Effects Of Selenium On Autophagy And Oxidative Damage Of Macrophages Infected By Staphylococcus Aureus

Macrophages play a key role in the immune system to protect the body against pathogens and regulate the development of tissue inflammation.Autophagy involves the degradation of organelles damaged by cells' internal functions or structures,misfolded proteins and pathogens invading cells,which are transported to lysosomes.It is an important mechanism for cells to resist the invasion of pathogens.Previous studies in our laboratory have found that Staphylococcus aureus(S.aureus),an important pathogen of cow mastitis,can induce autophagy of macrophages,a key cell of the immune system.Selenium is an important trace element for organism.The effect of Selenium on macrophage autophagy infected by S.aureus is not clear.Other studies have shown that selenium can effectively alleviate S.aureus-infected oxidative stress in dairy cow mammary epithelial cells.Whether selenium can alleviate S.aureus-infected oxidative stress in macrophages remains to be studied.In this study,RAW264.7,a mouse macrophage line,was used to study the effects of selenium on S.aureus-infected macrophage autophagy and NF-kappa B and MAPK signaling pathways,and the effects of selenium on S.aureus-infected oxidative stress system and PI3K/Akt/mTOR pathways.To study the effects of selenium on autophagy and related pathways of macrophages infected by S.aureus,the cells were divided into blank control group,S.aureus infection group,selenium supplementation group and co-treatment group of selenium and S.aureus.S.aureus was inoculated at the ratio of MOI=1:1 after 12 h of 2 ?M selenium preincubation.The treatment was terminated after 45 min.Western Blot was used to detect the expression of LC3,Atg5,Beclinland p62,key proteins of NF-kappa B and MAPK signaling pathway;laser confocal technique was used to detect the fluorescence intensity of LC3 protein foci in cells;transmission electron microscopy was used to observe the changes of cell ultrastructure,and plate counting method was used to study the change of S.aureus bacterial load in macrophages.The results showed that the expression levels of Beclinl,LC3II,Atg5 and p62 were significantly increased after S.aureus infection compared with the blank control group(p<0.01).Confocal laser microscopy showed obvious fluorescence foci of LC3 protein in cells.Transmission electron microscopy showed vesicles and autophages encapsulating S.aureus.The phosphorylation levels of key proteins of NF-kappa B and MAPK signaling pathways were extremely high.Compared with S.aureus infection group,the expression of LC3? increased(p<0.01),and the expression of p62 decreased(p<0.01)after selenium supplementation.The fluorescence foci of LC3 protein in cells increased by confocal laser microscopy.The encapsulating vesicles and autophages of S.aureus were observed by transmission electron microscopy.At the same time,compared with S.aureus infection group,the bacterial load in cells decreased significantly or very significantly at 1 h,2 h and 3 h after selenium supplementation(p<0.05 or p<0.01).The results showed that selenium could promote the autophagy of RAW264.7 macrophages infected by S.aureus,alleviate the blockage of autophagic flow,facilitate the removal of S.aureus by macrophages,inhibit its proliferation in cells,and significantly inhibit the activation of NF-kappa B and MAPK signaling pathway.In order to study the effect of selenium on PI3K/Akt/mTOR signaling pathway and antioxidant capacity of macrophages invaded by S.aureus,relevant studies were carried out after cell treatment.Western Blot was used to detect the key proteins expression of PI3K/Akt/mTOR signaling pathway.Flow cytometry was used to detect the changes of ROS and mitochondrial membrane potential.The activities of T-SOD,CAT,GSH-Px and the levels of MDA and T-AOC were detected by colorimetry.The results showed that the phosphorylation levels of PI3K?Akt and mTOR protein increased significantly after S.aureus infection compared with the blank control group(p<0.01).The intracellular ROS level increased significantly(p<0.01).The mitochondrial membrane potential decreased significantly(p<0.01).The activities of T-SOD,GSH-Px and CAT decreased significantly(p<0.01).Compared with S.aureus infection group,the phosphorylation level of PI3K Akt and mTOR protein decreased significantly after selenium supplementation(p<0.01).The ROS level in cells decreased significantly(p<0.01).The mitochondrial membrane potential increased significantly(p<0.05).The activities of CAT,GSH-Px and T-SOD increased significantly(p<0.01).The content of MDA decreased,and T-AOC increased significantly(p<0.01).The results showed that selenium could significantly increase the activity of RAW264.7 antioxidant enzymes,increase mitochondrial membrane potential,reduce ROS level and MDA production,and improve the antioxidant capacity of cells by inhibiting the expression of PI3K/Akt/m TOR signaling pathway proteins.In conclusion,selenium can promote autophagy of RAW264.7,alleviate the blockade of autophagic flow,inhibit the key proteins of MAPK and NF-kappa B signaling pathway,reduce inflammation,and inhibit PI3K/Akt/mTOR,improve cell antioxidant capacity,stabilize mitochondrial membrane potential,reduce ROS production and alleviate cell damage caused by S.aureus.This study provides a new idea and method for the prevention and treatment of clinical cow mastitis.