核黃素對(duì)北京鴨生長(zhǎng)發(fā)育和脂肪代謝的影響及其調(diào)控機(jī)制
發(fā)布時(shí)間：2018-09-11 19:43
【摘要】：本論文通過(guò)3個(gè)體內(nèi)試驗(yàn)研究了核黃素對(duì)北京鴨生長(zhǎng)發(fā)育和脂肪代謝的影響及其調(diào)控機(jī)制,并通過(guò)體外試驗(yàn)研究了核黃素對(duì)HepG2細(xì)胞增殖和線(xiàn)粒體功能的影響。試驗(yàn)一旨在研究日糧核黃素水平對(duì)15-35日齡北京鴨生長(zhǎng)發(fā)育的影響,并確定其需要量。本試驗(yàn)設(shè)6個(gè)核黃素水平(1.38、2.38、3.38、4.38、5.38、6.38 mg/kg),選取288只體重相近的15日齡雄性北京鴨,隨機(jī)分為6個(gè)處理組,每個(gè)重復(fù)8只鴨。與飼喂基礎(chǔ)日糧(核黃素含量為1.38mg/kg)相比,添加核黃素可顯著提高平均日增重、平均日采食量、胸肌率、腹脂率、血漿及肝臟核黃素含量,顯著降低料重比和肝臟指數(shù)。以生產(chǎn)性能和組織核黃素為評(píng)價(jià)指標(biāo),采用折線(xiàn)模型估測(cè)15～35日齡北京鴨核黃素需要量為2.33～3.57mg/kg。試驗(yàn)二旨在探究核黃素對(duì)生長(zhǎng)前期北京鴨生長(zhǎng)發(fā)育和脂肪代謝的影響及其調(diào)控機(jī)制。本試驗(yàn)選取360只1日齡健康的雄性北京鴨,隨機(jī)分為三個(gè)處理組:核黃素缺乏組、采食配對(duì)組(人為控制該組與核黃素缺乏組采食量一致)和自由采食對(duì)照組,每個(gè)處理組12個(gè)重復(fù),每個(gè)重復(fù)10只鴨,試驗(yàn)期為21天。與采食配對(duì)組和自由采食對(duì)照組相比,核黃素缺乏顯著降低了生長(zhǎng)前期北京鴨平均日增重,顯著提高了料重比和死亡率;顯著降低了血漿和肝臟核黃素含量;顯著提高了血漿和肝臟中甘油三酯和總膽固醇含量、肝臟總飽和脂肪酸、C6:0、C12:0、C16:0、C18:0含量和肝臟指數(shù)。這三組肝臟蛋白質(zhì)組學(xué)分析顯示,與采食配對(duì)組和自由采食對(duì)照組相比,核黃素缺乏導(dǎo)致63個(gè)蛋白質(zhì)表達(dá)量變化大于1.5倍,其中包括32個(gè)上調(diào)蛋白質(zhì)和31個(gè)下調(diào)蛋白。GO聚類(lèi)分析結(jié)果顯示差異蛋白主要富集在脂肪酸氧化和線(xiàn)粒體呼吸鏈電子傳遞過(guò)程。核黃素缺乏組中參與脂肪酸β氧化過(guò)程的ACADS、ACADM、ACAD9和ETFDH蛋白表達(dá)量下調(diào),提示脂肪酸β氧化受損,脂肪分解減少,進(jìn)而導(dǎo)致肝臟脂肪沉積。核黃素缺乏組中參與線(xiàn)粒體呼吸鏈電子傳遞過(guò)程的ACAD9、NDUFS1、NDUFA8和FXN蛋白表達(dá)量下調(diào),提示呼吸鏈電子傳遞過(guò)程受損,進(jìn)而導(dǎo)致ATP生成不足,影響動(dòng)物生長(zhǎng)。試驗(yàn)三旨在研究種母鴨核黃素對(duì)母鴨繁殖性能和子代胚胎發(fā)育的影響。本試驗(yàn)選取80只45周齡產(chǎn)蛋期北京鴨,隨機(jī)分為兩個(gè)組:核黃素缺乏組和對(duì)照組,分別飼喂核黃素添加量為0和10mg/kg的試驗(yàn)日糧,試驗(yàn)期為8周。種母鴨日糧核黃素對(duì)試驗(yàn)期母鴨體重、產(chǎn)蛋率、蛋重、種蛋受精率和子代初生重沒(méi)有顯著影響;從試驗(yàn)第2周開(kāi)始,核黃素缺乏組種母鴨血漿核黃素和種蛋黃核黃素含量顯著降低,種蛋孵化率急劇降低,試驗(yàn)第6周以后種蛋孵化率降為0。試驗(yàn)第8周種蛋孵化第13天胚胎肝臟蛋白質(zhì)組學(xué)分析結(jié)果顯示,種母鴨核黃素缺乏導(dǎo)致187個(gè)蛋白質(zhì)表達(dá)量變化大于1.5倍,其中包括67個(gè)上調(diào)蛋白質(zhì)和120個(gè)下調(diào)蛋白。KEGG通路分析顯示差異蛋白主要富集在三羧酸循環(huán)、脂肪酸β氧化和呼吸鏈電子傳遞等代謝過(guò)程。