| With the continuous improvement of people’s living standards and the increasing emphasis on health concepts,the national demand for milk quality is higher and higher,and the demand is also increasing.High quality milk and dairy derivatives rich in nutrients are favored by people.In the past decades in China,more and more attention has been paid to the improvement of milk composition and milk production performance of dairy cows through feed nutrition regulation,especially the improvement of protein and fatty acid composition of milk.Mammary epithelial cells can synthesize almost all medium and short chain fatty acids(MCFA and SCFA),α-casein andβ-casein.Casein is the main component of milk protein and a key indicator of milk protein content.Lithium,a silver-white alkali metal element,ranks27th in the earth.It is ubiquitous in the crust and various mineral waters,and also has a certain content in plants.So far,as a micronutrient,the effects of lithium on milk protein and milk fat synthesis in dairy cows during lactation are still scarce.Accordingly,based on bovine mammary epithelial cells(MAC-T)as a cell model,my research will probe into the effect of additional lithium chloride(LiCl)on the expression of milk fat and protein synthesis related genes in MAC-T cells and the expression of JAK2/STAT5,PI3K/AKT1/mTOR,Wnt/β-catenin and HIF-1αsignaling pathway related genes in lactation were detected.Firstly,the effect of LiCl on the growth of MAC-T cells was determined.MAC-T cells were seeded in 96-well plates at a density of 1×10~4cells/mL,200μL per well.After 24 h culture,96-well plates were divided into 6 groups with 10 replicates in each group.MAC-T cells were treated with different concentrations of LiCl(0,1,5,10,15 and 20 mM)for 24 h to detect the changes in cell viability.The adherent MAC-T cells were seeded in 6-well plates at a density of 2×10~4cells/mL and cultured in 2 mL growth medium per well.When the cell confluence reached more than 90%,the differentiation medium was replaced and continued to culture.At the same time,the 6-well plates were divided into 4 groups with 3 replicates in each group in random.After 4 days of treatment with 0,5,10 and 20 mM LiCl.Experiment 1:Effects of LiCl on the growth of MAC-T cells and expression of genes which are related to milk fat and protein synthesis.Compared with our control group,(1,5,10,15 and 20 mM)LiCl treatment groups could increased the activity of cells(P<0.05),and showed a trend of increased firstly and then decreased,and the activity of MAC-T cells was the highest at 10 mM.The effects of LiCl on the expression of those genes which are related to not only milk fat but also protein synthesis in MAC-T cells were further studied,and the concentrations of 0,5,10 and 20 mM LiCl were selected for subsequent experiments.The cells were collected to detect the genes and proteins related to milk fat and protein synthesis.The results made clear that,compared with our control group,the 5,10 and 20 mM LiCl treatment groups can increase the protein expression to a great extent ofβ-casein in MAC-T cells(P<0.05);the genes and proteins related to milk fat synthesis were detected.The results revealed that compared with our control group,5,10 and 20 mM LiCl treatment significantly increased the mRNA expression level of acetyl-Co Acarboxylase 1(ACC)(P<0.05),and 5 and 10 mM LiCl treatment significantly increased the mRNA expression level of fatty acid synthase(FASN)(P<0.05),and the promotion effect was the strongest at 10 mM;compared with the control group,5,10 and 20 mM LiCl treatment significantly increased the mRNA expression level of lipoprotein lipase(LPL)(P<0.05),and the promotion effect was the strongest at 10 mM,and the protein expression of stearoyl-Co Adesaturase(SCD),sterol regulatory element-binding transcription factor 1(SREBP1)and peroxisome proliferator-activated receptorγ(PPARγ)were significantly increased in 5,10 and 20mM LiCl treatment groups(P<0.05),and the expression was the highest at 20 mM.Experiment 2:Effects of JAK2/STAT5 and PI3K/AKT1/mTOR signaling pathways related to milk fat and protein synthesisJAK2/STAT5 and PI3K/AKT1/mTOR signaling pathway genes and proteins related to milk fat and milk protein synthesis were detected.The results showed that,for JAK2/STAT5 signaling pathway,the consequences proved that there was no remarkable difference in the mRNA expression