Study On Anti-inflammatory Activity Of The Derivatives Of Antimicrobial Peptide Temporin-1CEb

Lipopolysaccharide(LPS),also be called endotoxin,is a major structural component of outer membrane of gram-negative bacterial cells.It can enhance the phagocytic activity of phagocytes by inducing the release of inflammatory cytokines.On the one hand,inflammatory cytokines are important for the local inflammatory response,but excessive inflammatory cytokines will lead to septic shock,even to death.So there is a need to develop therapeutic strategies to prevent the activation of inflamma-tory cells by components of gram-positive cell walls Antimicrobial peptidess,an important component of innate immune system,are generally small,highly positively charged,and good hydrophobicity,and has the potential to neutralize the LPS.Antimicrobial peptides L-K6,L-K5,L-K6V1 and L-K6V2 were derivatives from the natural peptide Temporin-1CEb which be isolated and purified from the skin secretions of Rana chensinensis,with 6-7 net positive charges,respectively.Previous experiments have shown that the four antimicrobial peptides have good bactericidal activity and can significantly inhibit the release of cytokine TNF-a,IL-8,NO induced by LPS,In this present study,the anti-inflammatory mechanism of the four antimicrobial peptides on human monocyte macrophage U937 was studied.The results showed that the four kinds of antimicrobial peptides inhibited the release of chemokine MCP-1 and MIP-1 induced by LPS by reducing the expression level of MCP-1,MIP-1 and IL-8 gene.L-K6 exhibited the best inhibitory effect,followed by L-K5,and the inhibitory effect of L-K6V1 was weak.The results of Western Blot showed that the phosphorylation level of signal pathway protein My D88,I kappa B,NF-kappa B,ERK,p38 and JNK were inhibited by the four kinds of antimicrobial peptides at different level.Among them,the inhibitory effect of L-K6 on the phosphorylation level of My D88,I kappa B ERK,p38 and JNK was the most significant,L-K5 on the phosphorylation of NF-kappa B was the best,and L-K6V2 was relatively weak.It was demonstrated that the four antimicrobial peptides was actively taken up into U937 cells within 30 min and eventually localized to the perinuclear region by confocal laser scanning microscopy(CLSM).But cell membrane potential had not be changed,indicating that the four antimicrobial peptides did not affect the permeability of U937 cell membrane.When U937 cells were treated with the four antimicrobial peptides at 4 ℃,the uptake ofpeptides into significantly reduced compared to the uptake at 37 ℃,suggested that the antimicrobial peptides were uptake into the cells in an energy-dependent manner.After the pretreatment of U937 cells with LPS for 1 hour,only 80% of uptake of the peptides into the cells,compared to no pretreatment with LPS.Meanwhile,After the pretreatment of U937 cells with TAK-242 for 40 min,found the uptake of the peptides into U937 cells by TLR4.the production of TNF-α,IL-6 and chemokine MCP-1 and MIP-1 were in U937 cells were about70% of the normal treatment group,which indicated that peptides play an important anti-inflammatory effect into the cells.In addition,the pretreatment of U937 cells with peptides prior to the addition of LPS,the production of TNF-α,IL-6 and chemokines MCP-1and MIP-1 in U937 cells were more 75% than the normal treatment group,indicating that antimicrobial peptides play an anti-inflammatory activity by both intracellular target and neutralization of LPS.