摘要
目的:研究中药四季青水煎液对铜绿假单胞菌生物被膜生成、毒力因子分泌的抑制作用并探讨其作用机制。方法:将四季青水煎液与铜绿假单胞菌共同培养,观察四季青水煎液在亚抑菌浓度下对细菌生长的影响;将铜绿假单胞菌培养3天构建生物被膜模型,加入不同浓度的四季青水煎液共同培养,于不同时间取样,经结晶紫染色后570 nm处测定生物被膜的光密度(OD值);将不同浓度四季青水煎液与铜绿假单胞菌共同培养18 h,经氯仿提取后于540 nm处测定绿脓菌素光密度(OD值),与刚果红反应后,492 nm处测定弹性蛋白酶光密度(OD值);用荧光定量PCR法检测四季青水煎液作用于铜绿假单胞菌后Las I/R和Rhl I/R基因的表达;用LC-MS/MS法测定不同浓度四季青水煎液与铜绿假单胞菌共同培养48h,72 h后培养液中细菌信号分子3-oxo-C12-HSL和C4-HSL的浓度及与生物被膜生长及毒力因子分泌的关系。结果:在亚抑菌浓度下四季青水煎液不影响铜绿假单胞菌的生长,但能显著抑制铜绿假单胞菌生物被膜生成和毒力因子分泌。其减毒作用机制可能是四季青中含有铜绿假单胞菌群体感应系统抑制成分,抑制了该系统中上游信号分子3-oxo-C12-HSL与LasR基因的结合,从而阻断了下游基因的启动。结论:四季青水煎液能通过影响铜绿假单胞菌群体感应系统中信号分子对相关基因进行调控,从而降低铜绿假单胞菌的致病性。
Objective:To investigate the inhibitory effects and its mechanisms of Chinese herbal medicine Ilex chinensis Sims folium water decoction on the biofilm formation and virulence secretion of Pseudomonas aeruginosa.Methods:After co-cultured of Ilex chinensis Sims folium water decoction and pseudomonas aeruginosa,the bacterial growth was observed under the subinhibitory concentration.Pseudomonas aeruginosa was cultured for 3 days to establish a biofilm model,and then co-cultured with different concentrations of Ilex chinensis Sims folium water decoction.Samples are collected at different time points,OD value was measured at 570 nm after crystal violet staining.The medium treated with different concentrations of Ilex chinensis Sims folium water decoction and Pseudomonas aeruginosa was co-cultured for 18 hours.The concentration of pyocyanin,extracted with chloroform,was determined at 540 nm for OD value,the concentration of elastase was determined at 492 nm for OD value after reacted with ECR.The expressions of LasI/R and RhlI/R genes in pseudomonas aeruginosa treated with Ilex chinensis Sims folium water decoction were determined by fluorescence quantitative PCR.LC-MS/MS method was used to detect the concentrations of 3-oxo-C12-HSL and C4-HSL in the culture medium after treated with different concentrations of Ilex chinensis Sims folium water decoction and Pseudomonas aeruginosa for 48 h and 72 h,respectively,also the relationship between biofilm and virulence factors was analyzed.Results:The results showed that Ilex chinensis Sims folium water decoction had no effect on the growth of Pseudomonas aeruginosa,but significantly inhibited the biofilm formation and virulence secretion of Pseudomonas aeruginosa.The inhibitory effects of Ilex chinensis Sims folium water decoction might be related to the components in Ilex chinensis Sims folium water decoction which suppressed quorum sensing system of pseudomonas aeruginosa by inhibiting the binding of 3-oxo-C12-HSL and LasR in the upstream signal molecule of this system,resu
作者
程强
曾铭
鲁兰
羿国娟
贺英
周杨杨
Cheng Qiang;Zeng Ming;Lu Lan;Yi Guojuan;He Ying;Zhou Yangyang(Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province,Sichuan Industrial Institute of Antibiotic,Chengdu University,Chengdu 610052;Huaxi Institute of Biopharmaceutical,Chengdu 610052)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2019年第4期111-116,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
四川省科技计划项目(2016JY0077)
国家自然科学基金项目(81803812)
关键词
四季青水煎液
铜绿假单胞菌
生物被膜
毒力因子
群体感应系统
信号分子
Ilex chinensis Sims folium water decoction
Pseudomonas aeruginosa
biofilm
virulence
quorum sensing system
signal molecule
