Protective Effect And Mechanism Of Deoxycholic Acid（DCA） On Staphylococcus Aureus Mastitis In Mice

Dairy mastitis is a general term for the inflammation that occurs in the mammary glands,and it is one of the common and high incidence diseases in dairy cattle breeding.The occurrence of mastitis in dairy cows will not only lead to the decline of milk production and cause economic losses,but also easily cause food safety and other problems,threatening public health security.In a previous laboratory study,by constructing a subacute ruminal acidosis（SARA）model,the relationship between rumen flora disturbance and dairy cow mastitis was explored,and it was found that rumen flora disturbance could increase the susceptibility of Staphylococcal mastitis,but the relevant mechanism remains unclear.Based on the previous research in the laboratory,this study detected and screened the differential metabolites in the milk of SARA cows and healthy cows by means of metabolomics,and evaluated the protective effect of the screened metabolites on Staphylococcal mastitis.In order to elucidate the mechanism by which rumen flora disturbance increases susceptibility to Staphylococcal mastitis.First,milk samples from 8 healthy cows and 8 cows with SARA were collected for untargeted metabolomic testing.The results showed that compared with the metabolites in the milk of healthy cows,the milk of SARA-affected cows were mainly found in five significantly up-regulated differential metabolites,including N-cinnamoylglycine,thirty-nine significantly down-regulated differential metabolites,including Cinnamoylglycine and primary bile acid cholic acid（CA）and secondary bile acids and 4 metabolic enrichment pathways including bile acid secretion,saturated fatty acid synthesis,unsaturated fatty acid synthesis and Ferroptosis.By analyzing the results of differential metabolites and enriched pathways,we focused on the bile acid metabolism pathway,and speculated that bile acid metabolism disturbances may be related to the susceptibility of Staphylococcal mastitis to the disturbance of rumen flora.Further analysis found that the expressions of CA and DCA in the milk of SARA-affected cows were significantly decreased.The anti-mastitis effects and mechanisms of CA and DCA will be elucidated in the future.Secondly,by constructing a mouse model of Staphylococcal mastitis,to explore the protective effect of CA and DCA on Staphylococcal mastitis,in order to reveal the mechanism of rumen flora disturbance aggravating the susceptibility of Staphylococcal mastitis.The results showed that DCA could significantly reduce S.aureus-induced mammary tissue damage,MPO activity and the expression of inflammatory cytokines TNF-α and IL-1β.However,CA had no effect on S.aureusinduced mammary tissue injury,MPO activity and the expression of inflammatory cytokines TNFα and IL-1β.The above results indicated that DCA had a protective effect on S.aureus-induced mastitis,but CA had no protective effect on S.aureus-induced mastitis.Further,we clarified the effect and mechanism of DCA against Staphylococcal mastitis.By setting blank control group,DCA（30 mg/kg）treatment group,S.aureus model group,DCA（10 mg/kg）+S.aureus group,DCA（20 mg/kg）+S.aureus group,DCA（30 mg/kg）+S.aureus group,a total of 6 groups to explore the specific protective effect and mechanism of DCA on Staphylococcal mastitis,the results show that only high dose DCA injection has no side effects on normal mouse mammary gland tissue;DCA can inhibite the expression of inflammatory cytokines induced by S.aureus in a does-dependent manner,meanwhile,DCA could does-dependtly up-regulate the expression of tight junction proteins ZO-1,Occludin and Claudin-3 in a dose-dependent manner to maintain the integrity of the bloodmilk barrier.These results suggest that DCA protects against Staphylococcal mastitis by inhibiting the inflammatory response and blood-milk barrier integrity.Finally,the anti-mastitis mechanism of DCA was elucidated on mouse mammary epithelial cells.The cytotoxic effect of DCA on mouse mammary epithelial cells was detected by CCK8 assay;the expressions of inflammatory cytokines and signaling pathway proteins were detected by fluorescence quantitative PCR and Western blot experiments.The results showed that DCA had no effect on the activity of mammary epithelial cells within the concentration range of 10μM～30μM;DCA could dose-dependently inhibit the inflammatory cytokines TNF-α,IL-1β and IL-6 induced by Staphylococcus aureus At the same time,DCA can inhibit the expression of IκBα and p65 phosphorylated proteins in the NF-κB pathway induced by Staphylococcus aureus,as well as the protein expressions of inflammasome NLRP3,ASC,and IL-1β;Significantly upregulates the TGR5 receptor,and the inhibition of NF-κB and NLRP3 signaling pathways by DCA can be reversed by the TGR5 inhibitor SBI-115.The above results indicate that DCA exerts its anti-mastitis effect by up-regulating TGR5 receptor,thereby inhibiting NF-κB and NLRP3 signaling pathways.In conclusion,the metabolomic results showed that the disturbance of bile acid metabolism may be related to the increased susceptibility of Staphylococcal mastitis due to the disturbance of rumen flora,and the expression levels of CA and DCA in bile acid metabolism were significantly decreased in the milk of SARA cows.Further studies found that DCA had a protective effect on S.aureus-induced mastitis,while CA had no protective effect.DCA can alleviate the inflammation caused by S.aureus infection by activating the TGR5 receptor and then inhibiting the NFκB/NLRP3 inflammatory signaling pathway.The above results suggest that the decrease of DCA content in milk of SARA cows may be one of the reasons why the disturbance of rumen flora increases the susceptibility of Staphylococcal mastitis.The above results can provide a theoretical basis for the follow-up clinical screening of effective prevention and treatment of dairy cow mastitis drugs or feed additives research and development.