Complement C3 participates in the development of goose fatty liver potentially by regulating the expression of FASN and ETNK1

Non-alcoholic fatty liver disease ( NAFLD ) occurs in humans, domestic animals and poultry. Different from upregulation of complement C3 in human NAFLD, C3 expression is inhibited in goose fatty liver ( GFL ), implying a specific role of C3 in GFL. This study was mainly focused on uncovering the uniqueness of goose liver cells in the regulation of C3 expression and identifying the downstream genes of C3 to improve understanding on the specific role of C3 in GFL. The results showed that C3 expression was inhibited in the liver, muscle and fat tissues of the overfed versus control (normally fed) geese. Oleate and insulin could inhibit C3 expression in goose primary hepatocytes but induce it in mouse primary hepatocytes. A total of 1,123 differentially expressed genes ( DEGs ) were affected by C3 overexpression and were mainly enriched in immune response/inflammation and catabolism-related KEGG pathways. Additionally, the representative downstream genes (FASN and ETNK1) of C3 could mediate the role of C3 in the development of GFL. In conclusion, the suppression of C3 in GFL is at least partially attributed to hyperinsulinemia, hyperlipidemia and uniqueness of goose liver cells. Complement C3 does not only affect hepatic steatosis but also affect inflammation/immune response in GFL.     

卵巢肿瘤去泛素化酶7A基因与鹅肥肝形成关系的研究

【目的】 研究卵巢肿瘤去泛素化酶7A(OTUD7A)基因与鹅肥肝形成的关系。【方法】 选取健康、体重一致的70日龄朗德鹅公鹅40只,随机分为2组,对照组自由采食,试验组进行填饲试验,其中填饲第1~5天每日采食量为500 g,第6~12天每日采食量为800 g,第13~19天每日采食量为1 200 g。在填饲第7、14和19天时,每组随机选取6只鹅屠宰,取肝脏,采用实时荧光定量PCR测定不同填饲阶段肝脏中OTUD7A基因的表达水平。采用Ⅳ型胶原酶消化法分离23胚龄的鹅原代肝细胞,分别用浓度为0(空白组)、125、250 mmol/L的葡萄糖,0(空白组)、50、100、200 nmol/L的胰岛素,0(空白组)、0.125、0.250 mmol/L的油酸、亚油酸及0(空白组)、0.25、0.50 mmol/L棕榈酸处理鹅原代肝细胞,并用实时荧光定量PCR检测这些脂肪肝形成相关因子对OTUD7A基因表达水平的影响。构建过表达OTUD7A基因的载体pcDNA3.1-OTUD7A,并将构建好的过表达重组质粒转染鹅原代肝细胞,通过转录组学测序(RNA-Seq)进行差异表达基因的筛选,并对获得的差异表达基因进行GO功能富集分析。【结果】 在填饲第7、14天时,鹅肝脏中的OTUD7A基因表达显著高于对照组(P<0.05)。与空白组相比,250 mmol/L葡萄糖处理以及0.125 mmol/L和0.250 mmol/L棕榈酸处理均显著提高鹅原代肝细胞中OTUD7A基因的表达水平(P<0.05)。转录组学测序结果表明,OTUD7A基因过表达后共筛选到34个差异表达基因,其中有19个基因表达上调、15个基因表达下调。GO功能富集分析发现,差异表达基因注释到对微生物的防御反应、对外界生物刺激的反应、T细胞分化、细胞外外泌体等条目,其中CLMP、PROCR、SH3BP1、ARHGAP28、ACE、OTUD7A、LOC106045877、LOC106048002基因的表达上调,TNFSF8、DDX60、PLAC8、RSAD2、MX1、RSAD2、GBP1基因的表达下调。【结论】 鹅肥肝形成过程中OTUD7A基因的表达水平升高,OTUD7A基因可能通过调控TNFSF8、RSAD2、MX1、GBP1、CLMP和PROCR等基因的表达参与鹅肥肝的形成。     

Glucose participates in the formation of goose fatty liver by regulating the expression of miRNA-33/CROT

