Effects And Mechanisms Of Omega-3 Polyunsaturated Fatty Acids On The Inflammatory Reaction Of Mammary Epithelial Cells Induced By Lipopolysaccharide

Mastitis is a disease that affects the healthy development of the global dairy industry.Environmental pathogenic microorganism infection and physiological trauma or related metabolic pathway changes are the main causes of bovine mastitis.Escherichia coli can induce acute mastitis in bovine,and injection of its virulence component lipopolysaccharide(LPS),into the mammary gland could cause an immune response in the mammary gland.N-3 polyunsaturated fatty acids(n-3 PUFAs)are important lipid nutrients for the body,and as an essential fatty acids for humans and animals,it regulates intracellular signaling pathways through producing different lipid metabolites.It plays a beneficial role in the inflammation process of many diseases.This study aimed to study the inhibitory effect of n-3 PUFAs on LPS-induced inflammatory response and its possible molecular mechanism through in vivo and in vitro mastitis models.At the level of cellular experiments,LPS treatment to bovine mammary epithelial cells was used to establish a cell model of bovine mastitis.Protective effects of n-3 PUFAs pretreatment on the inflammatory induction of mammary epithelial cells induced by LPS were evaluated by detection of m RNA expression and protein secretion levels of inflammatory cytokines,including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6),through real-time fluorescence quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.Results showed that LPS can obviously induce mammary gland epithelial cells to increase the level of TNF-α,IL-1β and IL-6m RNA expression and protein secretion.Pretreatment with n-3 PUFAs can alleviate the secretion of these inflammatory cytokines induced by LPS.Through dual luciferase report experiment,immunofluorescence experiment and western blotting technique,we studied whether n-3 PUFAs can inhibit the inflammatory response of mammary gland epithelial cells induced by LPS by regulating the NF-κB signal pathway.Results of the dual luciferase report experiment showed that n-3 PUFAs pretreatment significantly inhibited the activation of NF-κB pathway by LPS.Results of immunofluorescence experiments showed that LPS can enhance translocated of phosphorylated P65 from cytoplasm into the nucleus,and n-3 PUFAs pretreatment can reduce this enhanced translocation.Results of western blot analysis showed that pre-intervention of n-3 PUFAs reduced the expression of My D88,p-IκB-α/IκB-α,p-P65/P65 and P50 pathway proteins in the LPS-induced inflammatory state.These results indicate that n-3 PUFAs inhibit the inflammatory response of mammary epithelial cells induced by LPS via inhibiting the activation of NF-κB signal pathway.At the level of animal experiments,LPS injection into mammary gland was used to establish a mouse model of mastitis.The protective effect and mechanism of n-3 PUFAs pretreatment to mice by gavage on the inflammatory response of the mammary gland induced by LPS were studied.A series of biochemical and molecular biology methods,including histopathological examination,detection of serum alkaline phosphatase(ALP),procalcitonin(PCT)and C-reactive protein(CRP)change levels,detection of the expression of NF-κB signaling pathway related proteins in mammary gland tissue were used in this part.Results showed that,LPS can obviously induce the extensive infiltration of leukocytes in the mammary glands in the mammary gland tissues of mice,the increase of the white blood cells numbers in the peripheral blood swells,the enhancement of activity of alkaline phosphatase decreases,and the activation of the inflammatory pathway NF-κB in the mammary gland tissues.The intervention of n-3 PUFAs significantly attenuated the LPS-induced mammary gland tissues inflammation in mice.In addition,the data of 16 S r DNA Amplicon sequencing analysis of mouse intestinal contents showed that LPS-induced significantly changed the structure of mouse intestinal flora,thus leading to structural disorder of subdominant bacteria.The pre-intervention of n-3 PUFAs improved the structural disorder of subdominant bacteria in the intestines of mice caused by LPS stimulation.In conclusion,n-3 PUFAs pretreatment can reduce the inflammatory response of mammary epithelial cells and mice mammary tissue induced by LPS by inhibiting the activity of the NF-κB signaling pathway and maintaining the homeostasis of the intestinal flora.