The Protective Effect And Mechanisms Of TIR Domain Derived Decoy Peptides On LPS-induced Mastitis In Mcie

Mastitis is one of the most important diseases led to huge loss of diary farming industry.Gram-negative bacterial like Escherichia coli(E.coli),is a major factor that cause mastitis,serves to activate an inflammation of mammary gland and so as to damage the immune system.Mastitis model induced by Lipopolysaccharide(LPS),the internal molecules within the outer membrane of Gram negative bacteria including E.coli,has been wildly accepted.Toll like receptor 4(TLR4)is activated by LPS.After being stimulated by LPS,TLR4 activate signaling pathways that lead to NF-?B phosphorylation and then causes the production of pro-inflammatory cytokines that stimulate the innate immune responses.The intracellular part of a TLR4 is comprised of the intracellular Toll-Interleukin-1 Receptor(TIR)homology domain.TIR domain,also found in TLR adapter proteins and Interleukin-1 Receptor family members,are protein interaction domains that function to mediate transient interactions of signaling proteins required for signal transduction.Stimulation of a TLR with its cognate agonist leads to recruitment of TIR domain-containing TLR adapters to TLRs through interactions of TIR domains present in TLR4 and TLR4 adapters.Therefore,in our study we synthesizes some cell-permeable TIR domain-derived decoy peptides and examines these substances for the ability to inhibit TIR : TIR interactions that are required for the assembly of function TLR4 signaling pathway.For this study,we synthesized and selected anti-inflammatory cell blocking peptides(TM6 and TR6)to investigate their protective function and mechanism toward mastitis model induced by LPS in mice.Mastitis model was induced by LPS.And the mice were treated with cell decoy peptides TM6 and TR6 of different concentration respectively.Twenty-four hours later,the mice were killed and the mammary gland tissues were collected.The pathological changes were observed using a light microscope after staining with H & E.Pro-inflammatory cytokines TNF-?,IL-1?,and IL-6 was tested by ELISA.The MPO activity was detected by MPO kits.The result suggested that compared with the control group,the mammary gland tissues from LPS groups showed significant pathologic changes,such as inflammatory cell infiltration,thickening of the alveolar wall and mammary gland destruction,as well,MPO activity and the levels of TNF-?,IL-1?,and IL-6 markedly increased.However,the treatment of TM6 and TR6 apparently reduced inflammatory reactions induced by LPS,which also played a dose-dependent manner.These data showed that TM6 and TR6 have a protective effect on mastitis induced by LPS in mice.In order to further invest the protective mechanism of cell blocking peptides toward LPS-induced mastitis,the primary mouse mammary epithelial cells were used for next step.The potential cytotoxic effects of TR6 or TM6(0-20 ?M)on primary mouse mammary gland epithelial cells were evaluated by MTT assay.The cells were pretreated with TM6 or TR6 of different concentrations before 1 h,and then stimulated with LPS(1 ?g/ml)for another 1 h.the samples were collected to test the levels of the pro-inflammatory cytokines and the expression of NF-?B and MAPK signaling pathways.The result suggested that TM6 and TR6 of different concentrations effectively repress the production of pro-inflammatory cytokines,expression of NF-?B and MAPK signaling pathway proteins in LPS-induced mouse mammary epithelial cells,therefore playing protective role against mastitis.In summary,this study suggested the TM6 and TR6 have a protective effect on mastitis in mice in vivo.Moreover,the primary mouse mammary epithelial cells were used to test the mechanism of it.The result showed that TMR and TR6 may inhibit the production of pro-inflammatory cyttokines by LPS-induced activation of NF-?B and MAPK signaling pathway,thereby avoiding LPS-induced mastitis.In the future,it may turn into a new possibility of developing anti-inflammatory medicine through synthesize various blocking peptides targeting on TLR4 innate immune signaling pathway and associating adaptor proteins so as to inhibit signal transduction.