丁酸梭菌对肉鸡脂肪代谢的影响及其机理研究
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摘要
本研究旨在探讨丁酸梭菌对肉鸡脂肪代谢的影响及其作用机理。分体内试验和体外试验两部
分进行研究。第一部分通过体内试验研究丁酸梭菌和屎肠球菌配伍对肉鸡生产性能、脂肪代谢和盲肠微生
物的影响。试验选用1日龄健康罗斯308肉公鸡264只,随机分为4个处理,每个处理6个重复,
每个重复11只鸡。处理1组饲喂基础日粮,其余3组分别在基础日粮的基础上添加丁酸梭菌1×109
CFU/kg,屎肠球菌2×109CFU/kg,丁酸梭菌1×109CFU/kg+屎肠球菌2×10-CFU/kg。试验期为
42d。结果发现,日粮中添加丁酸梭菌和屎肠球菌对肉鸡的生产性能、脂肪代谢和盲肠微生物均
不存在互作效应,但日粮中添加屎肠球菌显著降低了21d肉鸡血清瘦素水平(P=0.022),提高了
42d肉鸡肝脏中的脂肪酸合成酶(FAS)、苹果酸酶(ME)和乙酰辅酶A羧化酶(ACC)mRNA
水平(P<0.001)；日粮中添加丁酸梭菌显著提升了肉鸡的平均日采食量和日增重(P<0.05),提
高了21d肉鸡的血清胰岛素水平(P=0.016)和42d肉鸡的肌内脂肪含量、肝脏FAS和胸肌脂蛋
白酯酶(LPL)活性以及肝脏中FAS、ME、ACC和胸肌LPL mRNA表达(P<0.05),降低了21d
肉鸡盲肠中拟杆菌门的数量(P=0.030)。以上结果表明,丁酸梭菌组肉鸡肌内脂肪含量的增加与
肉鸡脂肪合成能力的增强密切相关。第二部分通过体外试验研究丁酸梭菌及其组份对脂肪代谢的影响。试验首先通过丁酸梭菌
1×106、1×107和1×108CFU/m1分别与Caco-2细胞共培养2h,研究了丁酸梭菌对Caco-2细胞的
细胞毒性和血管生成素样蛋白4(ANGPTL4)mRNA表达的影响。结果发现,丁酸梭菌在此添加
剂量范围内对Caco-2细胞成活率无影响,但丁酸梭菌1×107CFU/ml显著提高了Caco-2细胞的
ANGPTL4mRNA表达。之后就丁酸梭菌1×107CFU/ml的各组份对Caco-2细胞ANGPTL4的影
响进行了探究,结果发现,丁酸梭菌的细胞壁成分和其代谢产物均可以促进Caco-2细胞的
ANGPTL4mRNA表达和蛋白分泌。为了确定是否是丁酸梭菌的主要代谢产物丁酸起到了促进
Caco-2细胞ANGPTL4产生的作用,试验就单羧酸转运蛋白1(MCT1)在丁酸梭菌代谢产物影
响Caco-2细胞ANGPTL4中的作用和0.1mM丁酸对Caco-2细胞ANGPTL4的影响进行了研究。
最终确定了促进Caco-2细胞ANGPTL4产生的丁酸梭菌的代谢产物的主要成分就是丁酸。此外,
试验中还发现,活的丁酸梭菌、灭活的丁酸梭菌以及丁酸分别作用于Caco-2细胞2h后所产生的
可溶性成分均具有降低HepG2细胞脂肪合成代谢相关基因表达的作用。以上结果表明,丁酸梭
菌1×107CFU/ml可以通过其细菌壁成分和其主要的代谢产物如丁酸来降低脂肪生成。综上所述,丁酸梭菌本身具有的功能是降低脂肪沉积,其发挥作用的有效成分是其细菌壁成
分和其主要的代谢产物丁酸。而丁酸梭菌提高肉鸡肌内脂肪沉积的现象可能与丁酸梭菌改变了肉
鸡肠道中的菌群结构进而促进了肉鸡的脂肪合成有关。
Two experiments were conducted to investigate the effects of Clostridium butyricum on lipid metabolism of broiler chickens and its underlying mechanism.
