| Introduction Sepsis is a condition caused by a harmful or damaging host response to infection, which may result in septic shock, multiple organ failure(MOF) and ultimately death. A recent epidemiological study from North America found that the incidence was approximately 3.0 cases per 1,000 population, which translated into an annual burden of approximately 750,000 cases. The overall mortality is approximately 30%, rising to 40% in the elderly and is 50% or greater in patients with the most severe syndrome, septic shock. The most common cause of sepsis is an exposure to the structural component of the Gram-negative bacterial outer membrane, lipopolysaccharide(LPS; known also as endotoxin). LPS is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD 14 and toll-like receptor 4 (TLR4). Mastoparan (MP), a cationic peptide isolated from wasp venom, has been shown to have comprehensive biological activities. The biological activities of mastoparans include stimulation of GTP-binding protein (G protein), phospholipase A2, phospholipase C, and membrane-perturbing activity, etc. However, the role of mastoparans in sepsis was rarely reported in the literature. As the LPS molecule was characterized by negative charge, the mechanism of its biological activities involves specific binding of lipid A disaccharide headgroup to cationic residues of a receptor model, and several disparate agents that antagonize the effects of LPS exhibit extensive physicochemical similarities (hydrophobicity, cationic charge) within their binding domains such as polymyxin B (PMB), bactericidal permeability-increasing protein (BPI) and Limulus anti-LPS factor (LALF). Thus, we presumed that there might be a functional association between mastoparans and LPS. Based on this hypothesis, we synthesized seven kinds of mastoparans and screened one from them, mastoparan-l(MP-l), which displayed high-affinity binding to LPS and could protect mice from LPS challenge. In this article, we further confirmed the protection of MP-1 in sepsis model of mice and explored its possible mechanisms.Objective To investigate the effects of MP-1 on bacteria and LPS in vivo and in vitro.Methods 1. The antimicrobial effects of MP-1 about minimal inhibitory concentration(MIC) and minimal bactericidal concentration(MBC) of 18 tested strains were measured by a microdilution method; the morphologic changes of E. coli ATCC25922 were observed after incubated with MP-l(100ng/ml) for 5, K) and 15 min by transmission electron microscope(TEM). 2. The affinity of MP-1 for LPS and Lipid A was assayed by biosensor, and the activity of MP-1 neutralizing LPS was detected by kinetic turbidimetric limulus test; the expression of tumor necrosis factor alpha(TNF- a ), interleukin-6(IL-6 )and TLR4 mRNA in murine peritoneal macrophages (PMΦ) after exposure to LPS(100ng/ml) were measured by RT-PCR. 3. The influence of MP-1(0, 5, 10> 20 > 40umol/L) on respiratory burst in PM Φ simulated with LPS(400ng/ml) was investigated. The superoxide anion and the activity of NADPH oxidase in PMΦ were assayed with an ultraviolet spectrophotometer and the hydrogen peroxide released from PMΦ was detected with fluorescence spectrophotometer. 4. The effect of MP-l(3mg/kg) protecting mice from lethal E.coli ATCC25922(1 X 10nCFU /kg) and LPS(20mg/kg) challenge were observed.Results 1. MP-1 revealed antibacterial effect on a middle level against the Gram-positive and Gram-negative bacteria compared with other tested antibiotics. TEM images showed the rough membrane surface was presented after E.coli ATCC25922 was incubated with MP-1 for 5 min, asymmetry and vacuoles in the cytoplasm of bacteria for l0min and severe degeneration for 15 min. 2. MP-1 binded with high-affinity to LPS and Lipid A with dissociation equilibrium constant (Kd) of 484 nM and 456 nM, respectively; the limulus test showed that MP-1 could neutralize LPS in a dose-dependent manner; moreover, in LPS-stimulated murine PM3...
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