一种合成的新型内毒素结合肽突变体的体内外拮抗内毒素效应初步研究

A new synthetic mutant of endotoxin binding peptide attenuates endotoxin-induced inflammatory responses in vitro and in vivo

目的设计构建新型内毒素结合肽突变体(mutant of endotoxin binding peptide,mEBP),并研究其体内外拮抗内毒素效应。方法采用F-moc固相合成法获得内毒素结合肽(endotoxin binding peptide,EBP)及其突变体mEBP;CCK-8法检测mEBP对RAW264.7细胞增殖的影响;ELISA法检测mEBP对内毒素(lipopolysaccharide,LPS)诱导的RAW264.7细胞分泌TNF-α和IL-6的影响;Western blot法检测mEBP对LPS诱导的RAW264.7细胞表达p-p38 MAPK的影响;中性红法测定mEBP对LPS诱导的RAW264.7细胞吞饮功能的影响。复制烧伤合并内毒素血症小鼠模型;检测mEBP对建模后6 h小鼠血浆中TNF-α、ALT和AST浓度的影响;检测mEBP对模型小鼠48 h生存率的影响。结果 mEBP无可见的细胞毒性;mEBP可降低LPS诱导的RAW264.7细胞分泌TNF-α和IL-6(P<0.05);抑制p-p38 MAPK信号通路的激活;抑制RAW264.7细胞的吞饮功能(P<0.01);mEBP预处理可明显降低烧伤合并内毒素血症模型小鼠血浆中的TNF-α、ALT和AST浓度(P<0.05),提高模型小鼠48h生存率。结论 mEBP和EBP均具有明显的拮抗内毒素活性,其中mEBP拮抗活性更强。

Residues 91-108 of human lipopolysaccharide binding protein（Endotoxin binding peptide,EBP） have been proved with obvious anti-LPS effects.This study is designed to identify whether mutant of Endotoxin binding peptide（mEBP） possess stronger anti-LPS effects than EBP in vitro and in vivo.EBP and mEBP were synthesized by the solid phase peptide synthesis method on a peptide synthesizer by F-moc chemistry.The impact of mEBP on RAW264.7 cells proliferation was detected by cell counting Kit-8 assay,the effect of mEBP on the secretion of cytokines of LPS-induced RAW264.7 cells was detected by ELISA,the influence of mEBP on p-p38 MAPK expression of LPS-induced RAW264.7 cells was detected by Western blot analysis,and the influence of mEBP on pinocytosis of LPS-induced RAW264.7 cells was detected by neutral red pinocytosis test.Mouse model of burn combined with endotoxemia was established,and the plasma levels of ALT,AST,and TNF-α were determined 6 h after modeling,while survival rate was monitored within 48 h.Results showed that mEBP had no cytotoxicity;mEBP could inhibited significantly the secretion of cytokines by RAW264.7 cells,the activation of p-p38 MAPK signaling pathway,and the pinocytosis ability of RAW264.7 cells.Compared with the model group,the plasma concentrations of ALT and AST,and TNF-α were significantly reduced by mEBP,the survival rate of mouse model of burn combined with endotoxemia was improved by mEBP.These results provide evidence that mEBP possesses stronger anti-endotoxin bioactivity than EBP,suggested that mEBP may be useful in the treatment of LPS-associated inflammation.