天冬酰胺调控MAC-T合成α-酪蛋白相关基因及通路研究OA

[Objective]The nutrient value of milk is largely determined by its milk protein content.Amino acids,as precursors and critical regulatory factors in milk protein synthesis,have drawn significant attention for their role in regulating casein synthesis.However,the regulatory function of asparagine,a functional amino acid,in bovine mammary epithelial cells remains unclear.This study investigated how varying asparagine concentrations in the culture medium affect α-casein synthesis and secretion in MAC-T.The study,and explored the regulatory role of asparagine on α-casein encoding genes and key factors in PI3K-Akt-mTOR signaling pathway,providing a theoretical support for a deeper understanding of amino acid nutritional functions and the development of precise mammary gland nutritional strategies.[Method]Utilizing MAC-T as the model,cells cultured in serum-free medium were designated as the negative control group(0×),while cells cultured in 10%FBS medium were designated as the positive control group(PC).The experimental groups were supplemented with 0.982,1.964,3.927,7.855,15.710,and 31.418 mmol/L asparagine extract(1×,2×,4×,8×,16×,and 32×groups),respectively,with three replicates per group.After a 12-hour treatment period,the levels of cell α-casein synthesis and secretion were measured using an ELISA assay.2×,4×,8×,and 16×groups were selected based on these results.Real-time quantitative PCR was utilized to assess the expression of α-casein encoding genes(CSN1S1 and CSN1S2)and the genes located upstream(PI3K,AKT1,and TSC2)and downstream(mTOR,EIF4EBP1,EIF4E,S6K1,and RPS6)of PI3K-Akt-mTOR signaling pathway.The phosphorylation levels of key proteins upstream(PI3K,AKT,and TSC2)and downstream(m TOR,4EBP1,S6K1,and RPS6)in this pathway were detected using Western blotting.[Result]Compared with 0×group,① The α-casein synthesis in cells was significantly elevated in 2×,4×,and 8×groups(P<0.05),and α-casein secretion was significantly enhanced across 2×,4×,8×,and 16×groups(P<0.05).② CSN1S1 gene expression in cells was significantly upregulated in 4×group(P<0.05),and CSN1S2 gene expression was significantly increased in both 4×and 8×groups(P<0.05).③ The expression of PI3K gene in cells was significantly upregulated in 4×,8×,and 16×groups(P<0.05).The expression of AKT1 gene was significantly elevated in 2×,4×,and 8×groups(P<0.05),whereas TSC2 gene expression was significantly downregulated in all asparagine-supplemented groups(P<0.05),and the phosphorylation of PI3K was significantly enhanced(P<0.05).Furthermore,the phosphorylation of both AKT and TSC2 proteins was significantly increased in 2×,4×,and 8×groups(P<0.05).④ The expression of mTOR and EIF4EBP1 genes in cells were significantly increased in 4×and 8×groups(P<0.05).The expression of EIF4E and RPS6 genes were significantly increased in 2×,4×,and 8×groups(P<0.05).The expression of S6K1 gene was significantly increased in all asparagine-supplemented groups(P<0.05).The phosphorylation of mTOR protein in cells was significantly increased in 2×,4×,and 8×groups(P<0.05).The phosphorylation of 4EBP1 and S6K1 proteins were significantly increased in 4×,8×,and 16×groups(P<0.05),and the phosphorylation of RPS6 protein was significantly increased in all asparagine-supplemented groups(P<0.05).[Conclusion]Asparagine promoted the synthesis and secretion of α-casein in MAC-T with a dose-dependent manner by up-regulating the expression of CSN1S1 and CSN1S2 genes and activating PI3K-Akt-mTOR pathway.The addition of 1.964,3.927,and 7.855 mmol/L asparagine could significantly promote the above processes,while the addition of 15.710 mmol/L asparagine only promote secretion and some downstream signals.Among them,adding 3.927 mmol/L asparagine had the best effect.