Effect And Mechanism Of LPS On Lactose Metabolism Of Bovine Mammary Epithelial Cells

The mammary gland of bovine has the function of secreting milk and responding to inflammation.Lactation is the basic physiological characteristics of mammals,and mastitis has a great impact on all mammals,especially can lead to the decline of milk yield and milk quality.Lactose is only secreted by mammary epithelial cells,which can directly affect the milk yield by regulating the permeability.Pathogenic Escherichia coli is one of the main pathogens of mastitis.Lipopolysaccharide,the main component of its cell wall,can intervene in the process of glucose metabolism and further regulate the synthesis of lactose by inducing the inflammatory response of mammary epithelial cells.However,there are few reports at home and abroad.Therefore,in this study,we will use LPS treated bovine mammary epithelial cell line（MAC-T）as an experimental model to study the changes of MAC-T metabolic pathway and its effect on lactose synthesis during acute inflammatory response induced by LPS.In order to further understand the relationship between immunity and metabolism during the acute inflammatory response of bovine mammary epithelial cells,and to choose a more reasonable regulation scheme of immune metabolism to ensure the health of bovine mammary gland,a new idea was provided.In this study,MAC-T was treated with different concentrations of LPS,and the optimal stimulation concentration of LPS was determined by cell counting,microscope observation and transmission electron microscope observation;the activities of hexokinase,phosphofructokinase,pyruvate kinase,lactic acid content,glucose content,lactose content and AKT1 content were detected by ultraviolet/visible spectrophotometry;Protein kinase B,hypoxia inducible factor-1αand glucose transporter 1 were detected by Western blot and real-time PCR,and mitochondrial respiration was detected by seahorse XF 96.The optimal concentration of LPS was 10μg·ml^-1;The activity of hexokinase（P<0.0 001）,phosphofructokinase（P=0.0 005）and pyruvate kinase（P<0.0 001）in MAC-T cells were significantly increased after LPS treatment;The content of lactic acid was significantly increased（P<0.0 001）;The protein expressions of AKT（P=0.0 018）,p-AKT1（P=0.0 014）and HIF-1α（P<0.0 001）were significantly increased,and the m RNA expressions of AKT1（P=0.0 002）and HIF-1α（P<0.0 001）were significantly increased;In oxidative phosphorylation metabolism,ATP synthesis（P<0.0 001）and proton leakage（P=0.0 037）were significantly decreased,while basal respiration（P<0.0 001）,maximal respiration（P<0.0 001）and respiratory reserve capacity（P=0.0 017）were significantly decreased,while non mitochondrial respiration and mitochondrial coupling efficiency were not significantly changed;Glucose content（P<0.0 001）and GLUT 1expression（P<0.0 001）were significantly increased,while lactose content（P<0.0 001）was significantly decreased.Conclusion:LPS treatment can inhibit the mitochondrial respiration of MAC-T,increase the uptake and consumption of glucose,and most glucose is used for glycolysis metabolism,which may be related to the increase of AKT1 and HIF-1α.