The Protect Effect And Regulation Mechanism Of Stevioside On The Model Animal Of Staphylococcus Aureus Induced Mastitis

Mastitis is a serious threat to dairy cows. Dairy cows suffering from mastitisdeliver drastically reduced milk yield and product quality. This disease results in greateconomic losses in the dairy cow industry world-wide. Pathogenic microorganisms isthe main cause of mastitis. Staphylococcus aureus (S.aureus) is one of the mostcommon grampositive bacterial pathogens and causes mammary infections in dairyruminants.Both the clinical mastitis and the recessive mastitis can be caused by S. aureus,which release exotoxin and even cause death. Once S. aureus infections have beenestablished, they are extremely difficult to control and remove. In addition, treatmentrelies almost completely on the use of antibiotics, which is not very successful againstsome pathogens. The imprudent use of antibiotics cause the spread of drug resistant S.aureus strains. Moreover, the use of antibiotics could result in an increased risk ofantibiotic residues in milk. Thus far, the issues of antibiotic residues and treatmentresistance have practically no effective solutions. S. aureus infection leads to theinflammation and apoptosis of host cells, surviving in living organism of host bymeans of immune evasion. So the treatment of mastitis induced by S.aureus mayIncluding anti-inflammatory, reducing the apoptosis and enhancing the bactericidalaction of natural immune system.Traditional Chinese medicine or natural medicine treatment of dairy bovinemastitis attracted much attention in recent years because of its sterilization andbacteriostasis, anti-inflammatory, regulating the body’s immune function. Steviosideis obtained from leaves of stevia rebaudiana, which is commonly used in traditionalmedicine and in food additives as a natural sweetener. Stevioside is reported topossess some therapeutic benefits such as anti-hyperglycemic, anti-hypertensive,anti-apoptosis effects, anti-inflammatory, and immunomodulatory. In the present study,we sought to examine the pharmacological function of stevioside. It is expensive andinhuman to make an S.aureus induced mastitis on cattle. The animal model ofS.aureus induced mastitis in mouse was more appropriated to study mechanisms andexperiment. The S.aureus mastitis model of mouse was made via perfusion Staphylococcus aureus though Milk ducts. The method of histology HE staining,TUNEL staining, ELISA, western blot and qPCR were used to study the function ofstevioside in inflammation and apoptosis of the S.aureus mouse mastitis model. Theresults showed that stevioside reduce the S.aureus infection induce breast tissuedamage, inflammation, and cell death effectively. Stevioside significantly reduced theinflammatory cell infiltration and the levels of TNF-α, IL-1β, and IL-6and therespective expression of their messenger RNAs (mRNAs). Further studies revealedthat stevioside downregulated the NF-κB, and (mitogen-activated protein kinase)MAPK signaling pathways in the S. aureus-infected mouse mammary gland.Mammary epithelial cells was considered as the most important functional cell inbreast. in order to confirm the experimental results, we isolated the mouse mammaryepithelial cells（MMEC）and make test in vitro. The cells were treated with varyingdoses of stevioside before infection with S.aureus. The pro-inflammatory cytokineswere determined by ELISA. The mRNA of proteins related to NF-κB, MAPK andapoptosis were analyzed by q-PCR. The relative protein expression levels weredetermined by Western blot. The results indicated that stevioside inhibited the mRNAand protein expression of TNF-α, IL-6and IL-1β dose-dependently in S.aureus-stimulated MMECs. Stevioside suppressed the S.aureus-inducedphosphorylation of the NF-κB and MAPK pathways as well as apoptosis. The mRNAlevels of IκB, p38, ERK, JNK, p65, and caspase-3were not influenced by thestevioside treatment. Then, the live/dead cells were detected by immunofluorescencemicroscopy and found that stevioside protect cells from S.aureus induced cell death.To further analyze the pharmacological mechanism of stevioside in MMEC, weinvestigateTLR2, which is the main Pattern Recognition Receptors (PRRs) to S.aureus, can also adjust the downstream signaling pathways. TLR2can not onlyregulate inflammatory signaling pathways, but also regulate apoptotic proteins for theS. aureus infection. The results showed that stevioside inhibit the expression of TLR2and reduced activation of downstream signaling. Stevioside inhibited TLR2expression and reduce the corresponding nuclear transfer factor into the nucleus byinhibiting the phosphorylation of p38, ERK, JNK and p65, IκB in NF-κB and MAPKsignaling pathways to inhibits the expression of IL-1β, TNF-α and IL-6and othercytokines. Stevioside exertsanti-apoptotic properties by inhibiting the activation ofTLR2and reduced activation of caspase-3and Bax. The mRNA levels of IκB, p38, ERK, JNK, p65, and caspase-3were not influenced by the stevioside treatment. Onthe other hand, up regulating innate immune system function of killing bacteria isgood to control disease. The neutrophils was the most important immune cells killingpathogens and the contributable component of innate immune system. The furtherstudy about stevioside up regulating innateimmune system was investigate byConfirming the synergy of stevioside and NETs killing S. aureus. Neutrophils werepurified and induce to generate NETs, mixed with Stevioside and incubate the MMECfirst, followed by stimulating with S. aureus. This was to simulate a part of the innateimmune response when S. aureus infection happened. The results showed thatstevioside and NETs have an synergy effect on inhibiting inflammation and apoptosis,and meanwhile retarding bacterial growth. The combined effect was stronger than thetwo separate effects. This suggests that the stevioside and the NETs of innate immunesystem were synergy.In conclusion, we observed the anti-inflammatory and anti-apoptosis function ofstevioside on mouse model. The Further investigatewas made on MMEC model andanalysis the mechanism of its anti-inflammatory, anti-apoptosis and synergy withNETs up regulating innate immunity. We wished that our results would providetheoretical guidance and help for the application of stevioside in veterinary clinicaland the development of protective drugs for S. aureus-induced mastitis.