Glucose oversupply promotes formation of fatty liver, and fatty liver is usually accompanied with hyperglycemia. However, the mechanism by which glucose promotes formation of fatty liver is not very clear. In this study, fatty liver was successfully induced in Landes goose by 19 days of overfeeding with corn-based feed, the overfed geese had a significantly higher level of blood glucose than the normally fed geese (control group). In goose primary liver cells, high level of glucose promoted fat deposition and induced the expression of SREBF2(or SREBP2), a key regulator of lipid metabolism, and its intronic gene, miR-33. Moreover, overexpression of miRNA-33(miR-33) promotes lipid accumulation in goose primary liver cells. Consistently, miR-33 inhibitor suppressed glucose induced lipid accumulation in liver cells. Interestingly, the relative abundance of miR-33 in goose fatty liver was significantly higher than that in normal liver, while the relative mRNA and protein abundances of CROT, the target gene of miR-33, in goose fatty liver were significantly lower than those in goose normal liver. Taken together, these findings suggest that miR-33 mediates glucose promotion of lipid accumulation in goose primary liver cells, and that glucose participates in formation of goose fatty liver by regulating the expression of miR-33/CROT.     

鹅肥肝形成相关基因的研究进展

鹅肥肝与鲟鱼子酱、黑菌被西方人誉为世界三大美食。对于肥肝产业而言,加强对鹅遗传育种的研究,培育产肝鹅新品系有着重要的意义。作者依据肥肝形成机制,综述了近年来研究发现的影响肥肝形成的相关基因,包括主要参与脂肪合成的硬脂酰辅酶A去饱和酶(SCD-1)、脂蛋白脂酶(LPL)、脂肪酸合成酶(FAS)、肝X受体(LXRα)、脂肪酸长链延伸因子6(ELOVL-6)、固醇调节元件结合蛋-1c(SREBP-1c)、碳水化合物反应元件结合蛋白(CHREBP)基因,参与脂肪转运的脂肪型脂肪酸结合蛋白(A-FABP)、长链酯酰辅酶A合成酶1(ACSL1)基因,以及参与脂肪氧化的过氧化物酶体增殖物激活受体(PPAR)、胆固醇7a轻化酶(CYP7A1)基因等。     

Decrease in stearic acid proportions in adipose tissues and liver lipids in fatty liver of dairy cows

Samples of liver and perirenal, mesenteric and subcutaneous fat were collected from 16 sick necropsied dairy cows to evaluate the fatty acid profiles in the hepatic and adipose tissues associated with advanced fatty liver or hepatic lipidosis. Hepatic triglyceride and eight fatty acids were measured in the hepatic and adipose tissues. Six cows had more than 3% triglyceride on fresh weight in their livers and were classified as having fatty liver. Stearic and linoleic acid proportions in the liver decreased markedly with increased hepatic triglyceride levels, while the proportion of palmitic and oleic acids increased. The most striking fluctuations in hepatic lipidosis were manifested as decreased stearic acid in the adipose tissues including subcutaneous fat with the trend of decreasing stearic acid. Palmitic acid was elevated in hepatic and perirenal fat in fatty liver cows. In instances of advanced hepatic lipidosis, palmitoleic acid increased in only subcutaneous fat and not in perirenal or mesenteric fat. In addition to the proportions of hepatic fatty acids in fatty liver, this study also clarified the fluctuations observed in the profiles of fatty acids of the adipose tissues in cows with advanced hepatic lipidosis, particularly the decline in the proportions of stearic acid.     

Study on Changes of Blood Chemistry Indexes,Hepatic Routine Nutritional Composition and Expression of Lipid Metabolism-associated Genes during the Recovery of Geese with Fatty Livers