Exp.1To investigate the effects of Clostridium butyricum and Enterococcus faecium on growth performance, lipid metabolism and cecal microbiota of broilers,264one-d-old male Ross308broiler chicks were randomly allocated into4treatments with6replicates in a2x2factorial arrangement and fed4diets with2levels of C. butyricum (0or1×109CFU/kg) and2levels of E.faecium (0or2×109CFU/kg) for42d. There was no significant interaction between C. butyricum and E. faecium on growth performance, lipid metabolism and cecal microbiota of broilers. However, broilers supplemented with E. faecium had lower (P=0.022) serum leptin level at d21and higher (P<0.001) fatty acid synthase (FAS), malic enzyme (ME) and acetyl-CoA carboxylase (ACC) mRNA levels in the liver at d42. Supplementation of C. butyricum improved (P<0.05) average daily feed intake and average daily gain, increased (P=0.016) serum insulin level at21d of age, enhanced (P<0.05) content of intramuscular fat, activities of FAS in the liver and lipoprotein lipase (LPL) in the breast muscle, mRNA expression of FAS, ME and ACC in the liver and LPL in the breast muscle at42d of age, but reduced (P=0.030) cecal Bacteroidetes relative abundance at21d of age. The results of this study indicate that the increased intramuscular fat content of broilers fed C. butyricum as observed may be the result of enhanced lipogenesis.
Exp.2The objective of this study was to assess the possibility that C. butyricum and its potential components and metabolites could regulate lipogenesis. Co-culture experiments of Caco-2cells and1×106,1×107, and1×108CFU/ml of C. butyricum were set up to test the cytotoxicity of C. butyricum and the changes of angiopoietin-like protein4(ANGPTL4) mRNA expression. It was found that cell viability was not affected by C. butyricum and ANGPTL4mRNA expression in Caco-2cells was highly induced by1×107CFU/ml of C. butyricum. Co-culture experiment of Caco-2cells and potential components of C. butyricum was set up to monitor any ensuing alteration in ANGPTL4. It was observed that bacterial wall components and potentially secreted substances from C. butyricum could induce ANGPTL4mRNA expression and protein secretion. To determine whether butyrate could affect the ANGPTL4production in Caco-2cells, the role of monocarboxylate transporter1(MCT1) in mediating potentially secreted factors from C. butyricum-induced ANGPTL4production in Caco-2cells and the effect of0.1mM of butyrate on ANGPTL4production in Caco-2cells were investigated. It's confirmed that butyrate was the factor secreted by C. butyricum that stimulate ANGPTL4production. Besides, the soluble factors secreted by live C. butyricum-Caco-2cells interaction, bacterial wall components-Caco-2cells interaction, and the main metabolites butyrate-Caco-2cells interaction reduced lipogenic genes expression in HepG2cells. The results of this study indicate that1×107CFU/ml of C. butyricum could reduce lipogenesis through the bacterial wall components and the metabolites such as butyrate.
In conclusion, C. butyricum per se could reduce lipogenesis through both the bacterial wall components and the metabolites such as butyrate. However, the enhanced intramuscular fat of broilers in C. butyricum-supplemented groups observed in this study may be partially attributed to a changed microecological environment which promoted fat synthesis.
分进行研究。第一部分通过体内试验研究丁酸梭菌和屎肠球菌配伍对肉鸡生产性能、脂肪代谢和盲肠微生
物的影响。试验选用1日龄健康罗斯308肉公鸡264只,随机分为4个处理,每个处理6个重复,
每个重复11只鸡。处理1组饲喂基础日粮,其余3组分别在基础日粮的基础上添加丁酸梭菌1×109
CFU/kg,屎肠球菌2×109CFU/kg,丁酸梭菌1×109CFU/kg+屎肠球菌2×10-CFU/kg。试验期为
42d。结果发现,日粮中添加丁酸梭菌和屎肠球菌对肉鸡的生产性能、脂肪代谢和盲肠微生物均
不存在互作效应,但日粮中添加屎肠球菌显著降低了21d肉鸡血清瘦素水平(P=0.022),提高了
42d肉鸡肝脏中的脂肪酸合成酶(FAS)、苹果酸酶(ME)和乙酰辅酶A羧化酶(ACC)mRNA
水平(P<0.001)；日粮中添加丁酸梭菌显著提升了肉鸡的平均日采食量和日增重(P<0.05),提
高了21d肉鸡的血清胰岛素水平(P=0.016)和42d肉鸡的肌内脂肪含量、肝脏FAS和胸肌脂蛋
白酯酶(LPL)活性以及肝脏中FAS、ME、ACC和胸肌LPL mRNA表达(P<0.05),降低了21d
肉鸡盲肠中拟杆菌门的数量(P=0.030)。以上结果表明,丁酸梭菌组肉鸡肌内脂肪含量的增加与
肉鸡脂肪合成能力的增强密切相关。第二部分通过体外试验研究丁酸梭菌及其组份对脂肪代谢的影响。试验首先通过丁酸梭菌
1×106、1×107和1×108CFU/m1分别与Caco-2细胞共培养2h,研究了丁酸梭菌对Caco-2细胞的
细胞毒性和血管生成素样蛋白4(ANGPTL4)mRNA表达的影响。结果发现,丁酸梭菌在此添加
剂量范围内对Caco-2细胞成活率无影响,但丁酸梭菌1×107CFU/ml显著提高了Caco-2细胞的
