The Stability And Anti-endotoxin Effect Of Site-specific PEGylation In Mutant Of Endotoxin Binding Peptide(mEBP9)

Lipopolysaccharide (LPS),one of the major clinical causes resulting in systemicinflammatory response syndrome (SIRS) or sepsis, is the primary component of the outermembrane of gram-negative bacilli. There are still high morbidity and mortality caused bySIRS and sepsis currently.Prevention and cure of SIRS and sepsis are mainly dependent onantibiotics to control infection at present, but the curative effect for Endotoxemia is farfrom satisfactory.Lipopolysaccharide binding protein (LBP) amplifies the toxicity of LPS. The aminoterminus of LBP contains special LPS binding sites; residues91-108of human LBP(Endotoxin binding peptide, EBP) has been proved to be one with obvious anti-LPSeffects.In the previous studies of our group,in order to enhance the anti-LPS effects,mEBP9having better anti-LPS effects was selected out from11mutant of EBP (mEBP) bystructure reconstruction of EBP.As the biological half-life of common polypeptide wasshorter in vivo,and polyethylene glycol-modified polypeptide may have higher stability andbetter protective effects than unmodified polypeptide in vivo;a cysteine(mEBP9-SH) wasintroduced to the amino terminal or carboxy terminal of mEBP9,site-specific PEGylatedmEBP9were obtained by adding the thiol radical(mEBP9-SH) to the double bond ofmethoxy polyethylene glycol-maleimide.mEBP9,PEG-NH-mEBP9and PEG-COOH-mEBP9were obtained by means ofbiosynthesis.By observing the anti-LPS bioactivity of PEG-NH-mEBP9andPEG-COOH-mEBP9, PEG-NH-mEBP9with stronger anti-LPS effects was filtered out,onthe basis of which a comparative study of anti-LPS bioactivity in vitro and in vivobetween PEG-NH-mEBP9and mEBP9were carried out.The main findings are as follows:1. CCK-8assay shows that1,5,10,20μmol/L mEBP9, PEG-NH-mEBP9andPEG-COOH-mEBP9had no obvious toxicity on RAW264.7cells. 2. The inhibitional effect of PEG-NH-mEBP9on LPS-induced TAL activation wasdistinctly stronger than that with the same concentration of PEG-COOH-mEBP9(P＜0.01);the inhibitional effect of PEG-NH-mEBP9on LPS-induced RAW264.7cells’secretion of TNF-α and IL-6was better than that with the same concentration ofPEG-COOH-mEBP9.3. The inhibited LPS-induced TAL activation capacity of10μmol/L PEG-NH-mEBP9/mEBP9at the temperature of-20℃for30consecutive days was greater than that at37℃for the same30days; The inhibited LPS-induced TAL activation capacity of10μmol/LmEBP9at the temperature of-20℃for30consecutive days was greater than that ofPEG-NH-mEBP9at the same concentration.The inhibited LPS-induced TAL activationcapacity of10μmol/L PEG-NH-mEBP9at the temperature of37℃for30consecutivedays was substantially greater than that of mEBP9with the same concentration under thesame condition(P＜0.05).After stimulating RAW264.7cells for3h or6h or12h, theinhibitional effect of5μmol/L mEBP9on LPS-induced RAW264.7cells’ secretion ofTNF-α at the same time points was stronger than the same concentration ofPEG-NH-mEBP9;after stimulating RAW264.7cells for24h or36h or48h, however,theinhibitional effect of5μmol/L PEG-NH-mEBP9on LPS-induced RAW264.7cells’secretion of TNF-α at the same time points was superior to the same concentration ofmEBP9.4. A comparative study of PEG-NH-mEBP9and mEBP9on anti-LPS bioactivity invitro.The inhibitional effect of mEBP9on LPS-induced TAL activation was higher than thesame concentration of PEG-NH-mEBP9.The inhibitional effect of mEBP9on LPS-inducedRAW264.7cells’ secretion of TNF-α and IL-6was remarkably supperior to the sameconcentration of PEG-NH-mEBP9(P＜0.05).5. A comparative study of PEG-NH-mEBP9and mEBP9on anti-LPS bioactivity invivo.(1)Increasing the survival rate of mouse model:the survival rate of “LPS/D-GalN”group,“mEBP9+LPS/D-GalN” group and “PEG-NH-mEBP9+LPS/D-GalN” group wererespectively0%,40%and60%.(2) Reducing plasma TNF-α and IL-6level:afterintraperitoneal injection of LPS/D-GalN for90min, PEG-NH-mEBP9and mEBP9obviously inhibited the secretion of the plasma TNF-α/IL-6(P<0.01),and the anti-LPSeffect of PEG-NH-mEBP9was significantly stronger than mEBP9(P<0.05).(3) Reducing serum ALT and AST level: after intraperitoneal injection of LPS/D-GalN for6h,PEG-NH-mEBP9and mEBP9remarkably inhibited the secretion of the serum ALT/AST(P<0.01), and the anti-LPS activity of PEG-NH-mEBP9was obviously stronger thanmEBP9(P<0.05).(4) Reducing pathological changes of mouse liver:in the “LPS/D-GalN”group, it showed severe pathological abnormalities including hepatocytes necrosis,congestion, destruction of hepatic architecture,massive aggregation of inflammatory cells inthe hepatic sinusoids;in the “LPS/D-GalN+mEBP9” group,the degree of liver tissuenecrosis was decreased,while the liver histopathological changes of “LPS/D-GalN+PEG-NH-mEBP9” group was lower than “LPS/D-GalN+mEBP9”group.The main conclusions are as follows: the anti-LPS bioactivity of PEG-NH-mEBP9wasobviously supperior to the same concentration of PEG-COOH-mEBP9.The anti-LPS effectof PEG-NH-mEBP9was little weaker than mEBP9while the stability of PEG-NH-mEBP9was better than mEBP9.PEG-NH-mEBP9and mEBP9both protected the D-galactosaminesensitized mouse model of endotoxemia,and PEG-NH-mEBP9had better protective effectsthan mEBP9.