CAJ | 학술논문

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(5,10,20,40 μmol/L)干预后异硫氰酸荧光素标记LPS(FITC-LPS,100 μg/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;MTT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10 μmol/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.
목적 관찰봉독태MP-1적체외항내독소(LPS)활성병탐토기작용궤제. 방법 응용생물전감기검측MP-1대류지A(lipid A)적친합력,채용동태비탁법후시험검측MP-1대LPS(2μg/L)적중화작용,격광소묘공취초현미경관찰불동농도MP-1(5,10,20,40 μmol/L)간예후이류청산형광소표기LPS(FITC-LPS,100 μg/L)여소서RAW264.7세포적결합,면역세포화학법관찰MP-1대LPS(100μg/L)유도적RAW264.7세포TLR4표체적영향;실시형광정량RT-PCR화ELISA법검측불동농도MP-1작용하LPS(100μg/L)자격적RAW264.7세포TLR4、TNF-α화IL-6기인급단백적표체;MTT법검측MP-1대RAW264.7세포활력적영향. 결과 MP-1구유일정적결합lipid A급중화LPS적작용,재10 μmol/L농도가현저억제FITC-LPS여RAW264.7세포결합(P<0.05),병대LPS자격적소서RAW264.7세포TLR4、TNF-α화IL-6적기인급단백표체유억제작용(P<0.05혹<0.01),해작용구유일정적제량효응관계.MP-1체외실험농도대세포활력무영향(P>0.05). 결론 MP-1가능통과중화LPS작용,조단LPS여RAW264.7세포막수체적결합,종이억제LPS개도적세포활화.
Objective To investigate the mechanism of mastoparan-1 (MP-1) antagonizing lipopolysaecharide (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 fluorescin isothiecyanate (FITC) labeled LPS (FITC-LPS) to MP-1 at different concentrations (5, 10, 20, 40 μmol/L), the binding of FITC-LPS to murine 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 immunoeytochemieal 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.