The Anti-Inflammatory Mechanism Of Cationic Antimicrobial Peptides Containing Trp

LPS,the main composition of outer membrane of Gram-negative bacteria,can induce the inflammatory reaction.The abuse of antibiotics results in not only the emergence of multiple-drug resistant bacteria,also the release of LPS from the killed bacteria cell wall.Furthermore,LPS induces the produce of the inflammatory factors,such as IL-6 and TNF-?,and causes sepsis.Antimicrobial peptides(AMPs)with broad-spectrum antibacterial activity,have potential as novel antibiotics against microorganisms.L-K6 from the natural antibacterial peptides Temporin-1CEb extracted from skin secretion of Rana chensinensis exhibited high antimicrobial activity.Furthermore,a series of antibacterial peptides I1 W,I4W,L5 W,L11W,L12 W,I1WL5W and I4WL5 W were synthesized through the amino acid substitution at position of L-K6 amino terminal 1,4,5,6,11,and 12.Previous studies showed that these containing Trp peptides with 6 positive charges had significant bactericidal activity.This present study focus on the anti-inflammatory mechanisms of cationic antimicrobial peptides containing Trp on mice RAW264.7 cells induced by LPS.Firstly cell toxicity of cationic antimicrobial peptides containing Trp on mice macrophage RAW264.7 cells was determined using MTT method.Based on the IC50 value,the concentration 10-20?M antimicrobial peptides were used to explore the inflammatory response induced by LPS.The results showed that cationic antimicrobial peptides containing Trp inhibited the release of inflammatory factor TNF-? and IL-6 of RAW264.7 cell induced by LPS.Among of the antimicrobial peptides,I1 W,I4WL5W and L5 W exhibited the highest activity,and the activity of L-K6 was the worst.The previous results showed that the antimicrobial peptides containing Trp targeted on the cell membrane of bacteria and tumor cells,and killed bacteria or tumor cells by destroying the cell membrane permeability.In order to explore the anti-inflammatory mechanisms of cationic antimicrobial peptides containing Trp,I1 W,I4WL5W and L5 W were used as experiment material,and L-K6 as negative control,to explore the effects of peptides on the membrane permeability of RAW264.7 cell by PI staining,depolarization and observation of cell structure by confocal microscopy.The results showed that the antibacterial peptides didn't damage the cell morphology and membrane,but entered into the cytoplasm across the membrane.Then the gene expression of TLR4 / MyD88 /NF-?B and p38 / ERK / JNK were determined using RT-qPCR and western blot,respectively.The results of RT-qPCR showed that theseantimicrobial peptides inhibited gene expression at different level.Among the peptides,I4WL5 W and I1 W showed the highest inhibition on TLR4 andMyD88 and NF-?B,respectively.The results of western blot showed that these peptides also inhibited expression of the signal proteins.Among of them,I4WL5 W had the best inhibition on p38 and JNK,I1 W had the best inhibition on ERK and L-K6 had the relative weakest inhibition on channel protein expression to the others.The combining capacity of these 4 antimicrobial peptides and LPS was tested by Zeta potential and chromogenic substrate limulus reagent.Zeta potential showed that the 4antimicrobial peptides could combined with cell's outer membrane surfaces(LPS)from klebsiella pneumoniae,singular proteus-beta,escherichia coli,sewer e.coli and pseudomonas aeruginosa.I1 W not only combined with outer membrane,but also counteracted all the negative charges,which showed that they could be inserted into the outer membrane of antimicrobial peptides.In combination with the result of chromogenic substrate limulus reagent showed that the antibacterial peptides could be combined and neutralized with LPS,and inhibit inflammation produced by LPS combined with cells.