Study On The Antiseptic Effects Of Antimicrobial Peptides MS-PT And MS-PT1

Sepsis is the leading cause of death in patients with infectious diseases.Toll-like receptor 4(TLR4)triggers inflammation and sepsis by recognizing lipopolysaccharide(LPS).An ideal drug that effectively neutralizes LPS and competitively blocks the TLR4 pathway has not yet been developed.Antibacterial peptides are promising antibiotics due to their unique antibacterial mechanisms as killing bacteria and simultaneously neutralizing LPS released from dead bacterial cells.It has the potential to be developed as a new anti-inflammatory drug.1.Study on anti-inflammatory activity in vitro: The antibacterial peptide MS-PT is derived from the skin secretions of the south American frog Phyllomedusa tarsius,and MS-PT1 is a more potent derivate which is engineered based on the nautral MS-PT.MTT,WB,q RT-PCR,and ELISA experiment results on cells indicated that MS-PT and MS-PT1 could inhibit LPS-induced phosphorylation of Ikkβ and IκBα,TNF-α and IL-6 gene expression and corresponding protein expression.2.Study on the antiseptic effect in vivo: Both MS-PT and MS-PT1 had protective and therapeutic effects on LPS-induced sepsis mice,and could significantly improve the survival rate of mice.WB,q RT-PCR and ELISA assays showed that MS-PT and MS-PT1 significantly inhibited the phosphorylation of IκBα,the expression and release of TNF-α and IL-6 genes in lung tissues,and the release of TNF-α and IL-6 proteins in serum.Stained section experiments showed that MS-PT and MS-PT1 alleviated lung fibrosis and pathological changes caused by LPS in septic mice.3.Study on anti-inflammatory mechanism : The circular dichroism(CD)experiment showed that MS-PT1 was induced to form α-helix structure in LPS environment.Combined with the conformational change confirmed by in-situ infrared spectroscopy.Zeta potential experiment result indicated that the interaction force of MS-PT1 with LPS were mainly electrostatic attraction.Isothermal titration calorimetry(ITC)and limulus endotoxin neutralization test(LAL)showed that MS-PT1 could directly bind to LPS and had endotoxin neutralization effect.Further dynamic light scattering(DLS)experiments showed that MS-PT1 could depolymerize LPS aggregations.Fluorescence co-localization assay verified that MS-PT1 inhibited the action of LPS on macrophages by binding to LPS.Immunoprecipitation and immunofluorescence showed that MS-PT1 could competitively bind LPS with TLR4/MD2 protein,thereby inhibiting the production of TLR4-MD2-My D88 complex,and thereby reducing the intracytoplasmic p65 protein transfer into the nucleus of the NF-κB signaling pathway mediated by TLR4-MD2-My D88 complex.In summary,MS-PT1 neutralizes the toxicity of LPS by binding to LPS followed by inhibited TLR4-MD2-My D88 signal pathway to protect LPS-induced sepsis mice,which provides a particular theoretical and experimental basis for the development of anti-inflammatory drugs for the LPS-TLR4 pathway.