Mechanism Research On Heat-clearing And Detoxicating Drug Resisting Staphylococcus Aureus Adhesion To Holstein-bovine Mammary Epithelial Cell

In this study3decoction (Flos Lonicerae decoction, Fructus Forsythiae decoction, FlosLonicerae and Fructus Forsythiae decoction) were used to prevent and treat Mouse mastitisinfected by Staphylococcus aureus. The model of bacteria adhesion cells was made that usingEGFP marked E.coli and Staphylococcus aureus adhere bovine mammary epithelial cells toexplore the infection mechanism of Bovine Mastitis, the mechanism of Flos Lonicerae andFructus Forsythiae’s prevention and treatment for Mastitis, anti-adhesive mechanism of FlosLonicerae and Fructus Forsythiae.Tissues explant culture was used to culture bovine mammary epithelial cells, when80%of the plate would be covered with cells, purified mammary epithelial cells could be obtainedby trypsin digesting from time interval for2-3times. It was detected that the expression ofcytokeratin18genes was positive in mammary epithelial cells. The cow mammary epithelialemitted red fluorescence when excited at514nm.It proved that the mammary epithelial cellshad the characteristic of autofluorescence.Bacteria adhesion to mammary epithelial cells was established by using Staphylococcusaureus and EGFP labeled E.coli BL21infected mammary epithelial cells. By using Gramstaining, it could be observed that cow mammary epithelial cell cytoplasm was stained lightpink, nucleus were stained purple; pink E.coli BL21bacteria closely gathered around themammary epithelial cell, a lot of purple Staphylococcus aureus gathered in the surrounding ofmammary epithelial cells cytoplasm, near the nucleus; EGFP marked E.coli BL21bacteriaproduced green fluorescence at excitation488nm, cow mammary epithelial emitted redfluorescence at excitation514nm, therefore it could be clearly observed the sites thatbacterial adhesion in mammary epithelial cells. Staphylococcus aureus showed typical grapescluster clump adhered the mammary epithelial cell’s surface. It was proved that by using threemethods to observe Staphylococcus aureus and EGFP marked E.coli BL21successfullyadhered to mammary epithelial cells.The mouse mastitis pathological model was made injected different concentrationssuspension of Staphylococcus aureus(104CFU·L-1、105CFU·L-1、106CFU·L-1) into mouse mammary base, observe pathological changes by H.E. Staining. The results show that withincreasing concentrations of inoculation, mouse mammary pathology exacerbated: the lowconcentration104CFU·L-1inoculation group, had mild hyperemia of capillary, a small amountof epithelial cell shedding; high concentrations106CFU·L-1inoculation group, mammarygland flocculus necrosis, interstitial inflammatory edema, a large number of epithelial cellnecrosis and shedding;105CFU·L-1group, mammary gland lobular interstitial edema,neutrophil infiltration, acini partial collapse, a small amount of neutrophil infiltration, a smallamount of glandular epithelial cell necrosis and fall of acinus cavity, capillary a littlebleeding.It is the best choice to use105CFU·L-1suspension of Staphylococcus aureus as theinduction dose to infect Mouse mmmary gland.By using heat-clearing and detoxicating drug------Flos Lonicerae decoction, FructusForsythiae decoction to prevent and cure mouse mastitis, breast bacterial counts to testinfection of bacteria amount, H.E. stain to observe pathological changes, ELISA to determinethe expression level of TNF-α to explore the effect of Flos Lonicerae and FructusForsythiae’s prevention in mice mastitis. The results showed that Flos Lonicerae decoction,Fructus Forsythiae decoction could inhibit the bacterial infection, prevention group hadsignificant inhibitory effect than treatment group, and between three drugs group, FlosLonicerae group was better than the two kinds drugs, Fructus Forsythiae was the last.Bothprevention and treatment could reduce the lesion, made the mammary gland functionnormal.In treatment group the level of the serum TNF-α return to normal, in prevention grouplevel of the serum TNF-α was lower than normal, Flos Lonicerae group was better than thetwo kinds drugs, Fructus Forsythiae was the last. Mast cells showed the body’s immunity andanti-infection,in treatment group, the number of mast cells tended to be normal, indicatingthat the body inflammation relieved, bacterial infection weakened; in prevention group,although the number of mast cells reduced had not reached normal levels, ndicating that thebody was still in its anti-infection or inflammation. In summary,when using Flos Loniceraedecoction, Fructus Forsythiae decoction to prevent and cure mouse mastitis, the treatmenteffect was better than the prevention, but the effect between three drugs was not significant.