Bactericidal And Antiendotoxic Properties Of Synthetic Cyclic Peptides XS10/REMP1/REMP2 Derived From Limulus Anti-lipopolysaccharide Factor

Lipopolysaccharide (LPS), also called endotoxin, is a major component of the outer membrane of gram-negative bacteria. It is considered to be an important mediator of many of the responses manifested during the development of gram-negative bacterial sepsis and septic shock, which often leads to resultant multiple organ dysfunction and death in intensive care patients. The promising strategies targeting sepsis are preventing LPS from binding to its receptors, blockage of LPS intracellular signal transduction, antagonism of cytokine and application of glucocorticoids,but unfortunately all these therapies got little clinical benefits. There is no effective and safe drugs available till now.The Limulus anti-lipopolysaccharide factor and Tachypleus anti-lipopolysaccharide factor(LALF,TALF)from both limulus polyphemus and Tachypleus tridentatus Leach have been proved striking anti-gram negative bacillus as well as anti-LPS effects in vitro and in vivo. Based on the analysis of LALF and TALF structural features as well as their functional domains by way of bioinformatics methods, we designed and synthetized a series of peptides by computational molecular modeling. In this study we will investigate the bactericidal and antiendotoxic properties of synthetic cyclic peptides XS10, REMP1 and REMP2.Section 1. Bactericidal and antiendotoxic activities of XS10/REMP1/REMP2 in vitro.Methods:1. The LAL(Limulus Amebocyte Lysate)test was to determine the ability of XS10/ REMP1/ REMP2 to neutralize LPS in vitro. Various concentrations of the peptides and Polymyxin B (PMB) were incubated with LPS respectively , The kinetic turbidity was measured using a endoxin detecte system. The LPS neutralizing rates were calculated.2. Mouse RAW264.7 macrophages were cultured and then 10μl LPS (100 ng/ml) and equal volume peptides and PMB solutions (10, 20, 40 and 80μM) were added simultaneously. After cells were incubated for 4 h, the levels of TNF-αin the supernatants were analyzed using the ELISA kits.3. The minimal inhibitory concentration (MIC) of XS10 \REMP1 \REMP2 for the Escherichia coli, Pyocyaneum bacterium, Staphylococcus aureus, et al were determined by agar dilution.4. The morphological changes of E.coli ATCC 25922 affected by REMP1 and REMP2 were observed by transmission electron microscope.5. Cytotoxicity of XS10, REMP1 and REMP2 to the Mouse RAW264.7 macrophages was established using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.Results:1. XS10,REMP1,REMP2 and PMB exhibited neutralizing activities on LPS in a dose-dependent manner. The activities decrease progressively from REMP2, REMP1 to XS10. REMP2 was the best with the neutralizing rates from 5.66% to 88.66%, which were similar to PMB.2. Treatments of cells with XS10, REMP1, REMP2 and PMB both inhibited the TNF-αrelease in response to the presence of LPS in a dose-dependent manner. The TNF-αdecreased significantly compared with the control group (p<0.05).3. All the peptides have Bactericidal activities. the MIC were from 16μg/ml to 256μg/ml for the bacteria in test. The activities decrease progressively from REMP1, REMP2 to XS10. moreover REMP1 and REMP2 have bactericidal activities to Staphylococcus aureus which is belong to gram positive coccus.4. Under the electron microscope the outer and interior membranes of Escherichia coli. affected by REMP1 and REMP2 was obscure, the bacterium swelled,vacuoles formation and even ruptured.5. XS10, REMP1 and REMP was not observed to be toxic to mouse macrophages at the concentrations tested in the MTT test. Section 2. Antiendotoxic activities of XS10/REMP1/REMP2 in vivo.Methods:1. To generate the murine endotoxin shock model induced by D-galactosamine- sensitized KunMing mice, Kunming mice were stimulated with intraperitoneal(i.p.) injection of different dosage of D-galactosamine(D-Gal) and LPS one hour later. Mice lethality was recorded at 72 h. the dose of LD100 were chosed.2. Generate the murine endotoxin shock models. groups of 10 mice were injected with the peptides and PMB(0.5, 1.0 and 2.0 mg/kg) intraperitoneally, together with LPS. Survivals was recorded every 6 h during the period of 72 h.3. Generate the murine endotoxin shock models. groups of 10 mice received an i.p. injection of peptides and PMB (2.0 mg/kg) at indicated time points: (1) 1h before LPS; (2) 1h after LPS. Survivals was recorded every 6 h during the period of 72 h.4. Mice endotoxin shock models was made. Seventy-five minutes after intra-peritoneal injection of peptides, PMB(2.0 mg/kg) and LPS, blood samples were collected from the sacrificed mice. The levels of TNF-αin serum samples were analyzed using the ELISA kits.5. Mice endotoxin shock models was made. Mice were injected intraperitoneally with REMP1 and REMP2 (2.0 mg/kg) together with the LPS. Five hours later the mice were sacrificed and the lung, heart, liver, intestine and kidney removed for histopathology.Results:1. Treatment of mice with D-galactosamine increased remarkably their sensitivity to the lethal effects of LPS. The dosage of D-Gal 500mg/kg + LPS 250μg /kg or D-Gal 600mg/kg + LPS 50μg/kg could produc 100% mortality in 72 h.2. The survival rates of LPS- attacked mice increased significantly with peptides intervention at the three dosages (p < 0.05). REMP2 have show the highest rescue rates with 30%, 60% and 90% at the dosages of 0.5, 1.0 and 2.0mg/kg.3. The survival rates of LPS-challenged mice after 72 h were 30%, and 50% when receiving REMP2 injection 1h before or 1h after LPS respectivel. The protection effects were less than that of injection together with LPS.4. the TNF-αconcentrations of mice challenged by LPS alone increased fiercely to 6365±2087 pg/ml, but decreased significantly after treatment with the peptides. The inhibitory efficacy of REMP2(905±264 pg /m l) was nearly at the equal grade with PMB(p > 0.05).5. LPS-attacked mice produced acute inflammation in the lung, heart, liver, intestine and kidney in mice of control group. In contrast, with REMP1 and REMP2 treatment, there was significantly abatement of inflammation.Conclusions:1. The synthetic cyclic peptides XS10/REMP1/REMP2 based on LALF can directly neutralize the LPS in solutions.2. XS10, REMP1 and REMP2 can inhibit the TNF-αrelease in response to the presence of LPS in Mouse RAW264.7 macrophages.3. XS10, REMP1 and REMP2 have bactericidal activities to some of the bacteria obtaine from clinical patients including the Staphylococcus aureus which is belong to gram positive coccus.4. XS10, REMP1 and REMP2 have not Cytotoxicity.5. XS10, REMP1 and REMP2 can increase survival rates of mice challenged by lethal LPS.6. XS10, REMP1 and REMP2 can decrease the TNF-αlevels in endotoxin shock mice.7. REMP1 and REMP2 can decrease inflammation in the lung, heart, liver, intestine and kidney in mice of endotoxin shock.8. The bactericidal and antiendotoxic activities of XS10, REMP1 and REMP2 in vivo are in line with that in vitro. The antiendotoxic activities degree are REMP2 > REMP1 > XS10 and the bactericidal activities degree are REMP1 > REMP2 > XS10。9. REMP2 have the antiendotoxic activities nearly at the equal grade with PMB, but have less bactericidal activities than that of PMB.