黄芩苷对铜绿假单胞菌生物被膜体内和体外作用及机制

Pseudomonas aeruginosa is one of the most frequently isolated bacteria in nosocomial infections. As an opportunistic human pathogen, it colonizes immunocompromised hosts and mechanically ventilated patients and causes of mortality in this population. The capacity of this bacterium to cause such diverse infections is due to the formation of biofilm and the production of a plethora of virulence factors. It has been shown that P. aeruginosa employs quorum sensing in the regulation of genes encoding biofilm and extracellular virulence factors. .In the previous study, our group had detected that scutellaria aqueous-extracts inhibited P. aeruginosa biofilm formation. Baicalin is one of the active component of scutellaria. In the preliminary experiment, we found biofilm formation has been inhibited by Baicalin. Our group had established P. aeruginosa biofilm model in vitro and chronic P. aeruginosa lung infection in rats. .In the previous study, .1..We tested the Inhibition of biofilm in Pseudomonas aeruginosa by Baicalin. .2..We investigate the synergetic effect of Baicalin against pa biofilm combined with diverse antibiotics. .3..To assess the inhibition production of virulence factors and expression of QS gene by Baicalin..4..To explore the role of Baicalin in the Chronic P. aeruginosa lung infection in rats and the interference with related immune molecule expression. ..Investigating the effect of Baicalin on P. aeruginosa biofilm in vitro and in vivo, we attempt to analysis the mechanism in the level of molecule and gene.

铜绿假单胞菌（Pseudomonas aeruginosa，P.a）是院内感染主要致病菌之一。通常定植到免疫力低下的患者和机械通气患者并致其死亡。其所致的多种感染和其形成生物被膜（biofim BF）及产生毒力因子有关，相关基因都受到群体感应系统(Quorum sensing, QS)的调节。我课题组前期研究证明，黄芩水煎液能抑制BF的形成。黄芩苷是黄芩的活性成分之一，预实验中发现黄芩苷有抗P.a 的BF作用。我们已成功制备P.a体外BF模型和大鼠BF慢性肺部感染模型。本项目拟探讨：.1.观察黄芩苷对BF的抑制形成和破坏作用，及与其它多种抗生素的体外协同杀菌作用；.2.检测黄芩苷对相关毒性因子和QS分子和相关基因表达的抑制作用， .3.观察黄芩苷对大鼠体内P.a生物被膜形成的影响，并分析相关免疫分子的变化和干预效果。从而在分子水平和基因水平上，探讨黄芩苷对P.a生物被膜感染的作用机制。

铜绿假单胞菌（Pseudomonas aeruginosa，P.a）是院内感染主要致病菌之一。通常定植到免疫力低下的患者和机械通气患者并致其死亡。其所致的多种感染和其形成生物被膜（biofim BF）及产生毒力因子有关，相关基因都受到群体感应系统(Quorum sensing, QS)的调节。我课题组成功制备P.a体外BF模型和大鼠BF慢性腹腔感染模型。测定了黄芩苷对P.a的生长曲线及MIC值，用荧光染色、激光共聚焦显微镜和扫描电镜观察受试菌株生物膜形成过程中的形态学变化。.应用上述多种形态学方法及菌落计数证实黄芩苷能抑制和破坏静止模型中形成P.a生物膜。黄芩苷联合与其它抗生素如美罗培南、头孢他啶、头孢哌酮舒巴坦等对生物被膜内细菌显示出协同杀菌作用，总体效果与阿奇霉素效果相当。检测黄芩苷对铜绿假单胞菌动力学的影响，结果显示黄芩苷课抑制P.a 的Swimming泳动、Swarming运动及Twitching运动与阿奇霉素抑制水平相当。.对P.a生物被膜主要成分藻酸盐及其相关基因algu，algd的测定结果显示黄芩苷可以有效抑制藻酸盐的形成，抑制藻酸盐相关基因algu，algd的表达。测定黄芩苷对P.a的毒力因子的抑制作用，结果提示黄芩苷可有效抑制LPS、绿脓菌素﹑外毒素A﹑鼠李糖脂、弹性蛋白酶和蛋白水解酶等毒力因子的活性，抑制效果与阿奇霉素组相当，与QS基因缺陷株比较无明显差异。 HPLC-MS测定铜绿假单胞菌信号分子：结果显示黄芩苷显著抑制P.a的AHI信号分子合成。.建立大鼠慢性P.a的BF腹腔感染模型，经SEM、LCSM观察结果显示黄芩苷腹腔注射BF明显减少，BF内细菌的活菌技术明显减少，和阿奇霉素作用效果相当。局部感染炎症组织病理明显减轻，血清炎症介质（IFN-γ、IL-1a及IL-4、IL-6、IL-8、IL-12、TNF-α、MIP-2等）均有不同程度的减轻。.以上结果显示，黄芩苷通过抑制QS系统可以很好的控制和缓解铜绿假单胞菌生物被膜所致的相关感染，具有良好的临床应用前景。.