Nontargeted Metabolomics Of Bovine Mammary Epithelial Cells Induced By LPS

Bovine Mammary Epithelial Cells(bMECs)are the main cells of the dairy cows' mammary glands,in addition to milk production,they are also the effector cells of mammary immunity.When being exposed to stimuli,they can produce a strong immune response.Although bMECs have been shown to stimulate NF-?B signaling pathway via Toll-like receptors and NOD-like receptors after they are stimulated by lipopolysaccharide(LPS),and regulate inflammatory mediators,changes in metabolite levels have not been studied.Metabolomics can analyze all metabolites qualitatively and quantitatively,find the relationship between metabolites and physical or pathological changes,provide the most comprehensive biological information.Therefore,in order to reveal the metabolic pathway of LPS-stimulated bMECs,and provide scientific basis for further screening biomarkers of diagnosis and treatment of bovine mammary gland inflammation,this study includes the following parts:1.Using tissue adherent and enzyme digestion methods to isolate and culture primary bovine mammary epithelial cells in vitro.Microscope was used to observe the cell morphology,and then detected gene expression of ?-casein,the cells were immunofluorescently stained with Keratin-18 antibody and Vimentin antibody respectively as well.The results revealed that both methods could successfully culture bovine mammary epithelial cells.Although enzyme digestion method could obtain cells within a shorter time,two cells mixed together increased the difficulty of purification,while tissue adherent method could get more pure and a larger number of mammary epithelial cells.The results also showed that the cells expressed ?-casein gene,and were positive for Keratin-18,negative for Vimentin.Combined with the morphological observation,all results indicated that the cultured cells were mammary epithelial cells.2.Cells were randomly divided into 3 groups: Control group(Control),LPS-induced 12 h group(LPS12h)and LPS-induced 24 h group(LPS24h),3 replicates were set in each group.RT-qPCR method was used to investigate the changes of mRNA expression of IL-6,TNF-? and CCL-2,and COX-2,LOX-5,LOX-15,which are the key enzymes of producing lipid mediators.Furthermore,ELISA technology was used to explore the content changes of PGE2,IL-6 and TNF-? in the cell supernatant.The results showed that the mRNA expression of CCL-2,IL-6 and TNF-? in LPS12 h increased extremely significantly(P<0.01)compared with Control,when compared LPS24 h with LPS12 h,the mRNA expression of IL-6 and TNF-?(P<0.05)increased while CCL-2 decreased but still higher than that of Control(P<0.01),and mRNA expression of IL-6 in LPS24 h increased extremely significantly compared with Control(P<0.01).For enzymes in LPS12 h,the mRNA expression of COX-2 and LOX-5 significantly increased(P<0.05)and the mRNA expression of LOX-15 was extremely significantly up-regulated(P<0.01)compared with Control.In LPS24 h the mRNA expression of COX-2 decreased significantly(P<0.05)compared with LPS12 h,while the mRNA expression of LOX-5 and LOX-15 increased extremely significantly compared with Control(P<0.01).The results of ELISA showed that the secretion of IL-6(P<0.05),TNF-?(P<0.05)and PGE2 increased in LPS12 h compared with Control,and the secretion of PEG2 and IL-6 continued to increase significantly(P<0.05)in LPS24 h when compared with Control,while it showed a significant drop for TNF-?(P<0.05)in LPS24 h compared with LPS12 h,and there was no significant difference between that of LPS24 h and Control.3.Cells were randomly divided into 3 groups: Control group(Control),LPS-induced 12 h group(LPS12h)and LPS-induced 24 h group(LPS24h),with 6 biological replicates in each group.HPLC-Q-TOF MS detection technology was used in combination with data-dependent acquisition analysis methods to screen and identify differential metabolites after LPS stimulation of bMECs.The results showed that 63 differential metabolites were successfully screened and identified,belonging to nucleotides,amino acids,fatty acids and so on.There were 38 between Control and LPS12 h,of which there were 26 up-regulated metabolites and 12 down-regulated ones;between LPS24 h and LPS12 h,13 were up-regulated and 18 were down-regulated;at last,there were 35 differential metabolites between LPS24 h and Control,8 of them were up-regulated.Moreover,metabolic pathway analysis found that differential metabolites were mainly involved in eight pathways,including fatty acid metabolism,glutathione metabolism,amino acid biosynthesis and metabolism and so on.Taken together,LPS simulated the mRNA expression of TNF-??CCL-2?IL-6 and COX-2?LOX-5?LOX-15 and secretion of PGE2?IL-6?TNF-? in bMECs.Metabolomics study revealed 63 differential metabolites such as linoleic acid(LA),nicotinamide,betaine,myo-inositol and creatine,which were mainly involved in the 8 pathways like linoleic acid metabolism,glycerophospholipid metabolism and amino acid metabolism.