This experiment aimed to determine the changes of body weight, liver weight, abdominal fat weight, hepatic routine nutritional composition, blood biochemistry indexes, as well as mRNA expression of lipid metabolism-associated genes during the recovery of Landes geese with fatty liver, which provided the basis for the further elucidation of the recovery or protection mechanism of goose fat liver.18 Landes geese were divided into three groups (6 gees in each group).Group 1 was control group, in which the geese were fed with boiled maize;Group 2 was the overfeeding group without recovery, in which the geese were overfed with a maize-based diet for 19 days;And group 3 was the overfeeding group with 20-day recovery, in which the geese were overfed with the maize-based diet for 19 days followed by feeding boiled maize for 20-day recovery.The results showed that compared with group 2, the body weight and liver weight of the geese in group 3 decreased extremely significantly (P<0.01), and the abdominal fat weights undecreased significantly (P>0.05);Moreover, the percentages of nutrients including water, ash, CP of livers in group 3 increased significantly (P<0.05), while the percentage of EE decreased significantly (P<0.05), but there were no significant difference for these variables between groups 1 and 3 (P>0.05) except water.In regard to blood biochemistry indexes, the contents of blood ALT, AST, TG and HDL-C in group 3 decreased significantly (P<0.05), but there were no significant difference from those of group 1 (P>0.05).The mRNA expression levels of FADS1, SCD1 and CYP2C45 in group 3 decreased significantly compared with group 1 (P<0.05), but there were no significant difference between groups 1 and 3 (P>0.05).In summary, this study demonstrated the recoverability of goose fatty liver at different layers, and thus laid a foundation for further investigation on the recovery or protective mechanism of goose fatty liver and found the solution to resolve the problems related to animal fatty liver.     

肥肝鹅恢复期血液生化指标、肝脏常规营养成分及脂代谢相关基因表达水平的变化研究

试验旨在研究朗德鹅填饲形成肥肝及恢复后其体重、肝脏重、腹脂重、常规营养成分、血液生化指标和肝脏中与脂代谢相关基因的表达变化情况,为进一步阐明鹅肥肝的恢复或保护机制提供依据。将18只70日龄朗德鹅随机分为3组(每组6只):组1为对照组,用玉米饲料进行常规饲喂;组2为填饲组,填饲玉米饲料19 d;组3为填饲19 d后再用玉米饲料常规饲养20 d进行肝脏恢复。结果显示,与组2相比,组3鹅体重、肝脏重均极显著降低(P<0.01),而腹脂重下降不显著(P>0.05);与组2相比,组3肝脏水分、灰分和粗蛋白质均显著上升(P<0.05),而粗脂肪含量显著下降(P<0.05),但与组1相比,除水分外,各项指标均无显著变化(P>0.05);与组2相比,组3血浆中谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)浓度与组2相比均显著下降(P<0.05),但与组1相比,均无显著差异(P>0.05);与组2相比,组3肝脏中脂肪酸脱氢酶(FADS1)、硬脂酰辅酶A去饱和酶(SCD1)和细胞色素P450 2C45(CYP2C45)基因的表达量均显著下降(P<0.05),但与组1相比均无显著变化(P>0.05)。综上所述,本研究从不同层面揭示了鹅肥肝的可逆性,为深入研究鹅肥肝的恢复或保护机制,促进动物脂肪肝问题的解决奠定了基础。     

乙酸和β-羟丁酸对体外培养牛肝细胞脂代谢部分关键酶表达的影响

以体外原代培养的奶牛肝细胞为模型,添加不同浓度的乙酸（Aceticacid,AcOH）和β-羟丁酸（β-hydroxybu—tyrate,BHBA）共培养24h后,提取细胞总RNA。应用实时荧光定量PCR方法检测脂代谢关键酶长链脂酰辅酶A合成酶1（Long-chain acyl-CoA synthetase-1,ACSL1）、柠檬酸合成酶（Citrate synthase,CS）和乙酰辅酶A羧化酶α（Acetyl coenzyme A carboxylase α,ACCα）mRNA丰度的变化。结果显示,适当浓度的AcOH能够促进肝细胞脂肪酸活化及氧化途径关键酶ACSL1和CS的转录,而高浓度AcOH能够抑制脂肪酸从头合成途径关键酶ACCa的转录;高浓度BHBA能够抑制肝细胞ACSL1、CS和AcCα的转录。结果表明,血液中适当浓度的AcOH能够促进肝脏脂肪酸氧化并抑制脂肪酸从头合成,高浓度BHBA能够抑制肝脏脂氧化和合成,影响乳脂前体物的供应,进而影响乳脂合成。     

Inhibition of PI3K‐Akt‐mTOR signal pathway dismissed the stimulation of glucose on goose liver cell growth

