| This experiment adopts the method of combining in vivo and hepatic cell culture in vitro to explore the mechanism of 5-hydroxytryptophan’s influence on hepatic gluconeogenesis in perinatal ewe from body,tissue and cell level.It provides a theoretical basis for scientifically and effectively improving the energy negative balance,the health and production performance of ewes during perinatal period.The experimental research of this thesis includes four parts.Experiment 1,Effects of serotonin on perinatal ewe performance.Thirty ewes of bamei sheep with a body weight of 64.52±4.11 kg in the same perinatal period were randomly divided into three groups according to their body weight:the control group(saline infusion),the 5-HTP group(5-hydroxyl-tryptophan infusion,5-HTP)and the TRP group(tryptophan infusion),with 10 ewes in each treatment group.In the 5-HTP group and TRP group,jugular vein perfusion was conducted from the 7th day of antepartum to the 0th day of postpartum.The perfusion dose of 5-hydroxytryptophan and tryptophan was 0.178 mg/kg BW and the concentration was 0.1 mg/ml,to study the effects of serotonin on perinatal ewe weight and milk production.The results showed that:1)5-HTP can significantly improve lactation dry matter intake of ewes(P<0.05),Serotonin has a significant effect on postpartum weight gain in ewes(P<0.05).2)There was no significant effect of serotonin on milk yield and milk protein content of ewes(P>0.05),but the 3d milk fat content and non-fat solid content of ewes after delivery were reduced,and the lactose content in milk was significantly increased(P<0.05).Experiment 2,Effects of serotonin on blood physicochemical indexes related to glucose metabolism in perinatal ewes.The grouping and feeding management of animals in this experiment are the same as experiment 1.On the basis of experiment 1,jugular vein blood samples were collected on the 1st day of antepartum,the 3rd,6th,9th,15th and 30th day of postpartum respectively,to study the effects of serotonin on blood physicochemical indexes related to glucose metabolism in perinatal ewes.The results showed that:1)Infusion of 5-hydroxytryptophan and tryptophan could significantly increase the serotonin concentration from the 7th day of antepartum to the 9th day of postpartum during the ewe perinatal period(P<0.05).2)Serotonin can significantly increase the blood glucose concentration of perinatal ewe from prenatal to the 9th day of postpartum,and reduce the plasma NEFA concentration(P<0.05).INS and IGF-1 increased with the increase of blood glucose concentration,and serotonin significantly reduced the INS and IGF-1 concentrations from antepartum to 9th day of postpartum of perinatal ewe(P<0.05).Experiment 3,Effects of serotonin on mRNA expression and protein expression of key genes in hepatic gluconeogenesis.The grouping and feeding management of animals in this experiment are the same as experiment 1.In this study,on the basis of experiment 1,the animals were slaughtered after feeding experiment,and the mRNA and protein expressions of gluconeogenic rate-limiting enzyme genes in the liver were measured by real-time quantitative PCR and western blotting.The results showed that:1)Serotonin significantly increased the mRNA expression of PEPCK,G6P and PC of hepatic gluconeogenesis key genes in the 9th day of postpartum(P<0.05),and played a role in promoting hepatic gluconeogenesis.Perfusion of 5-HTP was more advantageous than perfusion of L-TRP in promoting the expression of hepatic gluconeogenesis key genes.2)Serotonin can significantly increase the protein expression of PEPCK,G6P and PC of hepatic gluconeogenesis key genes in the 9th day of postpartum(P<0.05).This suggests that serotonin plays a role in promoting the hepatic gluconeogenesis not only at the transcriptional level,but also at the translation level.Experiment 4,Effects of serotonin on PI3K-AKT-FOXO1 signaling pathway in perinatal ewe liver.This experiment were divided into two parts.The first part was the influence of serotonin on protein expression of PI3K-AKT-FOXO1 signaling pathway key factors in ewe hepatic tissues.The second part was the effect of serotonin on PI3K-AKT-FOXO1 signaling pathway in ewe hepatic cells.The first part of the experiment animal grouping and feeding management with experiment 1.On the basis of experiment 1,the animals were slaughtered after feeding and western blotting technique was used to determine the protein expression of PI3K-AKT-FOXO1 signaling pathway key factors in perinatal ewe hepatic tissues.Results show that:the 5-HTP and L-TRP perfusion significantly reduced the PI3K、AKT and p-FOXO1 activity of PI3K-AKT-FOXO1 signaling pathways key genes of the hepatic tissues in 9th of postpartum ewe(P<0.01),significantly reduced the protein expression of p-FOXO1(P<0.01),increased the protein expression of FOXOlof hepatic tissue(P<0.01),and to promote the hepatic gluconeogenesis.Perfusion of 5-HTP was more advantageous than perfusion of L-TRP in promoting the expression of hepatic gluconeogenesis key genes.The second part of the experiment was in vitro method.At first,the ewe hepatic cells were randomly divided into three treatment groups:basic culture medium group,NEFA group and 5-HT+NEFA group under different NEFA concentration and treatment time.Secondly,the ewe hepatic cells were divided into three treatment groups:control group,5-HT group and 5-HT+transfection group at different concentrations of 5-HT.Real-time quantitative PCR,western blotting and gene transfection techniques were used to determine the mRNA and protein expressionof gluconeogenesis and PI3K-AKT-FOXO1 signaling pathway key genes in hepatic cells.The results showed that:1)When the NEFA concentration was 2.4mmol/L and the action time was 24h,the proliferation rate decreased by 22%,and the PEPCK concentration increased significantly(P<0.01).2)When 5-HT concentration was 10-3 mol/L,the cell proliferation rate was 293.03%,NEFA concentration of hepatic cells was significantly decreased(P<0.01),and PEPCK concentration was significantly increased(P<0.01).3)Add NEFA significantly increased the protein expression of PEPCK,PC and G6P of hepatic gluconeogenesis(P<0.01),significantly increase the protein expression of t-FOXO1of PI3K-AKT-FOXO1 key genes(P<0.01),significantly reduced the protein expression of INSR,p-PI3K and p-AKT(P<0.01),significantly reduced the protein expression of phosphorylation FOXO1 and p-FOXO1/t-FOXO1(P<0.01);4)Add 5-HT can significantly increase the protein expression of PEPCK,G6P and FOXOl(P<0.01),a slightly higher of PC protein expression but no significant difference between groups(P>0.05),the protein expression of INSR,p-PI3K and p-AKTdecreased significantly(P<0.01),the protein expression ofr-FOXO1/significantly increased(P<0.01),phosphorylation FOXOl and p-FOXO1/t-FOXO1 significantly reduced(P<0.01);5)When PI3K gene was silenced,5-HT significantly increased the protein expression of t-FOXOIof PI3K-AKT-FOXO1 signaling pathway key gene(P<0.01),and significantly reduced the protein expression of INSR,p-PI3K,p-AKT,p-FOXO1 and p-FOXO1/t-FOXO1(P<0.01).In conclusion,serotonin can reduce the levels of NEFA that product by lipid mobilization,raise blood sugar levels,serotonin can regulate the protein expression of INSR,p-PI3K,p-AKT in PI3K-AKT-FOXO1 key genes,promote the protein expression of PEPCK,PC and G6Pof hepatic gluconeogenesis,improve energy negative balance has a positive role. |
