Effects Of Arachidonic Acid On Inflammatory Response Induced By Lipoteichoic Acid In Bovine Mammary Epithelial Cells And Non-targeted Lipidomics Studies

Lipoteichoic acid(LTA)plays a major role in the adhesion of Gram-positive bacteria and can cause inflammatory response in host cells.Arachidonic acid(AA),as one of the essential omega-6 polyunsaturated fatty acids in mammals,plays an important role in the inflammatory response.Mammary epithelial cells have the functions of synthesizing and secreting milk,and also play an important role in immune defense.Mammary epithelial cells cultured in vitro are widely used in the research of mastitis and breast cancer.However,there are few studies on the effects of exogenous AA on LTA-induced cow mammary epithelial cells.Therefore,in this experiment,an in vitro LTA-induced MAC-T inflammation model was constructed,and AA was used to intervene to preliminarily explore the effect of AA on it.Then,the non-targeted lipidomics method of UHPLC-QE-MS was used to screen differentially metabolized lipids.The aim of this study is to provide a scientific basis for further screening of potential therapeutic targets for dairy cow mastitis caused by Gram-positive bacteria.The following results were obtained:1.With the increase of LTA concentration,the activity of MAC-T cells can be decreased in a dose-dependent manner,and after different concentrations(0.1,1 and 10μg/m L)of LTA induced MAC-T for 24 h.The expressions of IL-1β,IL-2,IL-6,IL-8and TNF-α increased with the increase of LTA concentration in a dose-dependent manner.Among them,10 μg/m L LTA induced the most significant expression of MACT cytokines.Therefore,10 μg / m L LTA was selected for subsequent experiments.2.MAC-T was pretreated with 10 μg/m L LTA for 24 h,and then treated with 0.01,0.1 and 1 μg/m L AA for 12 h,respectively.AA was found to have dual effects on LTAinduced expression of cytokines IL-1β,IL-2,IL-6,IL-8,and TNF-α in MAC-T,and AA concentrations of 0.01 and 0.1 μg/m L significantly decreased The expression levels of IL-1β,IL-6,IL-8 and TNF-α in MAC-T induced by LTA were increased(P<0.05),and the expression of IL-2 was decreased,but not significantly(P>0.05);1 μg/m L AA significantly increased the expression of IL-1β,IL-2,IL-6 and IL-8(P<0.05),and it was still significant for the m RNA expression of TNF-α decreased(P<0.05).Combined with CCK-8 test,0.1 μg/m L AA was finally selected as the AA treatment concentration for non-targeted lipidomics test.3.The non-targeted lipidomics detection technology of UHPLC-QE-MS was used to screen and analyze the differential lipid molecules in bovine mammary epithelial cells in Control group(normal cultured MAC-T cells),AA group(0.1 μg/m L AA treatment for 12 h),LTA group(10 μg/m L LTA stimulation for 24 h)and LTA+AA group(10 μg/m L LTA stimulation for 24 h,followed by 0.1 μg/m L AA treatment for 12h).A total of 535 significantly different lipid molecules were successfully screened among the groups.In order to explore the changes of lipid molecules in the inflammatory model by AA,34 differential lipid molecules between LTA+AA group and LTA group were analyzed.LTA+AA group compared with LTA group,PC(18:2/18:2),TAG(16:0/18:1/20:4)and other 19 differential lipid expression increased;the expression of 15 differential lipids such as TAG(12:0/12:0/12:1),and PE(15:0/22:3)decreased,and there was a strong correlation between each other.Conclusion: After LTA stimulation of MAC-T cells,the expression levels of IL-1β,IL-2,IL-6,IL-8 and TNF-α m RNA were significantly increased,while 0.1 μg/m L exogenous AA could reduce the expression of these cytokines.Through non-targeted lipidomics studies,it was found that exogenous AA can change the lipid metabolism of MAC-T cells stimulated by LTA,among which 34 significantly different lipid molecules such as PC(18:2/18:2),TAG(16:0/18:1/20:4)and PE(15:0/22:3)have a strong correlation.The metabolic changes of these differential lipid molecules may be related to the regulation of LTA-induced MAC-T inflammatory response by exogenous AA,which provides a theoretical basis for finding exogenous AA-rich drugs in the future to change the metabolic changes of related differential lipids and regulate the inflammatory response,so as to restore the homeostasis of the internal environment.