In the formation of goose fatty liver induced by a high‐carbohydrate diet, it is characterized by the quick cell growth of liver. The carbohydrate is mostly digested and absorbed in the small intestine by the form of glucose. Recent studies have suggested a crucial role for PI3K‐Akt‐mTOR pathway in regulating cell proliferation, and then we speculate that PI3K‐Akt‐mTOR pathway may mediate glucose‐induced liver cell proliferation. Goose primary hepatocytes were isolated and incubated in either no addition as a control or glucose or PI3K‐Akt‐mTOR pathway inhibitors or cotreatment with glucose and PI3K‐Akt‐mTOR pathway inhibitors. The results firstly showed that 35 mmol/l glucose stimulated the mRNA level and protein content of factors involved in PI3K‐Akt‐mTOR signal pathway in goose primary hepatocytes. Secondly, 35 mmol/l glucose evidently changed the cell cycle PI index and protein expression of cyclin D1. Meanwhile, the upregulation of 35 mmol/l glucose on the DNA synthesis rate, cell cycle PI index, the mRNA expression, protein content and protein expression of factors involved in the cell proliferation was decreased significantly by the inhibitors of PI3K‐Akt‐mTOR pathway, LY294002, rapamycin or NVP‐BEZ235. In summary, glucose could stimulate the cell proliferation, and the PI3K‐Akt‐mTOR pathway inhibitors could dismiss glucose‐induced the upregulation of cell proliferation in goose primary hepatocyte.     

PPAR-α在酒精性脂肪肝发生中的作用研究进展

酒精性脂肪肝(AFLD)是由于长期大量饮酒导致肝脏脂肪代谢紊乱,并大量沉积的一种代谢性疾病。过氧化物酶增殖物激活受体-α(PPARα)是一类配体激活的核转录因子超家族成员之一,高表达于肝脏。研究发现,PPARα与乙醇代谢,以及肝脏脂肪代谢密切相关。乙醇影响PPARα的正常功能,而PPARα的缺失或表达受抑参与了酒精性肝损伤的过程,促进了肝细胞脂肪变性及炎症的发生、发展。另外,PPARα参与脂质分解代谢的多个方面,包括脂肪酸的透膜吸收、脂肪酸在细胞内的结合、脂肪酸的氧化及脂蛋白的合成与运输。PPARα及其特异性配体的运用将会成为AFLD治疗的一个新靶点。论文综述了PPARα的结构、生物学作用及其在AFLD发生机制中的作用,可为该病的治疗提供新的思路。     

不同种类脂肪酸对延边黄牛骨骼肌卫星细胞成脂转分化的影响

试验旨在探究用同一浓度不同种类的脂肪酸单体（油酸、硬脂酸、棕榈油酸及棕榈酸）处理延边黄牛骨骼肌卫星细胞（BSC）,研究其对脂肪生成相关基因表达及脂滴形成的影响。从18月龄延边黄牛半膜肌中分离提取骨骼肌卫星细胞进行体外培养,在分化培养基中分别添加100 μmol/L油酸（OA）、硬脂酸（SA）、棕榈油酸（POA）和棕榈酸（PA）培养96 h,油红O染色观察脂滴生成情况,并利用实时荧光定量PCR法检测与脂肪生成相关基因过氧化物酶体增殖物激活受体γ（PPARγ）、固醇调节原件结合蛋白1（SREBP1）、硬脂酰辅酶A去饱和酶（SCD）、CCAAT/增强子结合蛋白α（C/EBPα）的表达。油红O染色结果显示,与对照组相比,所有的脂肪酸处理组细胞均有脂滴形成,油酸和棕榈油酸处理组相对于棕榈酸和硬脂酸处理组在肌管内形成的脂滴数量更多,且脂滴的形态较大。实时荧光定量PCR结果显示,在延边黄牛骨骼肌卫星细胞中添加油酸和棕榈油酸等不饱和脂肪酸增加了与脂肪合成相关基因PPARγ、SREBP1、C/EBPα的表达,抑制了SCD基因的表达;添加饱和脂肪酸（硬脂酸和棕榈酸）则在促进PPARγ、SREBP1、C/EBPα基因表达的同时也显著增加了SCD基因的表达（P < 0.05）。结果表明,添加脂肪酸可以诱导延边黄牛骨骼肌卫星细胞向脂肪细胞转分化。     

