Effects Of TLR2-and TLR4-BBpeptides (BBPs) On Cytokine Secretion Of Mouse Peritoneal Macrophages

Toll like receptors(Toll-like receptors,TLRs)are a class of important pattern recognition receptors involved in innate immunity and play an important role in innate and acquired immune responses.When the bacteria and toxin pattern recognition molecule binds to TLRs and TLRs induced activation of chemokines,cytokines and mediators of inflammation and promote the secretion of inflammatory cells infiltration,occurrence of inflammation.The study found that TLR2 in the inflammatory process caused by bacterial infection and TLR4 play a key role,such as TLR2 and TLR4 can activate the control effect,can control the occurrence of excessive inflammatory response during bacterial infection.Therefore,according to the published sequences,we synthesized the specific blocking peptides of TLR2 and TLR4.We used fluorescence quantitative PCR,ELISA and Western Blot technique to detect specific TLR2 and TLR4 blocking peptide on mice peritoneal macrophage cytokine IL-1 beta,IL-10,TNF-alpha and RANTES expression and ERK,p38,JNK and P65 phosphorylation effect.TLR2 and TLR4 blocking peptide pretreatment(24h)in murine peritoneal macroph-ages stimulated by LPS in 4h,fluorescence quantitative PCR assay showed that comparison between CP control group and LPS stimulation group IL-1 beta,IL-10,TNF-alpha and RANTES mRNA expression had no significant difference(P>0.05),TLR2 and TLR4 blocking peptide group IL-1 beta,TNF-alpha,IL-10 and RANTES,the relative expression of mRNA was significantly decreased(P<0.01).The results indicated that TLR2 and TLR4 peptides could inhibit the expression of proinflammatory cytokines(IL-1 beta and TNF-alpha),anti-inflammatory cytokines(IL-10)and chemokines(RANTE S).ELISA test results showed that CP stimulation control group compared with LPS group IL-1 beta,IL-10,TNF-alpha and RANTES protein had no significant difference(P>0.05),TLR2 and TLR4 blocking peptide IL-1 group,TNF-alpha,IL-10 beta and RANTES protein expression was significantly decreased(P<0.01).The results indicated thatt TLR2 and TLR4 peptides had inhibitory effects on the expression of proinflammatory cytokines(IL-1 beta and TNF-alpha),anti-inflammatory cytokines(IL-10)and chemokine(RANTES)protein.Western Blot results showed that both TLR2 blocking peptide or TLR4 blocking peptide can suppress p65 activation and ERK significantly,and the TLR4 blocking peptide was better than TLR2 blocking peptide;TLR2 blocking peptide on the activation of p38 and JNK lack of obvious inhibitory effect,TLR4 effect and inhibition of p38 activation peptide closed JNK is more obvious,more than that closed peptide has certain inhibitory effect on LPS activation of NF-kappa B and MAPK signaling transduction.In summary,TLR2 and TLR4 blocking peptide can inhibit mouse peritoneal macrophage synthesis and secretion of proinflammatory cytokines(IL-1 beta and TNF-alpha)anti-inflammatory cytokines(IL-10)and chemokine(RANTES)by inhibiting activation of NF-kappa B and MAPK signal transduction pathway.For days after the TLR2 and TLR4 blocking peptide application as biological agents to lay a theoretical basis for veterinary clinical treatment of bacterial infectious diseases.