| Bovine mastitis is the number one disease that endangers the development of dairy industry.It can significantly reduce milk yield as well as milk quality and harm the production performance of dairy cows,thus causing huge economic losses worldwide every year.Staphylococcus aureus(S.aureus)is one of the main pathogens inducing mastitis in dairy cows.Due to its characteristics such as intracellular parasitism and immune escape,it is difficult to eliminate S.aureus once it invades the mammary gland,thus resulting in the continuous occurrence of mastitis in dairy cows.At present,antibiotics are still used in the clinical treatment of S.aureus-induced mastitis,but antibiotics are difficult to enter the cell,which makes this method ineffective and easy to cause bacterial resistance and drug residue,endangering food safety and human health.Recent studies have shown that the host immune status is crucial to the resistance to pathogen infection and inflammatory response.Therefore,inhibition of S.aureus infection by regulating the body’s immunity may be a potential way to alleviate S.aureus-induced mastitis in dairy cows.As an important component of the body,gut microbiota plays an important role in diseases of the intestinal tract and distal organs such as brain,liver,lung and mammary gland.Our previous study found that gut microbiota can affect the occurrence and development of mastitis in dairy cows by regulating the interaction between mammary gland and pathogenic bacteria,and regulating gut microbiota is an effective way to prevent and control mastitis.A large number of literatures have also shown that gut microbiota can regulate immune homeostasis.Therefore,targeting gut microbiota to regulate host immune response may be an important measure to prevent and treat mastitis in dairy cows in the future.Dietary fiber is an important precursor of Short chain fatty acid(SCFA)produced by gut microbiota,which can increase the abundance of gut microbiota and the level of SCFA in intestinal cavity,so as to maintain intestinal homeostasis and regulate host immunity.As a classic dietary fiber,Inulin(IN)has been shown to regulate gut microbiota and SCFA production and affect disease progression,but whether it contributes to the remission of etiological mastitis remains unclear.Therefore,this study aims to explore the protective effect of inulin against S.aureus-induced mastitis in mice and its potential mechanism.First,a mastitis model was established after feeding inulin to mice infected with S.aureus,and the effect of inulin on gut microbiota and S.aureus-induced mastitis was investigated.The results showed that inulin could significantly reduce S.aureus load,relieve the pathological damage of mammary gland tissue,reduce the concentration of inflammatory cytokines TNF-α and IL-1β,and increase the expression of tight junction(TJ)proteins ZO-1,Occludin and Claudin-3.In addition,16 S r RNA sequencing results showed that inulin could change the diversity,structure and function of gut microbiota in mice,mainly by increasing the abundance of SCFA-producing bacteria such as Bacteroides and decreasing the abundance of intestinal harmful bacteria such as Proteobacteria.These results suggest that inulin significantly alters gut microbiota composition and alleviates S.aureus-induced mastitis in mice.Secondly,giving antibiotic supplementation(ABX)to mice fed inulin to clear gut microbiota,followed by the mammary gland infection with S.aureus to establish a mastitis model,to explore whether inulin affects mastitis by regulating gut microbiota.The results showed that clearing gut microbiota inhibited the protective effect of inulin on the pathological damage of mammary gland tissue caused by S.aureus infection,the production of inflammatory cytokines and the increase of blood-milk barrier permeability in mice.After that,fecal microorganisms from the control group and inulin group were transplanted to ABX-cleared mice.16 S r RNA sequencing showed that compared with fecal microorganisms from the control group(CF)transplantion,fecal microorganisms from inulin mice(HIF)transplanted significantly increased the abundance of SCFA-producing bacteria and decreased the abundance of Proteobacteria and other pathogenic bacteria in the intestines of mice.At the same time,HIF could significantly reduce the inflammatory response of mammary gland tissue and the increase the blood-milk barrier permeability in mice.These results suggest that the protective effect of inulin against S.aureus-induced mastitis depends on gut microbiota.Finally,in order to explore the potential mechanism of inulin alleviating mastitis induced by S.aureus in mice,targeted metabolomics detection showed that inulin significantly increased the concentrations of acetate,propionate and butyrate in feces of mice.In addition,supplementation of propionate and butyrate can significantly relieve mammary gland inflammation induced by S.aureus and increase of blood-milk barrier permeability.The macrophages of different groups of mice were detected and the results showed that butyrate and propionate could significantly reduce the S.aureus load in macrophages and increase the expression of antimicrobial peptide-related genes,and the antibacterial response of macrophages in inulin fed and fecal microbial transplantation(FMT)mice was enhanced and dependent on gut microbiota.Mechanically,butyrate and propionate inhibit the expression of histone deacetylase 3(HDAC3),whereas RGFP966,a specific inhibitor of HDAC3,produced similar antibacterial effects.The above results showed that inulin inhibited HDAC3 activity and activated antibacterial immunity in macrophages through butyrate produced by gut microbiota metabolism,thus alleviating S.aureus-induced mastitis in mice.In summary,this study proved that inulin can relieve S.aureus-induced mastitis,and the specific mechanism is as follows: Inulin can promote the release of butyrate,propionate and acetate by increasing the abundance of SCFA-producing bacteria in the intestine,activate the HDAC3-mediated antibacterial immunity in macrophages,and relieve the mammary gland inflammation induced by S.aureus in mice.Therefore,regulating gut microbiota to promote SCFA production may be a potential strategy to protect against pathogenic mastitis and other infectious diseases. |
