| Sepsis,defined as life-threatening infection due to a dysregulated host response to infection.It is among the most expensive conditions treated in hospitals.Despite increasing clinical awareness,changes in diagnosis and better understanding of treatment measures,mortality remains high.An increasing incidence of antibiotic resistance and multiple-antibiotic resistance makes the treatment of sepsis a difficult problem.Furthermore,most antibiotics have no effect on the LPS released by bacteria,and sometimes,lysed bacteria release more LPS after the application of antibiotics,which in turn aggravates the clinical symptoms of patients and increases mortality.LPS is a major component of the outer membrane of Gram-negative bacteria,and LPS is composed of lipid A,a core polysaccharide chain and a variable polysaccharide chain called 'O-antigen'.In the extracellular space,LPS is recognized by the plasma membrane localized TLR4-MD2 coreceptor complex in combination with the coreceptor CD 14.TLR4 signaling,which is mediated by the adapters MyD88 and TRIF,then activates NF-?B and IRF3-mediated transcription of inflammatory molecules,including cytokines and chemokines.The overactivation of TLR4 by LPS and the ensuing cytokine storm are thought to contribute to endotoxic shock or sepsis.Antimicrobial peptides(AMPs),are produced by the majority of organisms on earth.The known AMPs exhibit diverse biological activities against bacteria,fungi,parasites,viruses and even microbial biofilms.Some AMPs participate in several defense-related processes by killing pathogens,binding or neutralizing endotoxins and modulating immune responses to infection.AMPs have become an alternative treatment for sepsis because of their potential biological activities and immune regulatory properties.Neutralization or elimination of LPS remains a promising approach for treating sepsis.The antibiotic polymyxin B was the first peptide shown to reduce IL-1 secretion and lethality by binding to LPS.However,nephrotoxicity limits the further application of polymyxin B,so AMPs with low toxicity and powerful bioactivities are still potential therapies for sepsis.AMPs are excellent candidates for developing peptide antibiotics,but there are some difficulties to be overcome in the use of naturally occurring AMPs as therapeutic agent.Lack of stability in vivo,potential toxicities to host cells,etc.In this study,the peak with LPS-neutralizing activity was purified from the skin secretions of Bombina maxima,the peptide sequences were determined by peptidomics,and 7 peptides with strong binding ability to LPS were screened by molecular docking.Two peptides screened with the strongest binding ability to LPS through the biolayer interference technology,named peptide 2,peptide 7.Peptide 2 is a 34-residue peptide of sequence DLLGQAGCTLVIGEEAGKEIADLTNSVIEDVLRK,peptide 7 is a 15-residue peptide of sequence NQPFTFTLGTGQVIK.Peptide 2 and peptide 7 have no direct antibacterial activity against common clinical bacterial strains,but they have strong binding ability with LPS,the affinity constant(KD)values are 1.05×10-9 M and 8.156×10-9 M,respectively;Peptide 2 and peptide 7 significantly inhibited the transcription and production of pro-inflammatory factors induced by LPS in RAW 264.7,including TNF-?,IL-1?,and IL-6.Peptide 2 and peptide 7 block the activation of MAPKs(ERK,JNK,and p38)and NF-?B signal pathways,exert an anti-inflammatory effect in LPS-stimulated RAW 264.7.In vivo,peptide 2 and peptide 7 could reduce the mortality of LPS-treated mice,and had a protective effect on sepsis mice.Further analysis of interaction between peptide 2/peptide 7 and LPS indicated that peptide 2,peptide 7 can partially neutralize LPS in a dose-dependent manner.Peptide 2 and peptide 7 may block the binding of LPS to LPS-binding protein,and hence suppresses the release of cytokines and other inflammatory mediators induced by LPS.Peptides 2 and 7 have immunomodulatory effects,can partially neutralize LPS and inhibit the production of pro-inflammatory cytokines. |
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