Effects Of Arginine And Arginine-containing Dipeptide On Casein Synthesis In Bovine Mammary Epithelial Cells

This thesis studies the effect of adding different concentrations of arginine(Arg)and four arginine-containing peptides replaceing a certain proportion of free Arg on casein synthesis in bovine mammary epithelial cells(BMECs),and explains the mechanism of key molecules and protein expression of mammalian target of rapamycin(m TOR)and tyrosine kinase 2-transduction activator 5(JAK2-STAT5),so as to establish a suitable Arg supply mode for BMECs and provide a theoretical basis for the regulation of dairy protein synthesis.This thesis includes three experiments.In experiment 1,the effect of different concentration of Arg(0.70,1.40,2.80,5.60,11.20 mmol/L)on BMECs activity,casein synthesis and arginine metabolism were studied.The results showed that: when the concentration of Arg was 2.80 mmol/L,the BMECs activity,gene expression levels of CSN1S1,AKT,S6K1,αs1-casein,arginase gene expression levels and activity were significantly higher than those in control group and other Arg groups(P<0.05).When the concentration of Arg was 2.80 mmol/L and 5.60mmol/L,the gene expression levels of CSN3 and 4EBP1,β-casein,m TOR protein phosphorylation and ornithine decarboxylase in BMECs were significantly higher than those in control group and 11.20 mmol/L Arg group(P<0.05).Especially 2.80 mmol/L had the best effect.In experiment 2,based on the optimal Arg concentration(2.80 mmol/L)selected in Experiment 1 for promoting the synthesis of casein in BMECs,to study the effect of four kinds of arg-containing dipeptides replaceing a certain proportion of free Arg on casein synthesis in BMECs.The addition amount of free Arg in control group was 2.80 mmol/L.The total amount of Arg in experimental group was the same as that in control group.four kinds of arg-containing dipeptides(arginine-arginine dipeptide,A-A;arginine-histidine dipeptide,A-H;arginine-lysine dipeptide,A-L;arginine-methionine,A-M)replacing free Arg was 10%,20% and 30%.The results showed that: When the proportion of A-A and A-L replacing free Arg was 10%,the gene expression levels of PI3 K,m TOR,4EBP1,S6K1 and CSN1S1 in BMECs were significantly increased(P<0.05).When the proportion of A-H and A-M replacing free Arg was 20%,the gene expression levels of PI3 K,m TOR and S6K1 in BMECs were significantly increased(P<0.05),and the gene expression levels of CSN1S1 in A-M replacement group were significantly increased(P<0.05).In conclusion,when the proportion of A-A,A-H,A-L and A-M replacing free Arg was 10%,20%,10% and 20%,the promotion effect on casein gene expression in BMECs was better.Experiment 3 used a single factor experimental design and was divided into five groups.The concentration of free Arg in the control group was 2.80 mmol/L,and the experimental group was the optimal replacement ratio group of four dipeptides that could promote the synthesis of BMECs casein obtained in the second study(10% A-A replacement group,20% A-H replacement group,10% A-L substitution group and 20%A-M replacement group)to compare the effect of four suitable substitution ratios of Arg-containing dipeptide in promoting casein synthesis in BMECs.The results showed that: When the ratio of arginine-arginine to replace free Arg was 10%,and the ratio of arginine-histidine and arginine-methionine to replace free Arg was 20%,the proliferation of BMECs was significantly promoted(P< 0.05),up-regulated the gene expression of Pep T2(P< 0.05).When the ratio of A-A to replace free Arg was 10% and the ratio of A-M to replace free Arg was 20%,by up-regulating the expressions of PI3 K and AKT upstream of m TOR,the protein phosphorylation level of m TOR was increased(P<0.05),and the downstream e IF4 E was up-regulated.The gene expression level(P< 0.05),and the phosphorylation level of the downstream S6 K protein was also increased(P < 0.05),which promoted the synthesis of casein in BMECs.