| Chapter 1 Molecular cloning and tissue expression analysis of HMGCR in Ctenopharyngodon idella3-hydroxy-3-methylglutaryl-Coenzyme A reductase(HMGCR) is a key rate-limiting enzyme which plays an important role in the maintenance of cholesterol homeostasis. In this study, using rapid amplification of c DNA ends(RACE) method, the complementary c DNA encoding HMGCR of Ctenopharyngodon idella was cloned. HMGCR was 3594 bp and encoded 842 amino acids with predicted molecular mass of 91.48 k Da, theoretical isoelectric point of 6.27, 5’ untranslated region(5’-UTR) long 77 bp, 3’ untranslated region(3’-UTR) long 988 bp, open reading frame(ORF) of 2529 bp. HMGCR protein included two domains: the amino terminal transmembrane domain complex complex(Asp62-Leu219) and the carboxy terminal cytosolic catalytic domain(Asn419-Arg826), the two sequences by a variable hydrophilic linker sequence(Val220-Ser418) are connected, and there are a phosphorylation site(Ser827) and a catalytically active sites(His821), the Met1-Ala39 amino acid residues were signal peptide. Amino acid homology analysis and phylogenetic analysis showed that the evolutionary relationship between Danio rerio, grass carp and Astyanax mexicanus were closed. The tissue expression analysis showed that the gene expression of HMGCR in grass carp detected in seven organizations, including the highest expression in the liver, followed by muscle, kidney and heart. Collectively, the present study demonstratesd the successful isolation of HMGCR from C. idellus, and its activity could be tightly regulated to ensure cholesterol homeostasis. Chapter 2 Molecular cloning and tissue expression analysis of CYP7A1 in Ctenopharyngodon idellaCholesterol 7α-hydroxylase(cholesteral 7 α hydroxylase, CYP7A1) is the critical rate-limiting enzyme in the classic bile acid synthesis pathway which plays an important role in maintaining bile acid homeostasis. In this study, using rapid amplification of c DNA ends(RACE) method, the complementary c DNA encoding CYP7A1 of Ctenopharyngodon idella was cloned. CYP7A1 was 1771 bp and encoded 509 amino acids with predicted molecular mass of 57.74 k Da, theoretical isoelectric point of 6.54, 5’ untranslated region(5’-UTR) long 22 bp, 3’ untranslated region(3’-UTR) long 219 bp, open reading frame(ORF) of 1530 bp. CYP7A1 protein included two domains: the amino terminal which comprising transmembrane helix(Leu5-Leu22) and the carboxy terminal which contains the conserved core sequence. The Met1-Ser24 amino acids were signal peptide. Amino acid homology analysis and phylogenetic analysis showed that the evolutionary relationship between Danio rerio, grass carp and Astyanax mexicanus were closed. The tissue expression analysis showed that the highest gene expression tissue of CYP7A1 detected in liver. Collectively, the present study demonstratesd the successful isolation of CYP7A1 from C. idellus, and its activity could be tightly regulated to ensure bile acid mechanisms.Chapter 3 The effect of cholesterol and bile acid metabolism synthesis pathway related enzymes in the liver and intestine under oxidized fish oil in Ctenopharyngodon idellusA 72-day experiment was carried out to investigate the effect of grass carp cholesterol and bile acid metabolism synthesis pathway in liver and intestine under oxidized fish oil. Five diets containing different levels of soybean oil, fish oil and oxidized fish oil(6% fish oil, 6% soybean oil, 2% oxidized fish oil, 4% oxidized fish oil, 6% oxidized fish oil) were evaluated at the same nitrogen and energy. Using q RT-PCR method for quantitative detection of gene expression levels about cholesterol synthetic and regulation enzyme HMGCR、SREBP2、CETP、ABCA1 and bile acid synthetic enzyme CYP7A1 in liver and intestine, in addition the content of total bile acid(TBA) and total cholesterol(TC) in serum, liver and intestine. The results showed that under oxidized fish oil,(1) in the liver, the gene expression of HMGCR was significantly increased(P<0.05), the gene expressions of ABCA1 and CYP7A1 were significantly reduced(P<0.05), at the same time, the concentrations of TC and TBA were significantly increased(P<0.05);(2) in the intestine, the