核黄素对北京鸭生长发育和脂肪代谢的影响及其调控机制

发布时间：2018-09-11 19:43

【摘要】：本论文通过3个体内试验研究了核黄素对北京鸭生长发育和脂肪代谢的影响及其调控机制,并通过体外试验研究了核黄素对HepG2细胞增殖和线粒体功能的影响。试验一旨在研究日粮核黄素水平对15-35日龄北京鸭生长发育的影响,并确定其需要量。本试验设6个核黄素水平(1.38、2.38、3.38、4.38、5.38、6.38 mg/kg),选取288只体重相近的15日龄雄性北京鸭,随机分为6个处理组,每个重复8只鸭。与饲喂基础日粮(核黄素含量为1.38mg/kg)相比,添加核黄素可显著提高平均日增重、平均日采食量、胸肌率、腹脂率、血浆及肝脏核黄素含量,显著降低料重比和肝脏指数。以生产性能和组织核黄素为评价指标,采用折线模型估测15～35日龄北京鸭核黄素需要量为2.33～3.57mg/kg。试验二旨在探究核黄素对生长前期北京鸭生长发育和脂肪代谢的影响及其调控机制。本试验选取360只1日龄健康的雄性北京鸭,随机分为三个处理组:核黄素缺乏组、采食配对组(人为控制该组与核黄素缺乏组采食量一致)和自由采食对照组,每个处理组12个重复,每个重复10只鸭,试验期为21天。与采食配对组和自由采食对照组相比,核黄素缺乏显著降低了生长前期北京鸭平均日增重,显著提高了料重比和死亡率;显著降低了血浆和肝脏核黄素含量;显著提高了血浆和肝脏中甘油三酯和总胆固醇含量、肝脏总饱和脂肪酸、C6:0、C12:0、C16:0、C18:0含量和肝脏指数。这三组肝脏蛋白质组学分析显示,与采食配对组和自由采食对照组相比,核黄素缺乏导致63个蛋白质表达量变化大于1.5倍,其中包括32个上调蛋白质和31个下调蛋白。GO聚类分析结果显示差异蛋白主要富集在脂肪酸氧化和线粒体呼吸链电子传递过程。核黄素缺乏组中参与脂肪酸β氧化过程的ACADS、ACADM、ACAD9和ETFDH蛋白表达量下调,提示脂肪酸β氧化受损,脂肪分解减少,进而导致肝脏脂肪沉积。核黄素缺乏组中参与线粒体呼吸链电子传递过程的ACAD9、NDUFS1、NDUFA8和FXN蛋白表达量下调,提示呼吸链电子传递过程受损,进而导致ATP生成不足,影响动物生长。试验三旨在研究种母鸭核黄素对母鸭繁殖性能和子代胚胎发育的影响。本试验选取80只45周龄产蛋期北京鸭,随机分为两个组:核黄素缺乏组和对照组,分别饲喂核黄素添加量为0和10mg/kg的试验日粮,试验期为8周。种母鸭日粮核黄素对试验期母鸭体重、产蛋率、蛋重、种蛋受精率和子代初生重没有显著影响;从试验第2周开始,核黄素缺乏组种母鸭血浆核黄素和种蛋黄核黄素含量显著降低,种蛋孵化率急剧降低,试验第6周以后种蛋孵化率降为0。试验第8周种蛋孵化第13天胚胎肝脏蛋白质组学分析结果显示,种母鸭核黄素缺乏导致187个蛋白质表达量变化大于1.5倍,其中包括67个上调蛋白质和120个下调蛋白。KEGG通路分析显示差异蛋白主要富集在三羧酸循环、脂肪酸β氧化和呼吸链电子传递等代谢过程。核黄素缺乏导致胚胎肝脏脂肪酸β氧化(ETFDH、CPT1A、ACSL1、ACADS、ACAT1、ACSL5、DECR1 和 ETFA)、三羧酸循环(DLD、SDHB、IDH1、SDHA 和 ACO1)和呼吸链电子传递(NDUFA9、NDUFS1、NDUFV1、NDUFA10和ACAD9)过程中蛋白表达下调,提示这些代谢过程可能受阻,进而导致ATP产生不足,导致胚胎发育不良甚至死亡。试验四分为3个体外试验,旨在研究核黄素对HepG2细胞增殖和线粒体功能的影响。试验1研究了不同核黄素耗竭时间对HepG2线粒体功能的影响。HepG2细胞在核黄素缺乏和核黄素充足的培养基中分别培养2、4、6、8、10和12天,试验结束时采用Seahorse测定细胞线粒体功能。与核黄素充足组相比,核黄素缺乏组HepG2细胞从试验第2天起最大耗氧量和呼吸潜力显著降低,随着试验周期的延长,最大耗氧量和呼吸潜力进一步降低,提示线粒体功能受损。试验2研究了不同核黄素水平对HepG2细胞增殖和线粒体功能的影响。HepG2细胞在核黄素添加水平为0、0.5、5、10、20、40、100和1064nmol/L的培养基中培养8天。在试验第8天,培养基中不添加核黄素组HepG2细胞数量、最大呼吸量和呼吸潜力显著低于核黄素添加组,随着核黄素添加水平的提高,这些指标逐渐提高,当核黄素水平分别提高到10、20和20nmol/L时到达平台期。试验3研究了补充核黄素对HepG2细胞增殖和线粒体功能的影响。HepG2细胞在不添加核黄素的培养基中培养8天,随后在添加不同水平的核黄素(0、0.5、5和1064nmol/L)的培养基中培养4天。培养基中添加5nmol/L核黄素对试验第4天细胞基础呼吸量、最大呼吸量和呼吸潜力均有显著的提高;培养基中添加1064nmol/L核黄素可显著提高细胞数量,显著提高基础呼吸量、最大呼吸量和呼吸潜力,完全恢复线粒体呼吸功能。以上结果表明,HepG2细胞在不添加核黄素的培养基中培养2天后线粒体呼吸功能受损,8天后细胞增殖速率降低。培养基中添加20nmol/L核黄素可维持HepG2细胞细胞正常生长和线粒体呼吸功能。HepG2细胞在不添加核黄素的培养基中培养8天,培养基中添加1064nmol/L核黄素培养4天可显著提高细胞增殖速率,并完全恢复线粒体呼吸功能。以上结果表明,核黄素缺乏可导致胚胎期和生长前期北京鸭生长发育不良、肝脏脂肪蓄积。肝脏蛋白质组学分析发现,核黄素缺乏导致肝脏线粒体脂肪酸β氧化(ACADS、ACAD9和ETFDH)、呼吸链电子传递(ACAD9和NDUFS1)和三羧酸循环(DLD)过程中关键蛋白表达量下调,阻碍脂肪分解导致脂肪蓄积;阻碍能量生成,导致动物生长发育不良。体外试验结果显示,核黄素缺乏可导致HepG2细胞线粒体功能受损,补充核黄素后可恢复线粒体功能。
[Abstract]:The effects of riboflavin on the growth and fat metabolism of Beijing ducks were studied by three in vivo experiments. The effects of riboflavin on the proliferation and mitochondrial function of HepG2 cells were studied in vitro. The first experiment was designed to study the effects of dietary riboflavin levels on the growth and development of Beijing ducks aged 15-35 days. Six riboflavin levels (1.38, 2.38, 3.38, 4.38, 5.38, 6.38 mg/kg) were used to select 288 15-day-old Beijing