The Effect And Mechanism Of Temporin-1CEb Analogues On LPS-induced Inflammatory Response

Lipopolysaccharide(LPS) is the major structural component of the cell wall of Gram-negative bacteria, which is also the important factor for causing inflammation. Excessive inflammation causes the organism suffering from sepsis and other diseases, and even lead to death of the organism. Antimicrobial peptides are proved to be an important component of the innate immune system of all organisms, they has a small molecular weight, stable physical and chemical properties, and no toxicity to mammals, especially they exhibit broad-spectrum bactericidal activity and are not easy to lead drug resistance and anti-inflammatory properties. Our previous study found that the four of temporin-1CEb analogues, L-K6, L-K5, L-K6V1, L-K6V2 had high bactericidal activity and poor hemolytic activity. These peptides with 5-6 net positive charges might bind into negatively charged LPS by electrostatic interaction, and neutralize LPS, which make them potency as the candidates of anti-inflammatory drugs. In this paper, the effects of these four antimicrobial peptides on LPS-mediated inflammatory response and their mechanism of action in human monocyte U937 cells were explore.The results of toxicity test on U937 cell showed that these antimicrobial peptides had nontoxic to U937 cells in low concentrations. While at high concentrations, L-K5, L-K6V1, L-K6V2 exhibited slightly toxic to cells and cell viability was about 85%, cell viability treated by L-K6 can be around 70%. After the invasion of LPS in vivo, immune cells can be activated, induces release of inflammatory cytokines such as TNF-α, IL-8, NO, etc. triggering inflammation. Our results show antimicrobial peptide L-K6, L-K5, L-K6V1, L-K6V2 can inhibit the release of LPS-induced inflammatory cytokines. Even at low concentrations 20μM, the four antimicrobial peptides can inhibit the release of TNF-α, IL-8, NO, in which L-K6 have the strongest inhibit ability.Circular dichroism(CD) and isothermal titration calorimetry(ITC) were used to examine the capacity of the four antimicrobial peptides bind to LPS. CD showed that four antimicrobial peptides exhibited a random coil structure in SP buffer solution, but in the presence of LPS, these peptides presented an α-helical structures. ITC showed that these antimicrobial peptides with net positive charged could bind to negatively charged LPS by electrostatic interaction, the binding abilities of L-K6 and L-K6V1 are stronger than that of L-K5 and L-K6V2. These peptides can depolymerize LPS aggregates, in which LPS with 6000 nm diameter size disappeared after L-K6 treatment. The depolymerization ability of L-K5 is relatively weaker. The results of FITC fluorescence experiments and cell-flow experiments proved that four antimicrobial peptides directly inhibit LPS to bind into CD14 which is a LPS receptor in cellular inflammatory signaling pathway. The inhibitory abilities of L-K6 and L-K6V1 are stronger than L-K6V2 and L-K5. The effect of antimicrobial peptides on the expression of MyD88, IκK, NFκB were also investigated by RT-qPCR. The results showed that these antimicrobial peptides, especially LK-6 inhibited the expression of MyD88, IκK and NFκB on different levels.