| Staphylococcus aureus(S.aureus)is a common pathogen causing mastitis in dairy cows,which often leads to persistent chronic intracellular infection.Autophagy is a key degradation mechanism in cells and is involved in the elimination of pathogenic microorganisms that invade cells.However,some microbes have evolved strategies to combat autophagy.Selenium is an essential trace element in mammals,which has a significant regulatory effect on animal immune function and inflammatory response.Therefore,this experiment studied the relationship between the autophagy of bovine mammary epithelial cells(BMECs)and the infection with S.aureus,and the effect of selenium on BMECs autophagy and intracellular bacterial proliferation of S.aureus,so as to provide theoretical basis for the prevention and treatment of S.aureus mastitis in dairy cows.The main results were as follows:(1)in order to investigate the effect of S.aureus intracellular infection on BMECs autophagy,the intracellular infection model was established by inoculating S.aureus with MO1=1:1 in BMECs,.The expression of autophagy related proteins were detected by Western blot at 1,2,3 and 4 h post infection(hpi),and the expression of LC3 was detected by immunofluorescence at 3 hpi.The formation of autophagy lysosomes was detected by co-localization of GFP-LC3 and lysosomal markers.The results showed that the level of LC3-Ⅱ in the S.aureus group was significantly higher than that in the control group at 2 h,3 h and 4 h pi(p<0.01),and the protein level of p62 in the S.aureus group was significantly higher than that in the control group at 3 h and 4 hpi(p<0.01).The results of immunofluorescence detection showed that there were more green LC3 accumulation points in cytoplasm and around nucleus in S.aureus group,but there was no obvious co-localization of LC3 green points and lysosomal red spots.(2)in order to investigate the effect of autophagy regulation on the intracellular proliferation of S.aureus in BMECs,autophagy regulators were added to S.aureus intracellular infection model.The expression of autophagy related proteins LC3-Ⅱ and p62 were detected at 3 hpi,and the proliferation of S.aureus in BMECs was detected by intracellular bacterial load at 1,2,3 and 4 hpi.The results showed that when rapamycin and S.aureus were co-processed,compared with the S.aureus group,there was no significant difference in the expression of LC3-II protein ip>0.05),but the expression of p62 protein was increased(p<0.05),and the number of intracellular bacteria increased significantly with the passage of time(p<0.05 orp<0.01).;In CQ and S.aureus group,the expression of LC3-Ⅱ protein was significantly higher than that of the S.aureus group(p<0.01),but there was no significant difference in the expression of p62 protein.At all test times,the number of intracellular bacteria was significantly higher than that in S.aureus group;In 3-MA and S.aureus co-treatment group,the number of intracellular bacteria was significantly higher than that in S.aureus infection group.Compared with the S.aureus group,the expression of LC3-II decreased significantly(p<0.01),but there was no significant difference in the expression of p62 protein and the number of bacteria in the cells at 2 h,3 h and 4 h(p<0.01)was significantly lower than that in S.aureus group.(3)in order to investigate the effect of selenium on the autophagy of BMECs and the proliferation of intracellular bacteria of S.aureus,BMECs was preincubated with different concentrations of selenium(2,4 and 8 μ M)to establish the infection model.The expression of autophagy related proteins were detected by Western blot at 3 hpi.And the degree of autophagic flow was observed by co-localization of autophagosome and lysosome.The proliferation of S.aureus in BMECs was detected by intracellular bacterial load.The results showed that the levels of LC3 protein and p62 protein in 4 and 8 μM selenium pretreatment groups were significantly lower than those in S.aureus group(p<0.05 or p<0.01),and the level of p62 protein was significantly lower in 4 and 8 μM selenium pretreatment group(p<0.01).Compared with the control group,there were more green fluorescence aggregation points in the cells treated with selenium and S.aureus,and the co-localization of lysosomes and LC3 was obvious compared with S.aureus group.The number of intracellular S.aureus in each selenium treatment group was significantly lower than that in S.aureus group(p<0.05 or p<0.01)and in a dose-dependent manner.In conclusion,autophagy was induced in BMECs after the infection of S.aureus,but autophagosome and lysosome could not fuse normally,autophagy flow was blocked.The accumulation of autophagy was helpful to the survival of S.aureus in BMECs.When autophagy formation was blocked,the intracellular proliferation of S.aureus decreased.Selenium could promote the binding of autophagosome and lysosome,improved the patency of autophagic flow,and reduced the proliferation of S.aureus in BMECs,thus participating in the regulation and improvement of S.aureus infection in BMECs.The results of this study provide a new idea for the prevention and treatment of S.aureus mastitis. |
