The Protective Effect And Molecular Mechanisms Of Selenium Against Inflammation Injury Of Primary Bovine Mammary Epithelial Cells And Macrophages Induced By Staphylococcus Aureus

From the point of pathology, bovine mastitis is characterized by the recruitment of immune cells to engulf pathogens and the up-regulation of inflammatory cytokines expression, as a result, the milk yield is decreased and the eliminate rate is increased, which resulted in tremendous losses in the dairy industry. In animals, selenium is exists in the form of selenoproteins, which is not only an essential trace element during the feeding process but also plays an important role in the inflammation defense system. The clinical studies showed that the incidence and the lesion degree of mastitis were significantly reduced by the addition of se in the feedstuff. In the current experiment, the the anti-inflammation effect of se and the relationship between se and inflammatory signaling pathway were studied to provide theoretical foundation and experimental date in the prevention and treatment of bovine mastitis.Staphylococcus aurens (S. aureus) is one of the main pathogens that induced bovine subclinical mastitis. To reveal the disease mechanism of S. aureus and the defense mechanism would be significant. First, an intramammary infusion of an acute bovine mastitis model caused by intramammary infusion of S. aureus was established. The effects of S. aureus on udder tissue were investigated by blood biochemistry analysis, blood routine test and pro-inflammatory genes analysis. After 24 h infection, blood routine test showed the amount of white blood cells count increased markedly, which proved that a serious infection happened. Blood biochemistry analysis results showed that the total protein (TP), globulin (GLO) and lactate dehydrogenase (LDH) levels in serum were above normal. The mRNA level of TNF-α、IL-1β、IL-6 and IL-8 were up-regulated to different degree (p<0.05 or p<0.01).These result proved that acute inflammatory injury was induced by S. aureus, therefore strong immune response was triggered.Mammary tissue was obtained from healthy mid-lactation holstein cows, bovine mammary epithelial cells were obtained by enzytamic digestion. Afterwards, an in vitro inflammatory model induced by S. aureus was performed to investigate the changes of inflammatory cytokines in bMECs. Results showed that mammary epithelial cells first formed monolayer island structure and after confluencing the cells formed typical epithelial cobblestone morphology and duct-like structure. With the density increased, the cells organized dome-like structure. Cytokeriatin analysis indicated that were epithelial cells. Fluorescent quantitative PCR results showed that the mRNA expression of TNF-α、IL-1β、IL-6 and IL-8 was increased in 4 to 6 h (p<0.05～p<0.001); all the four gene reached peak at 8 h. These date suggested that S. aureus could induce immune responses in bMECs in a time-dependent manner.To explore the effect of se on cell viability of bMECs, the cells were treated with se (0、1、2、 4、8、16、32。64 μmol/L) for 12 h. The cell viabilities were measured by MTT assay. The result showed that when the concentration of se is between 0～16 μM, the cell viabilities were not affected. Compared with control group the concentration of se in 32 and 64 μmol/L, the cell viabilities decreased significantly (p<0.001), which suggested that high concentration of se were toxic to bMECs.To further study the regulation of se on TLR2 and Nod2 during inflammation, the cells were treated with se (2、4、8 μmol/L) for different time periods. The effects of se on TLR2 and Nod2 signalling pathways were tested. The dates showed that se exhibit inhibitory effect on TLR2 signalling pathway. S. aureus markedly up-regulates TLR2 gene expression. After 8 h infection, the concentration of se in 4、8 μmol/L could significantly inhibit the effect (p<0.01). The gene expression of Myd88 was significantly increased after infection with S. aureus (p<0.001). After 8 h infection, the concentration of se in 2、4、8 μmol/L could significantly inhibit the expression of MyD88 induced by S. aureus. The expression of Irak4 and Irak1 were significantly up-regulated at various time points post S. aureus infection (p<0.01 or p<0.001) but se almost had no obvious inhibition effect on the expression of Irak4 and Irak1.S. aureus increased or significantly increased the gene expression of Traf6 (p<0.05 orp<0.001), se could inhibited or significantly inhibit the effect (p<0.01 or p<0.001)Se weaken the transduction of Nod2 signalling pathway. The expression of Nod2 was up-regulated after infection, se revealed a weak inhibitory effect on the gene expression of Nod2. The expression of RIP2 was increased or significantly increased after infection. Se could markedly inhibit the expression of RIP2 afte 8 h infection (p<0.01 or p<0.001)Se could also exhibit inhibitory effect on the gene expression of inflammatory cytokines. S. aureus significantly increased or increased the gene expression of TNF-α、1L-1β and IL-6. Se in significantly inhibit the expression of TNF-a. During all time points, se could inhibited or significantly inhibited the gene expression of all the three genes. Meanwhile, S. aureus could significantly increased the expression of AP-1/c-jun and AP-1/c-fos, and se could significantly inhibit the effect by reducing the formation of AP-1 dimers.To explore the the regulation effect of se on inflammatory signalling transduction pathway in protein level, the activation of NF-κB and MAPK were detected by using Western Blot. The cells were randomly divided into 5 group,the experimental groups were treated with three varying concentrations (2、4、8 μmol/L) of se for 12 h before infection with S. aureus for 0.5 h. While the positive control group was treated with S. aureus, the control group was left untreated. Then, the activation of IκBα、p65、p38 and Erk were measured. The result showed compared with the control group the phosphorylation of IκBα、p65、p38 and Erk were markedly increased after stimulation with S. aureus for 0.5 h (p<0.001). Se (4、8 μmol/L) markedly suppressed the phosphorylation of NF-κB IκBα、p65 and MAPK p38 and Erk. These data suggested that se (4、8 μmol/L) blocked TLR2 and Nod2 signalling pathways to achieve the inhibition effects on NF-κB and MAPK, and then down-regulated the releasing of cytokines, ultimately decreasing the inflammation injury induced by S. aureus.Macrophages, is an important part in immune systems. Finally inflammatory model was performed by S. aureus-induced macrophages. Varying concentrations of se (1、1.5、2 μmol/L) was added in cell culture media. The inhibitory effect of se on the release of inflammatory cytokines induced by S. aureus in RAW264.7 was tested by fluorescent quantitative PCR and ELISA. Result showed compared with control group, the expressions of TNF-α、IL-1β and IL-6 were markedly up-regulated (p　<0.001) after S. aureus infection, the effect was blocked or significantly blocked by se in different degrees (p<0.01 orp<0.001). To determine whether or not the NF-κB and MAPK mediates the se-inhibited inflammatory response, The NF-κB p65、 IκBα and MAPK p38、Erk、Jnk protein levels were tested by Western Blot. Result showed compared with control group, a significant (p<0.001) increase in p-IκBα、p-p65、p-p38、p-Erk and p-Jnk were detected after S. aureus were treated for 0.5 h, which indicated an obviously activation of NF-κB and MAPK pathway. Compared with positive group, the phosphorylation of IκBα、p65、p38、Erk and Jnk were suppressed by se, which revealed that se might has an inhibitory effect on the activity of NF-κB and MAPK.