Anti-inflammatory Effect Of Hydrogen On Mastitis Induced By Staphylococcus Aureus

Mastitis is characterized by inflammation in the mammary glands and has a detrimental impact on animal health and production.Staphylococcus aureus（S.aureus）is one of the most important pathogens of bovine mastitis.It can invade bovine mammary epithelial cells and cause continuous infection of udder.Hydrogen therapy is a rising technology in recent decades.It has excellent ability in antioxidant,anti-inflammatory,anti-apoptosis and regulation of cellular pathways.To identify the anti-inflammatory effect against bovine mastitis of H₂and define the mechanism of action,the models of in vitro and animal were performed respectively.To verify the protective effect of hydrogen in vitro,MAC-T cells were stimulated with S.aureus to establish an infection model.The experiments were divided into four groups:CON group,S.aureus group,H₂group and S.aureus+H₂group.After infection with S.aureus for 3 h,the cell culture medium of H₂group and S.aureus+H₂group were replaced with hydrogen-rich medium for 3h.The supernatants from each group were collected to detect Lactate dehydrogenase（LDH）content.Western blot was used to detect the expression levels of TLR2,Myd88,IKK,p65,caspase-1 and IL-1β.Immunofluorescence was used to analyze aggregation of ASC speck and the secretion of IL-1β,IL-6,TNF-αwas evaluated by ELISA.The following results are obtained that the content of LDH induced by S.aureus was significantly reduced in the S.aureus+H₂group.The activation of TLR2/NF-κB signals as well as ASC speck and inflammasome related protein expression was inhibited by H₂.Meanwhile,the release of pro-inflammatory factors such as IL-1β,IL-6 and TNF-αwas lower than that of the infected group after hydrogen treatment.To evaluate the clinical application of H₂in bovine mastitis,the mouse were stimulated with S.aureus to establish an infection model.The experiments were divided into four groups:CON group,hydrogen-rich glycerol（HRG）group,S.aureus group and S.aureus+hydrogen-rich glycerol（S.aureus+HRG）group.After infusing with S.aureus for 24 h,the skin of the fourth pair of udder ducts was smeared with HRG for a week.Hematoxylin and eosin（HE）staining was used for histopathological examination.Blood was analyzed by routine blood testing and the levels of IL-1β,IL-6 and TNF-αin serum were detected by ELISA.Western blot was used to assess the protein expression levels of TLR2,Myd88,p65and IKB.Results showed that application of HRG significantly relieved udder damage and inflammatory edema.And the interaction process of neutrophil infiltration and the release of cytokines was inhibited in the S.aureus+HRG group,which may be due to the inhibitory effect of HRG on TLR2/NF-κB signaling pathways.In conclusion,hydrogen ameliorates S.aureus-mediated inflammatory response associated with mastitis in vitro and animal models,suggesting that it may be a possible synergist to antibiotics against S.aureus-induced bovine mastitis for clinical application.