| Mastitis is one of the major diseases affecting the dairy industry.Staphylococcus aureus(S.aureus)is the main pathogen of subclinical mastitis in dairy cows.The innate immune system is an important factor of pathogen defense.Recent studies have found that autophagy is involved in the defense against pathogens.Selenium,as an essential trace element in the animal,plays an important role in anti-oxidation,immunity,and prevention of diseases.Therefore,this experiment aimed to explore the effects of selenium on autophagy and inflammation with macrophages infected with S.aureus.1.Effect of S.aureus infection on autophagy of bovine macrophages.S.aureus(MOI=1:1)was used to infect bovine macrophages for 0 h,0.5 h,1h,1.5h,2 h,2.5 h,3 h,and 4 h respectively.The expression of autophagy-related proteins LC3 and p62 were detected by western blot,and the expression of endogenous LC3 fluorescent aggregation spots was detected by laser confocal microscopy.The ultrastructural changes of bovine macrophages infected with S.aureus were observed by transmission electron microscope.The effect of S.aureus on the autophagy flux of bovine macrophages was examined by transfection of the ptfLC3 plasmid.The results show that,when S.aureus infected bovine macrophages for 1.5 h,2.5 h,3 h,and 4 h,the expression of LC3-II increased(p<0.05)and it was significantly increased at 2 h(p<0.01).The expression of p62 increased at 1.5 h(p<0.05)and increased significantly at 1 h and 2 h(p<0.01).Transmission electron microscope examination results showed that autophagy vesicles increased after bovine macrophages infected with S.aureus.The results of LC3 endogenous staining were consistent with the results of western blots.The expression of LC3 fluorescence aggregation sites was highest when S.aureus infected bovine macrophages for 2 h.Transfection results of ptfLC3 plasmid showed that autophagy flux was blocked after S.aureus infected bovine macrophages.In order to study the effect of S.aureus on bovine macrophages after autophagy was interfered,the cells were infected with S.aureus for 2 h with regulating medicines at different stages of autophagy.The expression of LC3 and p62 were detected by western blot.Laser confocal microscopy was applied to detect the expression level of endogenous LC3 aggregation points.Intracellular survival of S.aureus was detected by bacterial load examination.The results showed that compared with the 3-MA group,the expression of LC3 was increased in the 3-MA and S.aureus co-treatment groups(p<0.05),and the expression of p62 was not significantly different(p>0.05).Compared with the CQ group,the expressions of LC3 and p62 in the CQ and S.aureus co-treatment groups were not significantly different(p>0.05).Compared with the Rapamycin group,the expressions of LC3 and p62 in the Rapamycin and S.aureus co-treatment group were significantly increased(p<0.01).The results of LC3 endogenous staining were consistent with the results of western blot.After cells treated with CQ,intracellular survival of S.aureus increased gradually with time(p<0.05 or p<0.01).After Rapamycin treatment,intracellular survival of S.aureus decreased gradually with time(p<0.05).After 3-MA treatment,the intracellular survival of S.aureus did not change with time significantly(p>0.05).The results showed that the accumulation of autophagy was beneficial to the proliferation of intracellular bacteria.2.Effect of selenium on autophagy of bovine macrophages infected by S.aureus.Bovine macrophages were treated with 1?M,2?M and 4?M sodium selenite for 12 h,and then infected with S.aureus for 2 h.Western blot was used to detect the expression of LC3 and p62,and the expression of endogenous LC3 fluorescent aggregation spots was detected by laser confocal microscopy.The effect of selenium on the intracellular survival of S.aureus was measured by the method of bacterial load.The results showed that compared with the S.aureus group,there was no significantly different in the expression of LC3 in the selenium and S.aureus co-treatment groups(p>0.05),and the expression of p62 gradually decreased with the increase of selenium concentration(p<0.05 or p<0.01).And it was significantly reduced when pretreated with 4?M selenium(p<0.01).The endogenous staining results of LC3 were consistent with the western blot results.4?M selenium significantly reduced the intracellular survival of S.aureus.The above results showed that selenium could alleviate the inhibition of autophagic flow of bovine macrophages caused by S.aureus infection and reduced the proliferation of intracellular bacteria.3.Effect of selenium on MAPK/NF-?B pathway of bovine macrophages infected by S.aureus.The expressions of the key proteins in MAPK and NF-?B signaling pathway were detected by western blot.And the expression of related inflammatory factors was detected by qRT-PCR after bovine macrophages were infected by S.aureus for 6 h.The results showed that the phosphorylation of ERK,JNK and p38 were significantly increased after bovine macrophages infected by S.aureus(p<0.01).The 4?M selenium and S.aureus co-treated groups reduced the phosphorylation of ERK and p38(p<0.05),and significantly decreased the phosphorylation of JNK(p<0.01).S.aureus increased the phosphorylation of NF-?B(p<0.05),and significantly increased the phosphorylation level of IKB?(p<0.01).The 4?M selenium and S.aureus co-treatment group significantly reduced the phosphorylation level of NF-?B(p<0.01)and IKB?(p<0.05).The expression of IL-1?,IL-6 and IL-8 in bovine macrophages caused by S.aureus infection was also decreased with 4?M selenium(p<0.01).The above results showed that selenium suppressed the activation of MAPK and NF-?B signaling pathway induced by S.aureus infection,inhibited the phosphorylation of related proteins and the expression of inflammatory factors. |
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