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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。
Objective To design and construct a novel endotoxin binding peptide mutant (mEBP) and study its antagonism of endotoxin effect in vitro and in vivo. Methods The endotoxin binding peptide (EBP) and its mutant mEBP were obtained by Fmoc solid phase synthesis. The effect of mEBP on the proliferation of RAW264.7 cells was detected by CCK-8 assay. The effect of mEBP on endotoxin The effect of mEBP on the expression of p-p38 MAPK induced by LPS in RAW264.7 cells was detected by Western blot. The effect of mEBP on the secretion of TNF-α and IL-6 by LPS Effect of induced phagocytosis of RAW264.7 cells. The effect of mEBP on the plasma concentration of TNF-α, ALT and AST in mice at 6 h after modeling was examined. The effect of mEBP on the 48 h survival rate of model mice was examined. Results mEBP had no obvious cytotoxicity. MEBP could reduce the secretion of TNF-α and IL-6 by LPS-induced RAW264.7 cells (P <0.05), inhibit the activation of p-MAPK signaling pathway, and inhibit the swallowing of RAW264.7 cells (P <0.01). Pretreatment with mEBP could significantly decrease the concentration of TNF-α, ALT and AST (P <0.05) in the plasma of mice with burn combined with endotoxemia and increase the 48-h survival rate of model mice. Conclusion Both mEBP and EBP have obvious antagonistic endotoxin activity, of which mEBP antagonist activity is stronger.