ANGPTL4mRNA表达。之后就丁酸梭菌1×107CFU/ml的各组份对Caco-2细胞ANGPTL4的影
响进行了探究,结果发现,丁酸梭菌的细胞壁成分和其代谢产物均可以促进Caco-2细胞的
ANGPTL4mRNA表达和蛋白分泌。为了确定是否是丁酸梭菌的主要代谢产物丁酸起到了促进
Caco-2细胞ANGPTL4产生的作用,试验就单羧酸转运蛋白1(MCT1)在丁酸梭菌代谢产物影
响Caco-2细胞ANGPTL4中的作用和0.1mM丁酸对Caco-2细胞ANGPTL4的影响进行了研究。
最终确定了促进Caco-2细胞ANGPTL4产生的丁酸梭菌的代谢产物的主要成分就是丁酸。此外,
试验中还发现,活的丁酸梭菌、灭活的丁酸梭菌以及丁酸分别作用于Caco-2细胞2h后所产生的
可溶性成分均具有降低HepG2细胞脂肪合成代谢相关基因表达的作用。以上结果表明,丁酸梭
菌1×107CFU/ml可以通过其细菌壁成分和其主要的代谢产物如丁酸来降低脂肪生成。综上所述,丁酸梭菌本身具有的功能是降低脂肪沉积,其发挥作用的有效成分是其细菌壁成
分和其主要的代谢产物丁酸。而丁酸梭菌提高肉鸡肌内脂肪沉积的现象可能与丁酸梭菌改变了肉
鸡肠道中的菌群结构进而促进了肉鸡的脂肪合成有关。
Two experiments were conducted to investigate the effects of Clostridium butyricum on lipid metabolism of broiler chickens and its underlying mechanism.
Exp.1To investigate the effects of Clostridium butyricum and Enterococcus faecium on growth performance, lipid metabolism and cecal microbiota of broilers,264one-d-old male Ross308broiler chicks were randomly allocated into4treatments with6replicates in a2x2factorial arrangement and fed4diets with2levels of C. butyricum (0or1×109CFU/kg) and2levels of E.faecium (0or2×109CFU/kg) for42d. There was no significant interaction between C. butyricum and E. faecium on growth performance, lipid metabolism and cecal microbiota of broilers. However, broilers supplemented with E. faecium had lower (P=0.022) serum leptin level at d21and higher (P<0.001) fatty acid synthase (FAS), malic enzyme (ME) and acetyl-CoA carboxylase (ACC) mRNA levels in the liver at d42. Supplementation of C. butyricum improved (P<0.05) average daily feed intake and average daily gain, increased (P=0.016) serum insulin level at21d of age, enhanced (P<0.05) content of intramuscular fat, activities of FAS in the liver and lipoprotein lipase (LPL) in the breast muscle, mRNA expression of FAS, ME and ACC in the liver and LPL in the breast muscle at42d of age, but reduced (P=0.030) cecal Bacteroidetes relative abundance at21d of age. The results of this study indicate that the increased intramuscular fat content of broilers fed C. butyricum as observed may be the result of enhanced lipogenesis.
Exp.2The objective of this study was to assess the possibility that C. butyricum and its potential components and metabolites could regulate lipogenesis. Co-culture experiments of Caco-2cells and1×106,1×107, and1×108CFU/ml of C. butyricum were set up to test the cytotoxicity of C. butyricum and the changes of angiopoietin-like protein4(ANGPTL4) mRNA expression. It was found that cell viability was not affected by C. butyricum and ANGPTL4mRNA expression in Caco-2cells was highly induced by1×107CFU/ml of C. butyricum. Co-culture experiment of Caco-2cells and potential components of C. butyricum was set up to monitor any ensuing alteration in ANGPTL4. It was observed that bacterial wall components and potentially secreted substances from C. butyricum could induce ANGPTL4mRNA expression and protein secretion. To determine whether butyrate could affect the ANGPTL4production in Caco-2cells, the role of monocarboxylate transporter1(MCT1) in mediating potentially secreted factors from C. butyricum-induced ANGPTL4production in Caco-2cells and the effect of0.1mM of butyrate on ANGPTL4production in Caco-2cells were investigated. It's confirmed that butyrate was the factor secreted by C. butyricum that stimulate ANGPTL4production. Besides, the soluble factors secreted by live C. butyricum-Caco-2cells interaction, bacterial wall components-Caco-2cells interaction, and the main metabolites butyrate-Caco-2cells interaction reduced lipogenic genes expression in HepG2cells. The results of this study indicate that1×107CFU/ml of C. butyricum could reduce lipogenesis through the bacterial wall components and the metabolites such as butyrate.
In conclusion, C. butyricum per se could reduce lipogenesis through both the bacterial wall components and the metabolites such as butyrate. However, the enhanced intramuscular fat of broilers in C. butyricum-supplemented groups observed in this study may be partially attributed to a changed microecological environment which promoted fat synthesis.
引文
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