鹅肝细胞的高纯分离及培养

采用简单的＂胶原酶直接消化法＂分离鹅胚胎肝细胞,得到的肝细胞纯度高,活性好;HE染色显示大量双核或多核细胞,细胞具有旺盛增殖分裂能力;肝脏特异性基因白蛋白基因检测显示所分离细胞为成熟肝脏细胞。该法操作简单、高效,是获得鹅原代肝细胞的一种较好的方法,有利于以鹅原代肝细胞为模型的体外研究工作的开展。     

Effects of a high energy and low protein diet on hepatic and plasma characteristics and Cidea and Cidec mRNA expression in liver and adipose tissue of laying hens with fatty liver hemorrhagic syndrome

Cidea and Cidec are two members of Cell death‐inducing DNA fragmentation factor‐alpha‐like effector family proteins, which could be involved in lipid or fat metabolism. To better understand the roles of Cidea and Cidec in fatty liver hemorrhagic syndrome (FLHS), 150 healthy 155‐day‐old Hyline Brown laying hens were randomly divided into control group (fed with basic diet) and experimental group (fed with high‐energy low‐protein [HELP] diet). Analysis of the liver by tissue sectioning and hematoxylin and eosin staining showed that the HELP diet induced micro‐vesicular steatosis in laying hens. Subsequently, based on the liver color scores and the range of lipid accumulation observed in histological examination, we classified livers with <50% vacuolization as mild FLHS and >50% as severe FLHS. The results showed that the levels of Cidea and Cidec mRNA expression were markedly elevated in the liver and adipose tissues with FLHS and the levels of Cidea and Cidec mRNA expression in the liver with severe FLHS were significantly higher than that in the liver with mild FLHS. Thus, the present study revealed that the Cidea and Cidec genes may be involved in pathways of FLHS formation.     

乙酸和β－羟丁酸对体外培养牛肝细胞脂代谢部分关键酶表达的影响

本研究以体外原代培养的奶牛肝细胞为模型,添加不同浓度的乙酸（AcOH）和p羟丁酸（BHBA）,探讨其对奶牛肝细胞脂肪酸代谢关键酶基因表达的影响。添加不同浓度乙酸和β－羟丁酸,培养24h后,提取细胞总RNA。应用实时荧光定量PCR方法,检测脂代谢关键酶长链脂酰辅酶A合成酶1（ACSLl）、柠檬酸合成酶（CS）和乙酰辅酶A羧化酶α（ACCα）mRNA丰度的变化。结果显示,适当浓度的AcOH能够促进肝细胞脂肪酸活化及氧化途径关键酶ACSLl和CS的转录,而高浓度AcOH能够抑制脂肪酸从头合成途径关键酶ACCα的转录;高浓度BHBA能够抑制肝细胞ACSLl、CS和ACCα的转录。说明血液中适当浓度的AcOH能够促进肝脏脂肪酸氧化并抑制脂肪酸从头合成,高浓度BHBA能够抑制肝脏脂氧化和合成,影响乳脂前体物的供应,进而影响乳脂合成。     

不同开填体重朗德鹅产肝性能的比较试验

对不同开填体重的朗德鹅产肝性能进行了比较分析。结果发现:4.2 kg体重组的平均肝重最大,为827.50 g,极显著高于3.8 kg、3.0 kg体重组(P<0.01);4.7 kg体重组的肝重明显低于4.2 kg体重组,但没有达到显著水平(P=0.20);相关分析发现开填体重与肝重之间呈极显著的正相关;通过建立回归曲线得出国内朗德鹅12周龄的最佳开填体重为4.5 kg。对肥肝重与腹脂重、皮脂厚等指标进行了相关性分析,发现肝重与腹脂重呈正相关且达到显著水平(P<0.05),说明脂肪组织的脂肪沉积与肝脏的脂肪沉积有密切的关系。     

