Gram-Negative Bacteria Sepsis Antagonism And Possible Mechanism Of A Synthetic Cyclic Peptide CLP-19 Derived From Limulus Anti-lipopolysaccharide Factor

Background and ObjectiveSepsis with resultant multiple organ dysfunction syndrome (MODS) continues to be a serious problem in clinic, which often contributes to death in intensive-care patients. Lipopolysaccharide (LPS), also called endotoxin, the principal component of the outer membrane of gram-negative bacteria, is considered to play an important role in the pathogenesis of gram-negative bacterial sepsis and septic shock. Molecules that possess the abilities to directly neutralize LPS,the primary stimulus for the proinflammatory cytokine cascade may be promising agents in the treatment of sepsis. Limulus anti-LPS factor (LALF), a small basic peptide found in hemocytes from Limulus polyphemus, showed a high affinity binding to LPS and accordingly block the bioactivity of LPS. Based on analysis of LALF structural features, a peptide composed of 19 amino acid residues was designed and synthesized, designated as CLP-19, and was subsequently treated by amidation and cyclization. From our preliminary results, we found CLP-19 exhibited LPS binding property and resultant significant LPS neutralization. In the present study, the activity of CLP-19 to antagonize sepsis and its possible mode of action were investigated.Methods:Part I: Sepsis antagonism of CLP-191. The protective effects of CLP-19 on D-galactosamine sensitized endotoxemia mice: The survival rates of endotoxemia mice (D-Gal 600mg/kg + LPS 50μg/kg) simultaneously received various dosages of CLP-19 (0.5, 1.0 and 2.0mg/kg) or administrated with CLP-19 (2.0mg/kg) at different time were investigated. The histopathological examination and the evaluation of serum TNF-αlevel of model mice were also carried out.2. The protective effects of CLP-19 on Escherichia coli peritoneal sepsis mice: The survival rates and the bacterial load in tissues of model mice were investigated which received the prophylactic administration of CLP-19 at different time. The serum TNF-αconcentrations in infected mice injected with CLP-19 20h prior to inoculation of E.coli were also determined.Part II: Possible anti-sepsis mechanism of CL-191. Competitive ELISA assay to investigate the potential of CLP-19 to block the binding of LBP to LPS: LPS was first coated in 96-well ELISA plate, and then the dose-dependent manner of CLP-19 (10~100μg/ml) to interfere the LPS-LBP binding was evaluated when CLP-19 was added together with LBP (100ng/ml). The time-dependent property of CLP-19 (20, 40 and 80μg/ml) to block the binding was also examined.2. Evaluation of LPS-induced TNF-αrelease from PBMC: The dose-dependent manner of CLP-19 (10~100μg/ml) to inhibit the TNF-αproduction was evaluated when CLP-19 was pre-incubated with LPS (100ng/ml). The time-dependent relationship of CLP-19 (100μg/ml) to reduce the TNF-αrelease from PBMC was also investigated.3. Measurement of IL-1βstimulated TNF-αsecretion from RAW264.7 macrophages: The TNF-αsecretion from cells was determined when various concentrations of CLP-19 (1, 10 and 100μg/ml) were added before, after or together with IL-1β(100ng/ml).4. Investigation of CLP-19 to influence LPS binding to cells: The visualization of CLP-19 and LPS binding to cells in serum free medium was performed using fluorescence confocal microscopy.5. Effects of CLP-19 on LPS induced NF-κB activation in RAW264.7 cells: The NF-κB activation of RAW264.7 cells incubated with CLP-19 (1, 10 and 100μM), followed by LPS (1μg/ml) stimulation, were determined quantitatively using commercial NF-κB p65 ELISA kits.Results:Part I: Sepsis antagonism of CLP-191. The survival rates of model mice administrated with 3 dosages of CLP-19 (0.5, 1.0 and 2.0mg/kg) were 20%, 50% and 90%, respectively. When mice received CLP-19 (2.0mg/kg) before, after and together with LPS (50μg/kg), the survival rates of animals were 20%, 60% and 90%, respectively. The injury of five important organs including lung, liver, heart, kidney and small intestine in mice treated with CLP-19 (2.0mg/kg) and LPS (50μg/kg) simultaneously were lightened substantially as well as markedly decreasing serum TNF-αlevel.2. CLP-19 (2.0mg/kg) exerted a moderate protective effect (survival rate 40%) on mice when administrated 20h prior to a mortiferous inoculation of E.coli (0.6×108 CFU/mouse) , accompanied with the marked depression of TNF-αrelease. During 5~20 h, there were conspicuous rise in mice survival rates and decrease of bacterial load in tissues as the prolongation of time interval between the infection and peptide administration.Part II: Possible anti-sepsis mechanism of CL-191. CLP-19 could significantly block the binding of LBP to LPS in a dose-response manner within 10 ~ 40μg/ml when added together with LBP (100ng/ml). Moreover, no difference was found whenever CLP-19 added simultaneously with LBP or pre-incubated with LPS for 30min before the LBP addition. The inhibitory effects of CLP-19 went weaker gradually as the prolongation of time interval between CLP-19 and LBP addition.2. It was shown that CLP-19 possessed the abilities to suppress the TNF-αproduction from LPS-stimulated PBMC in a dose-response relationship within 10 ~ 40μg/ml when added together with LPS (100ng/ml). The best inhibitory activity was displayed when CLP-19 was premixed with LPS before adding to cells in serum contained medium, but when CLP-19 was added after LPS, little inhibition was observed.3. The TNF-αsecretion from RAW264.7 cells stimulated by IL-1β(100ng/ml) was significantly repressed by CLP-19 (1, 10 and 100μg/ml) in a dose-response manner.4. CLP-19 could directly bind to mice macrophages and resultantly facilitate LPS binding to cells in serum free medium.5. CLP-19 exhibited a direct inductive effect on the activation of NF-κB in RAW264.7 cells. When cells were treated with LPS (1μg/ml) in the presence of CLP-19 at lower concentrations (1μM and 10μM), the significant activation of NF-κB was observed. Oppositely, CLP-19 at high concentration (100μM) showed an excellent inhibitory effect on NF-κB activation when peptide was pre-incubated with cells followed by the LPS stimulation.Conclusion:1. Peptide CLP-19 possessed significant anti-sepsis activities manifested as the protection on mice from lethal LPS or bacteria challenges.2. The possible mechanisms of CLP-19 on LPS-mediated inflammatory cascade responses were as follow:(1) The blocking effects of CLP-19 on the binding of LPS to LBP;(2) The"inactive carrier effect"of CLP-19 on the binding of LPS to target cells.(3) The two-ways regulation of CLP-19 on LPS-mediated NF-κB activation in mouse macrophages and its modulatory effects on host immune system.