苦柯胺B拮抗细菌内毒素和CpG DNA的实验研究

Antagonistic effect of kukoamine B on bacterial endotoxin and CpG DNA

目的观察中药活性成分苦柯胺B(kukoamine B,KB)体内外拮抗脓毒症的药理活性。方法采用双偏振极化干涉测量技术测定KB与大肠埃希氏菌LPS、CpG ODN 1826的亲和力,并用鲎试剂法检测KB对大肠埃希氏菌LPS的体外中和作用,采用ELISA法体外检测KB对LPS和CpG DNA单独刺激RAW 264.7细胞释放炎症介质TNF-α和IL-6的抑制作用,ELISA法检测热灭活大肠埃希菌和热灭活金黄色葡萄球菌刺激RAW 264.7细胞释放炎症介质TNF-α和IL-6的抑制作用。在腹腔注射热灭活细菌菌液小鼠脓毒症模型中,将BALB/c小鼠分为对照组、KB(低、中、高剂量)组、和临床药物组(血必净和乌司他丁),观察比较7 d内各组动物死亡率。结果 KB与LPS和CpG ODN1826的KD值分别为7.38 nmol/L和197.2 nmol/L,在体外KB能够明显抑制LPS对鲎试剂的促凝作用(P<0.05,P<0.01),对热灭活细菌刺激RAW 264.7细胞释放TNF-α和IL-6具有显著的抑制作用,并呈现明确的量效关系(P<0.05,P<0.01)。在体内实验中,KB对重度和极重度脓毒症模型均具有明确的保护作用,KB组小鼠生存率显著高于对照组和临床药物组(P<0.05,P<0.01)。结论 KB作为一种多病原分子的中和剂,能够抑制炎症介质的释放,发挥对脓毒症模型小鼠的保护作用,且效果显著优于现有临床用药血必净和乌司他丁。

Objective To observe the in vitro and in vivo pharmacological antagonistic activity of kukoamine B（KB） on sepsis.Methods Affinity of KB to lipopolysaccharide（LPS） and CpG DNA in E.coli was detected by dual polarization interferometry,in vitro ability of KB to neutralize LPS in E.coli was assayed by limulus amebocyte lysate（LAL） test,and inhibitory effect of KB on release of TNF-α and IL-6 from RAW 264.7 cells induced by either LPS and CpG DNA alone or by heat-inactivated E.coli and Staphylococcus aureus was detected by ELISA.A mouse sepsis model was induced by injecting heat-inactivated E.coli and Staphylococcus aureus into abdominal cavity of BALB/c mice.The BALB/c mice divided into control group,low dose KB group,medium KB dose group,high KB dose group,and clinical drug treatment group with their mortality observed for 7 days and compared.Results The affinity of KB to LPS and CpG ODN1826 was 7.38 and 197.2 nmol/L,respectively.KB significantly inhibited the in vitro coagulation effect of KB on LPS（P〈0.05 or P〈0.01） and the release of TNF-α and IL-6 from RAW 264.7 cells induced by heat-inactivated E.coli and Staphylococcus aureus in a dose-dependent manner.KB played a significant role in protecting mice against severe and extremely severe sepsis.The survival rate of mice was significantly higher in KB group than in control group and clinical drug treatment group（P〈0.05 or P〈0.01）.Conclusion KB,as a neutralizer of several pathogen molecules,can inhibit the release of inflammatory cytokines and plays a role in protecting mice against sepsis.Its effect is significantly better than that of Xuebijing and ulinastatin.