核黃素缺乏導(dǎo)致胚胎肝臟脂肪酸β氧化(ETFDH、CPT1A、ACSL1、ACADS、ACAT1、ACSL5、DECR1 和 ETFA)、三羧酸循環(huán)(DLD、SDHB、IDH1、SDHA 和 ACO1)和呼吸鏈電子傳遞(NDUFA9、NDUFS1、NDUFV1、NDUFA10和ACAD9)過(guò)程中蛋白表達(dá)下調(diào),提示這些代謝過(guò)程可能受阻,進(jìn)而導(dǎo)致ATP產(chǎn)生不足,導(dǎo)致胚胎發(fā)育不良甚至死亡。試驗(yàn)四分為3個(gè)體外試驗(yàn),旨在研究核黃素對(duì)HepG2細(xì)胞增殖和線(xiàn)粒體功能的影響。試驗(yàn)1研究了不同核黃素耗竭時(shí)間對(duì)HepG2線(xiàn)粒體功能的影響。HepG2細(xì)胞在核黃素缺乏和核黃素充足的培養(yǎng)基中分別培養(yǎng)2、4、6、8、10和12天,試驗(yàn)結(jié)束時(shí)采用Seahorse測(cè)定細(xì)胞線(xiàn)粒體功能。與核黃素充足組相比,核黃素缺乏組HepG2細(xì)胞從試驗(yàn)第2天起最大耗氧量和呼吸潛力顯著降低,隨著試驗(yàn)周期的延長(zhǎng),最大耗氧量和呼吸潛力進(jìn)一步降低,提示線(xiàn)粒體功能受損。試驗(yàn)2研究了不同核黃素水平對(duì)HepG2細(xì)胞增殖和線(xiàn)粒體功能的影響。HepG2細(xì)胞在核黃素添加水平為0、0.5、5、10、20、40、100和1064nmol/L的培養(yǎng)基中培養(yǎng)8天。在試驗(yàn)第8天,培養(yǎng)基中不添加核黃素組HepG2細(xì)胞數(shù)量、最大呼吸量和呼吸潛力顯著低于核黃素添加組,隨著核黃素添加水平的提高,這些指標(biāo)逐漸提高,當(dāng)核黃素水平分別提高到10、20和20nmol/L時(shí)到達(dá)平臺(tái)期。試驗(yàn)3研究了補(bǔ)充核黃素對(duì)HepG2細(xì)胞增殖和線(xiàn)粒體功能的影響。HepG2細(xì)胞在不添加核黃素的培養(yǎng)基中培養(yǎng)8天,隨后在添加不同水平的核黃素(0、0.5、5和1064nmol/L)的培養(yǎng)基中培養(yǎng)4天。培養(yǎng)基中添加5nmol/L核黃素對(duì)試驗(yàn)第4天細(xì)胞基礎(chǔ)呼吸量、最大呼吸量和呼吸潛力均有顯著的提高;培養(yǎng)基中添加1064nmol/L核黃素可顯著提高細(xì)胞數(shù)量,顯著提高基礎(chǔ)呼吸量、最大呼吸量和呼吸潛力,完全恢復(fù)線(xiàn)粒體呼吸功能。以上結(jié)果表明,HepG2細(xì)胞在不添加核黃素的培養(yǎng)基中培養(yǎng)2天后線(xiàn)粒體呼吸功能受損,8天后細(xì)胞增殖速率降低。培養(yǎng)基中添加20nmol/L核黃素可維持HepG2細(xì)胞細(xì)胞正常生長(zhǎng)和線(xiàn)粒體呼吸功能。HepG2細(xì)胞在不添加核黃素的培養(yǎng)基中培養(yǎng)8天,培養(yǎng)基中添加1064nmol/L核黃素培養(yǎng)4天可顯著提高細(xì)胞增殖速率,并完全恢復(fù)線(xiàn)粒體呼吸功能。以上結(jié)果表明,核黃素缺乏可導(dǎo)致胚胎期和生長(zhǎng)前期北京鴨生長(zhǎng)發(fā)育不良、肝臟脂肪蓄積。肝臟蛋白質(zhì)組學(xué)分析發(fā)現(xiàn),核黃素缺乏導(dǎo)致肝臟線(xiàn)粒體脂肪酸β氧化(ACADS、ACAD9和ETFDH)、呼吸鏈電子傳遞(ACAD9和NDUFS1)和三羧酸循環(huán)(DLD)過(guò)程中關(guān)鍵蛋白表達(dá)量下調(diào),阻礙脂肪分解導(dǎo)致脂肪蓄積;阻礙能量生成,導(dǎo)致動(dòng)物生長(zhǎng)發(fā)育不良。體外試驗(yàn)結(jié)果顯示,核黃素缺乏可導(dǎo)致HepG2細(xì)胞線(xiàn)粒體功能受損,補(bǔ)充核黃素后可恢復(fù)線(xiàn)粒體功能。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S834.5
本文編號(hào)：2237683
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S834.5
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