ducks with similar body weight and randomly divided into six treatment groups with 8 ducks in each repetition. The riboflavin requirement of Beijing ducks aged 15-35 days was estimated by a broken-line model with the production performance and tissue riboflavin as the evaluation indexes. Experiment 2 was designed to explore the effects of riboflavin on the growth and lipid content of pre-growth Beijing ducks. In this study, 360 1-day-old healthy male Peking ducks were randomly divided into three groups: riboflavin deficiency group, feeding matching group (the same amount of food as riboflavin deficiency group) and free-feeding control group. Each group had 12 replicates, 10 ducks per replicate for 21 days. Riboflavin deficiency significantly reduced the average daily gain, increased the feed-to-weight ratio and mortality, decreased the riboflavin content in plasma and liver, increased the triglycerides and total cholesterol content in plasma and liver, and the total saturated fatty acids in liver, C6:0, C12, respectively. The three groups of liver proteomics analysis showed that the deficiency of riboflavin resulted in 63 protein expression changes more than 1.5 times, including 32 up-regulated proteins and 31 down-regulated proteins. The expression of ACADS, ACADM, ACAD9 and ETFDH proteins involved in the oxidation of fatty acid beta in the riboflavin deficient group was down-regulated, suggesting that fatty acid beta oxidation was impaired and fat decomposition was reduced, leading to liver fat deposition. The down-regulation of ACAD9, NDUFS1, NDUFA8 and FXN proteins suggests that the electron transport process in the respiratory chain is impaired, which leads to insufficient ATP production and affects animal growth. Riboflavin deficiency group and control group were fed with riboflavin supplement of 0 and 10 mg/kg for 8 weeks, respectively. Riboflavin had no significant effect on body weight, egg laying rate, egg weight, fertilization rate of breeding eggs and birth weight of offspring. From the second week of the experiment, plasma riboflavin and species of breeding ducks in riboflavin deficiency group were fed with riboflavin supplement of 0 and 10 mg/kg respectively. The content of riboflavin in egg yolk decreased significantly and the hatchability of eggs decreased sharply. The hatchability of eggs dropped to 0 after the 6th week of the experiment. Proteomic analysis of the liver of the embryos on the 13th day of the 8th week of the experiment showed that the riboflavin deficiency caused 187 protein expression changes more than 1.5 times, including 67 up-regulated proteins and 120 up-regulated proteins. KEGG pathway analysis showed that the differentially expressed proteins were mainly enriched in the tricarboxylic acid cycle, fatty acid beta oxidation and electron transport of respiratory chain. Riboflavin deficiency led to fatty acid beta oxidation (ETFDH, CPT1A, ACSL1, ACADS, ACAT1, ACSL5, DECR1 