gene expression of HMGCR was significantly increased(P<0.05), the gene expression of CYP7A1 was significantly reduced(P<0.05),at the same time, the concentration of TC was significantly increased(P<0.05), and the concentration of TBA was significantly decreased(P<0.05);(3) the content of Σ PUFA was significant positive correlation with the ABCA1 gene expression(P<0.05) in the liver. The MDA content was significant negative correlation with the ABCA1 gene expression(P<0.05) in the intestine. Collectively, the present study demonstratesd under the fish oil nutritional and damaged interaction effects, the ability of cholesterol synthesis and intracellular transport cholesterol get enhance, and the capacity of extracellular transport cholesterol and bile acid synthesis get diminish in the liver and intestine. The result led to the cholesterol level increased in the serum, liver and intestine and the bile acid content decreased in the serum and intestine. As for the bile acid content increased in liver, it showed the trend of bile acid deposed in the liver. It indicated that the fish may need more cholesterol to meet the needs of the physiological metabolism, and bile acids may appear insufficient supply in fish.Chapter 4 The spatial and temporal expression pattern of cholesterol and bile acid metabolism synthesis pathway key enzyme in the liver and intestine under oxidized fish oil in Ctenopharyngodon idellusIn order to study the effect of the cholesterol and bile acid metabolism synthesis in liver and intestine under oxidated fish oil at the time and explore the relationship between intestine injuried and liver and intestine cholesterol and bile acid metabolism synthesis under the influence of oxidized fish oil. Four diets containing different levels of soybean oil and oxidized fish oil(6% soybean oil, 2% oxidized fish oil, 4% oxidized fish oil, 6% oxidized fish oil) were evaluated at the same nitrogen and energy. Using q RT-PCR method for quantitative detection of gene expression levels about cholesterol synthetic limited enzyme HMGCR and bile acid synthetic enzyme CYP7A1 in the liver and intestine, in addition the content of total bile acid(TBA) and total cholesterol(TC) in the serum, liver and intestine. The results showed that during 10-30 days under oxidized fish oil,(1) in the serum, liver and intestine, the concentrations of TC and TBA were significantly decreased(P<0.05), at the same time, the gene expression of HMGCR was significantly increased(P<0.05) and the gene expression of CYP7A1 was significantly increased(P<0.05) in the liver and intestine;(2) the Occludin gene expressions in the intestine were significantly reduced(P<0.05) and the activitys of DAO in the serum were significantly lower(P<0.05);(3) the content of MDA was positive correlation with the HMGCR gene expression and was negative correlation with the CYP7A1 gene expression in the liver and intestine;(4) the content of ΣPUFA was negative correlation with the HMGCR gene expression and was positive correlation with the CYP7A1 gene expression in the liver and intestine. During 10-30 days, under oxidized fish oil, in the case of cholesterol content reduced in the liver and intestine, as for the fish oil nutritional and damaged interaction effects, caused the ability of cholesterol synthesis get enhance and the capacity of bile acid synthesis get diminish in the liver and intestine. The result led to the bile acid level decreased in the liver and intestine. Oxidized fish oil could depend on this way by which intestinal mucosa injuried to affect the cholesterol and bile acid metabolism synthesis in the intestine firstly, and then the cholesterol and bile acid metabolism synthesis in the liver affected.Chapter 5 The effect of cholesterol and bile acid metabolism synthesis pathway related enzymes in the liver and intestine under MDA in Ctenopharyngodon idellusA 72-day experiment was carried out to investigate the effect of grass carp cholesterol and bile acid metabolism synthesis pathway in liver and intestine under added MDA in diets. The diets of control group and three experiment groups were evaluated at the same nitrogen and energy. Using q RT-PCR method for