Effect And Its Mechanisms Of Stearic Acid On Synthesis Of Milk Fat And Milk Protein In Bovine Mammary Epithelial Cells

With the growing prosperity of my country and the continuous rapid development of the dairy industry,the national demand for milk has gradually increased.How to further improve the lactation performance of dairy cows and the quality of milk to meet the needs of national consumption has become a hot issue concerned by many researchers.Bovine mammary epithelial cells（BMECs）are the only cells in the mammary gland that have the function of producing and secreting milk.The milk production performance of dairy cows mainly depends on the ability of BMECs to proliferate and synthesize milk.Milk fat and milk protein are the main nutrients in milk and are also considered as important indicators for evaluating milk production performance and milk quality of dairy cows.The synthesis of milk fat and milk protein in BMECs is affected by many factors such as environment,hormone,growth factor and so on.Fatty acids（FAs）are substrates for the synthesis of various lipids,and also participate in the regulation of various biosynthetic processes in the body from multiple levels such as signal transduction,transcription,and translation.Stearic acid（SA）is a saturated long-chain fatty acid containing 18 carbons.As a rumen inert fatty acid,SA has great potential in regulating the lactation of dairy cows,and is often used as a feed additive in the dairy industry.At present,the research on the regulation of dairy cow lactation by SA is mainly focused on clinical application.However,the results of related studies are not consistent,and the specific mechanism of SA regulating lactation is not clear.Therefore,to clarify the effect of SA on synthesis of milk fat and mlik protein and reveal its potential regulation mechanism is of great significance for the dairy industry to reasonably apply SA to regulate cow lactation,ensure the sustained and healthy development of the milk industry,and improve the core competitiveness of China’s milk industry in the international market.In order to clarify the effect and its potential mechanism of SA on synthesis of milk fat and milk protein,the primary BMECs of Holstein cows during lactation was isolated by tissue block inoculation method,the isolated cells were purified by differential digestion method,and cytokeratin18（CK-18）of the epithelial cell marker protein was stained with immunofluorescence to identify that the cells obtained in this study were indeed BMECs.Then,BMECs was treated with different concentrations of SA（0,12.5,25,50,100,200 μM）.The results showed that SA within this concentration range had no significant effect on the viability of BMECs,but significantly increased the expression levels of β-casein and triglycerides（TGs）in BMECs,with the most significant effect at a concentration of 100 μM,which confirmed the positive effect of SA on lactation of dairy cows.The subsequent mechanism study found that SA significantly activated the activity of signal molecules such as PI3 K,m TOR and SREBP-1 in BMECs.In order to clarify the role of these signal molecules in the regulation of synthesis of milk fat and milk protein by SA,m TOR inhibitors-rapamycin（RAPA,200 n M）and PI3 K inhibitorsLY294002（15 μM）were used to inhibit the activity of m TOR and PI3 K signal molecules,respectively.The results showed that after m TOR was inhibited,the activities of 4EBP1,S6 K and SREBP-1 were significantly reduced,and the promoting effect of SA on synthesis of milk fat milk protein in BMECs was significantly blocked.After inhibiting the activity of PI3 K,the activation of m TOR-4EBP1/S6 K and m TORSREBP-1 signaling axes by SA was significantly attenuated,indicating that PI3 K plays a key role in SA-stimulated BMECs to activate m TOR and SREBP-1 signaling.Previous studies have found that cyclin-dependent kinase 1（CDK1）participates in the regulation of protein synthesis by affecting the activity of translation factors.In this study,SA significantly up-regulated the protein level of CDK1 in BMECs.Therefore,in this study,CDK1-si RNA and CDK1-pc DNA were constructed to explore the role in the regulation of synthesis of milk fat milk protein in BMECs by SA.The results showed that CDK1 deletion blocked the activation of SA on the above signal pathways and the synthesis of milk protein and milk fat;after overexpression of CDK1,even without SA stimulation,PI3K-m TOR-4EBP1/S6 K and m TOR-SREBP-1 signal axes were significantly activated.These results suggest that CDK1 responds to the stimulation of SA before the above signal molecules and plays a positive role in the milk synthesis of BMECs.In addition,SA belongs to long-chain fatty acids（LCFAs）and needs the assistance of transporters to enter cells.Therefore,this study examined the expression of fatty acid transport proteins（FATPs）in BMECs stimulated by SA.It was found that the m RNA levels of FATP3 and FATP4 in FATPs were significantly upregulated when BMECs were stimulated with 100 μM SA,and the basal m RNA expression abundance of FATP4 was found to be the highest.Many previous studies have shown that FATP3 is mainly responsible for regulating the function of long-chain fatty acyl-Co A synthase,and has a weak ability to transport LCFAs,while FATP4 has a much higher ability to transport LCFAs than FATP3,and is the main LCFAs transporter.Therefore,this study focused on FATP4,through the construction of FATP4-si RNA to detect the effect of SA on the regulation of synthesis of milk fat and milk protein BMECs.The results showed that in the absence of FATP4,the effect of SA on up-regulating CDK1 protein level and PI3K-m TOR-4EBP1/S6 K and m TORSREBP-1 signaling axis activities in BMECs was blocked,and the promoting effect on synthesis of milk fat and milk protein disappeared.This suggests that FATP4 is the main transporter mediating the transmembrane transport of SA into BMECs.To sum up,this study found that SA promotes the expression of milk fat and milk protein in BMECs,which confirms that SA has a positive effect on the lactation of dairy cows,and revealed that SA is transported into BMECs through FATP4,and activates PI3K-m TOR-4EBP1/S6 K and m TOR-SREBP-1 signal axis by up-regulated the protein level of CDK1,promotes the synthesis of β-casein and TGs in BMECs.The findings of this study provide a theoretical support for the rational use of SA in dairy industry to regulate cow lactation,and are of great significance to the rational and healthy development of dairy industry in my country.