level of Janus kinase 2(JAK2)between the groups(P>0.05);the mRNA level of E74-like factor 5(ELF5)in 10 mM LiCl treatment group was expressively higher than the same thing in other treatment groups(P<0.05);the mRNA levels of signal transduction and transcription activator5β(STAT5-β)in the 5,10 and 20 mM LiCl treatment groups were much more higher than those in the control group(P<0.05);what’s more,the mRNA expression of STAT5-βwas the highest at 10 mM;compared with the control group,the protein phosphorylation levels of STAT5-βin the 5,10 and 20 mM LiCl treatment groups were significantly increased(P<0.05),and 5,10 and 20 mM LiCl treatment significantly inhibited the mRNA expression of JAK2/STAT5 pathway inhibitors suppressor of cytokine signaling(SOCS2 and SOCS3)(P<0.05)and inhibited SOCS2protein expression(P<0.05);5 and 10 mM LiCl significantly inhibited SOCS3 protein expression compared with the control group(P<0.05).For PI3K/AKT1/mTOR signaling pathway,there was no significant difference in the mRNA expression levels of phosphatidylinositol 3-kinase(PI3K),mammalian target of rapamycin(mTOR)and eukaryotic initiation factor 4E(e IF-4E)among the groups(P>0.05);the mRNA level of RAC-alpha serine/threonine-protein kinase(AKT1)was significantly up-regulated in 5,10 and 20 mM LiCl treated groups compared with our control group(P<0.05),mRNA expression of AKT1 was highest at 10 mM treatment and the mRNA expression levels of ribosomal protein S6 kinase-1(S6K1)in 10 and 20 mM LiCl treatment groups were significantly increased(P<0.05),highest expression at 10 mM,there was no significant difference in S6K1 mRNA expression between 5 mM LiCl treatment and control group(P>0.05);the mRNA expression levels of eukaryotic translation initiation factor 4E binding protein 1(4EBP1)in 5,10 and 20 mM LiCl treatment groups were much more lower than those in control group(P<0.05),the inhibition effect of 20 mM LiCl was the most obvious.There was no significant difference in mTOR protein expression between groups(P>0.05).However,compared with the control group,5,10 and 20 mM LiCl significantly increased the expression of mTOR phosphorylation protein(P<0.05),and 10 and 20 mM LiCl treatment significantly increased the expression of S6K1 and its protein phosphorylation(P<0.05),there was no significant difference in the expression levels of S6K1 and its protein phosphorylation between the 5 mM LiCl group and the control group(P>0.05).Experiment 3:Effects of LiCl on Wnt/β-catenin and HIF-1αsignaling pathways.Detection of Wnt/β-catenin and hypoxia inducible factor-1α(HIF-1α)signaling pathway related genes and proteins.The results we gained from the experiments showed that compared with the control group,10 and 20 mM LiCl treatment groups significantly increased the mRNA expression of HIF-1α(P<0.05),and the expression was the highest at 20 mM;compared with other groups,10 mM LiCl treatment significantly increased the mRNA expression of erythropoetin(EPO)in the downstream gene of HIF-1α(P<0.05),there was no significant difference in EPO mRNA expression between other LiCl treatment groups and control group(P>0.05);5and 10 mM LiCl treatment significantly increased the mRNA expression of erythropoietin receptor(EPOR)in the downstream gene of HIF-1αcompared with the control group(P<0.05),and the expression was the highest at 5 mM,and there was no significant difference in EPOR mRNA expression between 20 mM LiCl treatment group and also control group(P>0.05);compared with our control group,the proteinβ-catenin and phosphorylation ofβ-catenin was significantly increased in 5,10 and 20mM LiCl treatment groups(P<0.05),and theβ-catenin expression was the highest at10 mM,and the protein expression of HIF-1αwas significantly increased in 10 and 20mM LiCl treatment groups(P<0.05),and the protein expression of HIF-1αwas the highest in 20 mM LiCl treatment,there was no significant difference in the protein expression of HIF-1αunder 5 mM LiCl treatment(P>0.05).In summary,this study has revealed that LiCl can effectively activate HIF-1α,β-catenin,and theβ-catenin downstream signaling pathways.Additionally,when LiCl is used at 10 mM,it inhibits SOCS2 and SOCS3 protein expression through modulation of the JAK2/STAT5,PI3K/AKT1/mTOR,and SREBP1 signaling pathways,thus enhancing synthesis of milk fat and protein. |
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