脂联素对犊牛肝细胞脂氧化关键酶表达的影响

体外原代培养犊牛肝细胞,添加0、16、64和128μg/L脂联素（adiponectin,ADPN）,分别培养4、8h后,提取细胞总RNA。应用实时荧光定量PCR方法,检测脂氧化关键酶脂酰辅酶A氧化酶（ACO）、肝脏脂肪酸结合蛋白（L-FABP）、肉碱脂酰转移酶-Ⅰ（CPTⅠ）、肉碱脂酰转移酶-Ⅱ（CPTⅡ）mRNA表达变化。结果显示,在ADPN作用4h后,ACO、L-FABP、CPTⅠ、CPTⅡ基因mRNA水平均随ADPN浓度的增加而增加,且均显著高于对照组,呈剂量依赖关系;作用8h时,ACO表达也增加,但差异不显著,L-FABP在中高剂量组显著高于对照组,CPTⅠ和CPTⅡ在各ADPN处理组均显著高于对照组。结果表明,ADPN可显著促进脂氧化关键酶的表达,增强肝脏脂氧化作用,减少肝脂储存。     

Effects of full-fat rice bran inclusion in diets on growth performance and meat quality of Sichuan goose

1. This trial was conducted to study the effect of full-fat rice bran inclusion in diets on growth performance, carcass and meat quality and fatty acid composition in Sichuan goose.

2. A total of 204 Sichuan white male geese (28-d-old, 984 ± 15 g) were used in the 42-d assay. Full-fat rice bran inclusion in diets was 0%, 6%, 12% and 18%, respectively.

3. On d 70, two geese from each pen were randomly selected and killed for measuring the carcass and meat quality and the meat fatty acid composition.

4. The results showed that full-fat rice bran inclusion had no effect on average daily gain during 28–56 d, but rice bran inclusion at 18% increased average daily gain during 57–70 d. In addition, the full-fat rice bran supplementation decreased the subcutaneous fat yield, and the inclusion of full-fat rice bran in amounts of 12% and 18% decreased the half-eviscerated carcass yield, eviscerated carcass yield and crude fat content in goose meat. Moreover, full-fat rice bran supplementation had no effect on the content of total saturated fatty acid (SFA), but decreased the content of total monounsaturated fatty acid (MUFA). The inclusion of full-fat rice bran in amounts of 12–18% increased the content of total polyunsaturated fatty acid (PUFA) and total n-6 in goose meat and in the amount of 18% increased n-3 fatty acids content in goose meat.

5. The results indicated that the rice bran inclusion had a positive effect in geese by stimulating growth performance and improving meat quality and fatty acid composition of goose meat.

     

超饲诱导水禽肥肝形成的机制

一些蹼足类动物(如鹅和鸭)在长期的进化过程中,因适应性需要,肝脏具有沉积大量脂肪形成肥肝的遗传特性。生产上利用鹅和鸭的这种特殊遗传特性,进行短期人工强制超饲(填饲),形成营养因素所致的可逆的、获得性的脂肪变性,即鹅(鸭)肥肝。本文综述了肥肝形成的过程及可能的机制,同时提出了有待研究的问题。     

cDNA cloning, genomic structure and expression analysis of the goose (Anser cygnoides) MHC class I gene

To provide data for studies on avian disease resistance, goose MHC class I cDNA (Ancy-MHC I) was cloned from a goose cDNA library, it's genomic structure and expression analysis were investigated. The mature peptides of Ancy-MHC I cDNA encoded 333 amino acids. The genomic organization is composed of eight exons and seven introns. Based on the genetic distance, six Ancy-MHC I genes from six individuals can be classified into four lineages. A total of nineteen amino acid positions in peptide-binding domain showed high scores by Wu-kabat index analysis. The Ancy-MHC I amino acid sequence displayed seven critical HLA-A2 amino acids that bind with antigen polypeptides, and have an 85.4-98.9% amino acid homology with each genes, and a 59.8-66.0% amino acid homology with chicken MHC class I. Expression analyses using Q-RT-PCR to detect the tissue-specific expression of Ancy-MHC I mRNA in an adult goose. The result appeared that Ancy-MHC I cDNA was expressed in the liver, spleen, intestine, kidney, lung, pancreas, heart, brain, and skin. The phylogenetic tree appears to branch in an order consistent with accepted evolutionary pathways.