蜂毒肽MP-1体外拮抗内毒素作用及其机制

Effect and mechanism of mastoparan-1 antagonizing lipopolysaccharide in vitro

目的观察蜂毒肽MP-1的体外抗内毒素（LPS）活性并探讨其作用机制。方法应用生物传感器检测MP-1对类脂A（lipid A）的亲合力，采用动态比浊法鲎试验检测MP-1对LPS（2μg／L）的中和作用，激光扫描共聚焦显微镜观察不同浓度MP-1（5，10，20，40μmol／L）干预后异硫氰酸荧光素标记LPS（FITC—LPS，100μg／L）与小鼠RAW264．7细胞的结合，免疫细胞化学法观察MP-1对LPS（100μg/L）诱导的RAW264．7细胞TLR4表达的影响；实时荧光定量RT—PCR和ELISA法检测不同浓度MP-1作用下LPS（100μg／L）刺激的RAW264．7细胞TLR4、TNF-α和IL-6基因及蛋白的表达；MTF法检测MP-1对RAW264．7细胞活力的影响。结果MP-1具有一定的结合lipid A及中和LPS的作用，在10μmol／L浓度可显著抑制FITC—LPS与RAW264．7细胞结合（P〈0．05），并对LPS刺激的小鼠RAW264．7细胞TLR4、TNF-α和IL-6的基因及蛋白表达有抑制作用（P〈0．05或〈0．01），该作用具有一定的剂量效应关系。MP-1体外实验浓度对细胞活力无影响（P〉0．05）。结论MP-1可能通过中和LPS作用，阻断LPS与RAW264.7细胞膜受体的结合，从而抑制LPS介导的细胞活化。

Objective To investigate the mechanism of mastoparan-1 （ MP-1 ） antagonizing lipopolysaeeharide （LPS） in vitro. Methods The affinity of MP-1 for lipid A was assayed by biosensor, and the neutralization of MP-1 on LPS （2 μg/L） was detected by kinetic turbidimetric limulus test. After exposing fluoresein isothiocyanate （FITC） labeled LPS （FITC-LPS） to MP-1 at different concentrations （5, 10, 20, 40 μmol/L） , the binding of FITC-LPS to routine RAW264.7 cells was analyzed by laser scanning confocal microscopy. The influence of MP-1 on TLR4 expression in RAW264.7 cells stimulated by LPS （100 μg/L） was detected by immunocytochemical staining. The expressions of TLR4, TNF-α and IL-6 at the gene and protein level were detected by RT-PCR and ELISA after exposing LPS （ 100 μg/ L） stimulated RAW264.7 cells to MP-1 at different concentrations. The effect of MP-1 on the viability of RAW264.7 cells was detected by MTT assay. Results MP-1 had high affinity to lipid A and could neutralize LPS. MP-1 at 10 μmol/L significantly inhibited not only binding of FITC-LPS to RAW264.7 （P 〈 0. 05 ）, but also protein and gene expressions of TLR4, TNF-α and IL-6 in LPS stimulated RAW264.7 cells in a dose-dependent manner （ P 〈 0. 05）. No toxic effect of MP-1 on the viability of RAW264.7 cells was found （P 〉 0. 05 ）. Conclusions MP-1 inhibits cell viability mediated by LPS, which may be related to its neutralization of LPS and inhibition of binding of LPS to RAW264.7 cell membrane receptors.