quantitative detection of gene expression levels about cholesterol synthetic and regulation enzyme HMGCR、SREBP2、CETP、ABCA1 and bile acid synthetic enzyme CYP7A1 in the liver and intestine tissues, in addition the content of total bile acid(TBA) and total cholesterol(TC) in the serum, liver and intestine. The results showed that under the effect of MDA,(1) in the liver, the gene expression of HMGCR、SREBP2、CETP were significantly increased(P<0.05), the gene expressions of ABCA1 and CYP7A1 were significantly reduced(P<0.05),at the same time, the concentrations of TC was significantly increased(P<0.05), and the concentration of TBA was significantly decreased(P<0.05);(2) in the intestine, the gene expression of HMGCR、SREBP2、CETP、ABCA1 、CYP7A1 was significantly reduced(P<0.05),at the same time, the concentration of TC was significantly increased(P<0.05), and the concentration of TBA was significantly decreased(P<0.05);(3) the content of MDA was positive correlation with the HMGCR、SREBP2、CETP gene expression, and was negative correlation with the CYP7A1、ABCA1 gene expression in the liver;(4) the content of MDA was negative correlation with the HMGCR、SREBP2、CETP、ABCA1 、CYP7A1 gene expression in the intestine. Collectively, the present study demonstrated the MDA content of diets caused the ability of cholesterol synthesis and intracellular transport cholesterol get enhance in the liver, the case were contrast in intestine, at the same time, the capacity of extracellular transport cholesterol and the capacity of synthesized bile acid get diminish in the liver and intestine. The result led to cholesterol levels increased and bile acid content decreased in the serum, liver and intestine. It indicated that the fish may need more cholesterol to meet the needs of the physiological metabolism, and bile acids may appear insufficient supply in the effect of MDA.Chapter 6 The spatial and temporal expression pattern of cholesterol and bile acid metabolism synthesis pathway key enzyme in the liver and intestine under MDA in Ctenopharyngodon idellus In order to study the effect of the cholesterol and bile acid metabolism synthesis in liver and intestine under MDA at the time and explore the relationship between intestine injuried and liver and intestine cholesterol and bile acid metabolism synthesis under the influence of MDA. The diets of control group and three experiment groups were evaluated at the same nitrogen and energy. Using q RT-PCR method for quantitative detection of gene expression levels about cholesterol synthetic limited enzyme HMGCR and bile acid synthetic enzyme CYP7A1 in the liver and intestine, in addition the content of total bile acid(TBA) and total cholesterol(TC) in the serum, liver and intestine. The results showed that during 10-30 days under MDA,(1) in the serum, liver and intestine, the concentrations of TC and TBA were significantly decreased(P<0.05), at the same time, the gene expression of HMGCR was significantly increased(P<0.05) and the gene expression of CYP7A1 was significantly increased(P<0.05) in the liver and intestine;(2) the Occludin gene expressions in the intestine were significantly reduced(P<0.05) and the activitys of DAO in the serum were significantly lower(P<0.05);(3) the content of MDA was positive correlation with the HMGCR gene expression in the liver and was negative correlation with the CYP7A1 gene expression in the liver and intestine;(4) the content of Σ PUFA was negative correlation with the HMGCR gene expression and was positive correlation with the CYP7A1 gene expression in the liver and intestine. During 10-30 days under the effect of MDA, in the case of cholesterol content reduced in the liver and intestine, the ability of cholesterol synthesis get enhance and the capacity of bile acid synthesis get diminish in the liver and intestine. The result led to the bile acid level decreased in the liver and intestine. MDA could depend on this way by which intestinal mucosa injuried to affect the cholesterol and bile acid metabolism synthesis in the intestine firstly, and then the cholesterol and bile acid metabolism synthesis in the liver affected. |
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