and ETFA), tricarboxylic acid cycle (DLD, SDHB, IDH1, SDHA and ACO1), and respiratory chain electricity in embryonic liver. The down-regulation of protein expression during Subtransmission (NDUFA9, NDUFS1, NDUFV1, NDUFA10, and ACAD9) suggests that these metabolic processes may be blocked, resulting in ATP deficiency, resulting in embryonic dysplasia and even death. Three in vitro trials were conducted to investigate the effects of riboflavin on the proliferation and mitochondrial function of HepG2 cells. HepG2 cells were cultured in riboflavin-deficient and riboflavin-rich medium for 2,4,6,8,10 and 12 days, respectively. Mitochondrial function was measured by Seahorse at the end of the experiment. Compared with riboflavin-deficient group, HepG2 cells in riboflavin-deficient group had the maximum oxygen consumption from the second day of the experiment. The effects of riboflavin levels on the proliferation and mitochondrial function of HepG2 cells were studied in experiment 2. HepG2 cells were cultured in medium with riboflavin levels of 0,0.5,5,10,20,40,100 and 1064 nmol/L. On the 8th day, the number of HepG2 cells in the medium without riboflavin was significantly lower than that in the riboflavin supplementation group. With the increase of riboflavin supplementation, these indexes gradually increased, reaching the plateau when the riboflavin levels increased to 10, 20 and 20 nmol/L, respectively. Effects of lutein on proliferation and mitochondrial function of HepG2 cells were studied. HepG2 cells were cultured for 8 days without riboflavin, and then for 4 days in medium with different levels of riboflavin (0,0.5,5 and 1064 nmol/L). The basal respiration, maximum respiration and respiratory potential of HepG2 cells were measured by adding 5 nmol/L riboflavin to the medium on day 4. The results showed that the mitochondrial respiratory function of HepG2 cells was impaired after 2 days of culture without riboflavin and 8 days after culture. The normal growth and mitochondrial respiratory function of HepG2 cells were maintained by adding 20 nmol/L riboflavin to the medium. HepG2 cells were cultured in the medium without riboflavin for 8 days and 1064 nmol/L riboflavin for 4 days. These results suggest that riboflavin deficiency can lead to maldevelopment and liver fat accumulation in Beijing ducks during embryonic and early growth stages. Proteomic analysis of liver revealed that riboflavin deficiency is the key factor in the process of fatty acid beta oxidation (ACADS, ACAD9 and ETFDH), respiratory chain electron transport (ACAD9 and NDUFS1) and tricarboxylic acid cycle (DLD) in liver mitochondria. The results of in vitro experiments showed that riboflavin deficiency could lead to mitochondrial dysfunction in HepG2 cells, and mitochondrial function could be restored after riboflavin supplementation.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S834.5

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