathogenesis of NASH and provides a comprehensive lipidomic profiling of NEFA and TFA homeostasis in serum and liver. Our findings provide novel mechanistic insights for the role of WAT in progression of MCD-induced liver injury.     

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity‐induced non‐alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity‐induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high‐fat diet (HFD) with or without 1‐week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI‐MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit β, glucosamine‐fructose‐6‐phosphate aminotransferase 1, zine finger protein 2, s‐adenosylmethionine synthase isoform type‐1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti‐metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.