| This experiment compared the effects of different doses of melatonin on cell viability,inflammatory cytokines production,the expression of key proteins involved in inflammatory signaling pathways,and autophagy of bovine mammary epithelial cells induced by lipopolysaccharide.The details are as follows:Experiment 1: The objective of this study was to investigate the effects of different doses of melatonin on cell viability and inflammatory cytokines in bovine mammary epithelial cells.Healthy bovine mammary epithelial cells were selected.It can be used after 3-8 generations.Bovine mammary epithelial cells were treated with different doses of melatonin(0,1,5,10,50 and 100 μM)for 24 or 48 hours,respectively.The cell viability of bovine mammary epithelial cells was detected by methyl thiazoyl terazolium assay,and tumor necrosis factor-α,interleukin-6 and interleukin-1β levels were measured with enzyme linked immunosorbent assay.The results showed that:(1)Compared with the control group,supplementation with melatonin had no significant effect on the cell viability of bovine mammary epithelial cells.(2)Compared with the control group,the levels of inflammatory cytokines in bovine mammary epithelial cells treated by different doses of melatonin were not increased,indicating that melatonin did not induce inflammation on bovine mammary epithelial cells.(3)Among different treatment groups,there was no significant difference on the cell viability of bovine mammary epithelial cells.It indicated that the melatonin doses designed in this study could be used in subsequent experiments.Experiment 2: The objective of this study was to investigate the effects of different doses of lipopolysaccharide on cell viability and inflammatory cytokines in bovine mammary epithelial cells.Healthy bovine mammary epithelial cells were selected.It can be used after 3-8 generations.Bovine mammary epithelial cells were treated with different doses of lipopolysaccharide(0,0.1,0.5,1,5 and 10μg/m L)for 6 or 12 hours,respectively.The cell viability and inflammatory cytokines of bovine mammary epithelial cells was detected.The results showed that:(1)Compared with the control group,the cell viability of bovine mammary epithelial cells induced by lipopolysaccharide for 6 or 12 hours were suppressed,and the effect of induction for 12 hours was more severe.(2)Compared with the control group,the levels of inflammatory cytokines in bovine mammary epithelial cells induced by different doses of lipopolysaccharide were increased,and the dose of 10 μg/m L was appropriate.(3)As mentioned above,lipopolysaccharide at 10 μg/m L to inducing bovine mammary epithelial cells for 12 hours was selected to establish cell inflammatory reaction model in subsequent study.Experiment 3: The objective of this study was to investigate the effects and mechanisms of different doses of melatonin on inflammation of bovine mammary epithelial cells induced by lipopolysaccharide.Healthy bovine mammary epithelial cells were selected.It can be used after 3-8 generations.Bovine mammary epithelial cells were divided into 7 treatments: the control group that was not treated with lipopolysaccharide and melatonin;lipopolysaccharide group that was induced with 10 μg/m L lipopolysaccharide for 12 hours;lipopolysaccharide and melatonin groups that were firstly induced with10 μg/m L lipopolysaccharide for 12 hours and then treated with different doses of melatonin(1,5,10,50 and 100 μM)for 24 or 48 hours,respectively.Each dose was repeated for 4 times.The results showed that:(1)Melatonin decreased the levels of inflammatory cytokines in bovine mammary epithelial cells induced by lipopolysaccharide,and the appropriate dose was 10 μM under the conditions of the present study.(2)Compared with lipopolysaccharide group,melatonin was found to suppress the expression of Toll-like receptor 4 treated for 48 hours,and the doses of 50 μM and 100 μM had significant effects(P<0.05).The protein expression of nuclear factor kappa B inhibitory protein-α was increased by melatonin,except for 100 μM treatment(P<0.05).The decrease of protein expression of nuclear factor kappa B homologous protein p65 induced by lipopolysaccharide was inhibited by melatonin treated for48 hours,and melatonin dose at 10 μM was extremely significant(P<0.01).(3)Compared with lipopolysaccharide group,melatonin increased the m RNA expression of sirtuin 1 in bovine mammary epithelial cells treated for 48 hours,and melatonin increased the m RNA expression of nuclear factor erythroid 2-related factor 2(P<0.05).(4)Compared with lipopolysaccharide group,the amount of autophagosome in bovine mammary epithelial cells treated with melatonin for 48 hours was increased.Taken together,melatonin could regulate the levels of inflammatory cytokines in bovine mammary epithelial cells,and the protein expressions of Toll-like receptor 4,nuclear factor kappa B inhibitory protein-α and homologous protein p65,and the gene expressions of sirtuin 1 and nuclear factor erythroid2-related factor 2,and the quantity of autophagosome.In this study,melatonin at 10 μM was appropriate,and it could provide a scientific basis for the application of melatonin in bovine mastitis. |
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