Abstract:

【Objective】Whether selenium (Se) could regulate the inflammatory damage of bovine mammary epithelial cells (bMECs) induced by S. aureus through Nod2/MAPK/mTOR pathway remains to be further studied. So in the study, the effects of Se on the key proteins in the Nod2/MAPK/mTORs signaling pathway in the bovine mammary epithelial cells (bMECs) infected by S. aureus was studied in order to provide a theoretical basis for elucidating the immune regulation mechanism of Se.【Method】 Firstly, the bMECs were inoculated into the 6 well plates with 10 ⁶cells/well. When more than 80% of the cells were confluent, the medium was replaced with the one containing different concentrations of Se (2, 4 and 8 μmol·L ^-1) and continued to culture for 12 h. Then after washing each well for 3 times with PBS, S.aureus was added into 6-well plates at a ratio of MOI=1:1 and continued to culture for 0.5 h. The bMECs were collected for further detection of related proteins expression. The experiment was divided into three groups: control (Con) group (bMECs), model (Mod) group (bMECs+S. aureus) and experimental group. The experimental group was divided into three sub-dose groups, namely Low group (bMECs+2 μmol·L ^-1 Se+S. aureus), Mid group (bMECs+4 μmol·L ^-1 Se+S.aureus) and Hig group (bMECs+8 μmol·L ^-1 Se+S. aureus), with three replicates each group. Total protein was extracted from the above bMECs using a bicinchoninic acid (BCA) protein assay kit. The expressions level of Nod2 and RIP2 and the phosphorylation level of JNK, AKT and mTOR proteins in bMECs were detected by Western blotting. The protein samples were loaded into 10% SDS polyacrylamide gel for electrophoresis, and the uniform volume of protein was 20 μg/hole. Then the protein was transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5 mL 5% nonfat milk for 2 h, then skimmed the nonfat milk and washed the membranes with TBST, subsequently the membranes were incubated overnight with 5 mL primary antibodies including Nod2, RIP2, JNK, AKT, mTOR and β-actin. The primary antibodies were recovered, and 5 mL second antibodies were added to the membranes and incubated for 2 h at room temperature. Subsequently the second antibodies were recovered the membranes were washed with TBST for 5 times. Finally the membranes were developed with chemiluminescent substrate under darkroom conditions.【Result】S. aureus could significantly increase the expression of Nod2 and RIP2 proteins and the phosphorylation of JNK, AKT and mTOR proteins in bMECs (P<0.01). At 0.5 h after S. aureus infection, the level of Nod2 protein increased significantly (P<0.01). The expression of Nod2 protein was significantly inhibited by adding 2 μmol·L ^-1 Se to the medium (P<0.01), and the expression of Nod2 was significantly inhibited by adding 8 μmol·L ^-1 Se to the medium (P<0.05); at 0.5 h after S. aureus infection, RIP2 protein level was significantly increased (P<0.05), while the expression of RIP2 protein was significantly inhibited by adding 8 μmol·L ^-1 Se to the medium (P<0.05); at 0.5 h after S. aureus infection, the phosphorylation level of JNK protein in model group was significantly higher than that in control group (P<0.01). The phosphorylation of JNK protein was significantly inhibited by adding 4 μmol·L ^-1 Se to the medium (P<0.05), and the phosphorylation of JNK protein was significantly inhibited by adding 8 μmol·L ^-1 Se to the medium (P<0.01); at 0.5 h after S. aureus infection, the phosphorylation level of AKT protein in the model group was significantly higher than that in the control group (P<0.01). The phosphorylation level of JNK protein was significantly inhibited by adding 4 μmol·L ^-1 Se to the medium (P<0.01). The phosphorylation level of AKT protein was significantly inhibited by adding 8 μmol·L ^-1 Se to the medium (P<0.05). After 0.5 h of S. aureus infection, the phosphorylation level of mTOR protein was significantly increased in the model group (P<0.01). The phosphorylation of mTOR protein was significantly inhibited by adding 4 and 8 μmol·L ^-1 Se to the medium (P<0.05). 【Conclusion】Se could alleviate the inflammatory response of bMECs induced by S. aureus by inhibiting the protein expression of key factors in the bMECs Nod2/MAPK/mTORs signaling pathway.

Key words: selenium, S. aureus, Nod2/MAPK/mTORs, bMECs