The Molecular Mechanism Of Mastitis Induced By Enterogenic Stenotrophomonas Maltophilia

Mastitis is one of the most important diseases of dairy cows.It not only causes huge economic losses to the development of animal husbandry,but also seriously endangers food safety.According to the traditional concept,mastitis is a local inflammatory reaction caused by bacteria and other pathogenic microorganisms infecting the mammary gland,while the traditional Chinese medicine theory believes that "mastitis is caused by stagnation of liver qi and stomach heat",that is,due to excessive consumption of refined food,the stomach heat is blocked,the qi and blood are blocked,and then the mastitis is caused.In the early stage,the research team established the animal model of subacute rumen acidosis(SARA),a typical rumen flora disorder,by feeding dairy cows with high concentrate for a long time.It was found that the rumen microbiota disorder could induce mastitis in dairy cows,and the abundance of Stenotrophomonas maltophilia(S.maltophilia)in the rumen of dairy cows with mastitis was significantly increased.At the same time,the bacteria appeared in a large number in the mammary gland,and the lactating mice were successfully induced mastitis by oral administration.In combination with the literature report that gut microbiota and their bacterial components can migrate to the mammary gland through endogenous pathways such as blood and/or lymph circulation,we boldly propose the theoretical hypothesis that S.maltophilia and/or their bacterial components in the rumen can migrate to the lactating mammary gland through endogenous pathways such as blood and/or lymph circulation,and induce mastitis.Firstly,fluorescence labeling was performed on S.maltophilia and they were administered orally to mice to detect their effects on mastitis in mice.The results showed that oral administration of S.maltophilia resulted in pathological damage of mammary gland,and the activity of inflammatory cytokines TNF-α,IL-1β,MPO and the expression of inflammatory signal pathways p-p65 and p-p38 were significantly increased.At the same time,the expression of blood-milk barrier related tight junction proteins ZO-1,Occludin and Claudin-3 was significantly decreased.Further examination of the pathological changes and barrier function of the intestinal tissue showed that oral administration of S.maltophilia resulted in inflammatory cell infiltration and crypt structure changes in the intestinal tissue.At the same time,the expression of p-p65 and p-p38 were significantly increased,and tight junction protein ZO-1 and Claudin-3 were significantly decreased.The above results showed that the oral administration of S.maltophilia could destroy the intestinal barrier and the bloodmilk barrier,and induce mastitis.Enterogenic S.maltophilia can destroy the intestinal barrier and blood-milk barrier and induce mastitis.We speculate that enterogenous S.maltophilia may cause mastitis due to its migration from the intestine to the mammary gland.Subsequently,the S.maltophilia in mesenteric lymph nodes,breast lymph nodes,mammary glands and other tissues and organs and serum samples of mice fed with S.maltophilia were detected.The results of agar gel electrophoresis showed that S.maltophilia after oral administration appeared in mammary gland and other tissues.Through the selective culture of S.maltophilia in the mammary gland,the detection of the protein expression of sf-GFP in mammary gland,and the localization of S.maltophilia in mammary gland,it is proved that enterogenous S.maltophilia can indeed migrate to the mammary gland and induce mastitis.Secondly,in order to analyze the molecular mechanism of mastitis induced by S.maltophilia,the transcriptome analysis was performed on the mammary gland tissue of mice fed with S.maltophilia.The results showed that the process of cell adhesion and cell differentiation in mouse mammary gland were significantly down-regulated by S.maltophilia.The results of KEGG analysis showed that compared with the control group,the MAPK signaling pathway,calcium signal pathway and AMPK signaling pathway were significantly up-regulated in the the mammary gland of mice fed with S.maltophilia,while the expression of tight junction protein cell process was decreased.These results suggest that S.maltophilia may induce mastitis by regulating calcium and AMPK signaling pathways.Finally,the mouse mammary epithelial cells(MMECs)infected by S.maltophilia were used as models to explore whether S.maltophilia could induce mastitis by activating the calcium-ROS-AMPK-m TOR-autophagy pathway.The results showed that the concentration of inflammatory cytokines TNF-α and IL-1β secreted by MMECs was significantly increased due to the infection of S.maltophilia.The expression of tight junction protein in the blood-milk barrier decreased significantly,and the signaling was significantly up-regulated.Further,through the pretreatment of MMECs with calcium chelator EGTA,it was found that EGTA significantly inhibited the activation of calcium-ROS-AMPK-m TOR-autophagy signaling by S.maltophilia.At the same time,it decreased the concentration of inflammatory cytokines and increased the expression of tight junction protein.In addition,pretreatment of MMECs with autophagy inhibitor 3-MA showed that inhibition of autophagy also alleviated the inflammatory reaction induced by S.maltophilia and increased permeability of bloodmilk barrier.These results suggest that S.maltophilia may induce mastitis by activating the calcium-ROS-AMPK-m TOR autophagy pathway.In conclusion,the results showed that enterogenic S.maltophilia could migrate from the gut to the mammary gland via the gut-mammary axis and activate the calciumROS-AMPK-m TOR-autophagy pathway to induce mastitis.Targeting the gutmammary gland axis may also represent an effective method to treat mastitis.