维拉帕米对铜绿假单胞菌群体感应抑制作用的体外研究

In Vitro Inhibition of Verapamil Quorum Sensing against Pseudomonas aeruginosa

考察维拉帕米对铜绿假单胞菌(Pseudomonas aeruginosa,PA)群体感应(Quorum sensing,QS)的抑制作用。测定维拉帕米最小抑菌浓度(Minimal inhibit concentration,MIC);构建培养环境,考察维拉帕米对PA生长的影响,绘制生长曲线;毒力因子表达实验中,分别考察维拉帕米对PA弹性蛋白酶表达、蛋白水解酶表达和绿脓毒素表达的影响。结果表明,维拉帕米MIC_(50)为128μg/mL,MIC_(90)为512μg/mL,最低抑菌质量浓度范围为128~512μg/mL时,具有较好抑菌活性;生长曲线表明,维拉帕米质量浓度为16μg/mL时,开始抑制PA的生长,随着浓度的增加,抑制作用逐渐增大;当维拉帕米质量浓度为512、256、128、64、32、16μg/mL时,明显抑制弹性蛋白酶表达(P<0.01),质量浓度为8μg/mL时,对弹性蛋白酶有一定抑制作用(P<0.05),维拉帕米质量浓度为512、256、128、64、32μg/mL时,明显抑制蛋白水解酶的活性和绿脓毒素的表达(P<0.01),当质量浓度为16μg/mL时,对蛋白水解酶活性和绿脓毒素的表达有一定抑制作用(P<0.05);维拉帕米对QS的抑制有浓度依赖。维拉帕米对PA的QS有明显抑制作用,体外可明显抑制PA生长,可作为抗菌增效的潜在开发药物。

Inhibition of verapamil on quorum sensing(QS)against Pseudomonas aeruginosa(PA)was observed.The minimal inhibit concentration(MIC)of verapamil was determined to establish its culture environment,observed the effect of verapamil on PA growth,and drew the growth curve;The expressions of PA elastase,proteolytic enzymes,and pyocyanin were observed respectively during virulence factor experiment.The results showed that In vitro verapamil showed good antibacterial activity against PA when MIC_(50)of verapamil was within 128μg/mL and MIC_(90)512μg/mL;the MIC_(Range) was 128-512μg/mL,suggested that it had comparative good inhibition activity;and the growth curve suggested that when the verapamil was at 16μg/mL it started to present inhibition against PA growth,as the increment of the concentration,the inhibition was gradually increased;when the concentration of verapamil was at 512μg/mL,256μg/mL,128μg/mL,64μg/mL,32μg/mL,and 16μg/mL,the activity of elastase was significantly inhibited(P<0.01),and elastase had certain inhibition at 8μg/mL(P<0.05).The concentration of verapamil was at 512μg/mL,256μg/mL,128μg/mL,64μg/mL,and 32μg/mL significantly inhibited the expression of pyotoxin and proteolytic enzymes(P<0.01),and it also had certain inhibition at 16μg/mL(P<0.05).Inhibition of verapamil had concentration dependence against PA.Verapamil had significant effect on QS of PA growth in vitro,and can be developed as potential synergist medicine of antibacterial one.