氨溴索对铜绿假单胞菌生物被膜密度感应系统毒力因子及相关基因的影响

Effect of ambroxol on virulence factors and expression of relative genes of Quorum-sensing system in pseudomonas aeruginosa biofilm

目的:探讨氨溴索对铜绿假单胞菌生物被膜(BF)密度感应(QS)系统所调控的毒力因子编码基因以及各自对应毒力因子表达的影响.方法:通过平板培养法培养铜绿假单胞菌建立成熟的BF体外模型.采用不同浓度氨溴索干预后,应用实时荧光定量PCR(rRT-PCR)检测其对QS系统各毒力因子编码基因表达的影响;采用ELISA法检测外毒素A和弹性蛋白酶水平;采用地衣酚—硫酸法检测鼠李糖脂水平;采用Folin—酚比色法检测碱性蛋白酶活性.结果:经氨溴索3.750g/L作用后基因toxA,rhlA,lasB,aprAmRNA相对于培养基对照组的表达量均显著减少,由1.000分别变化为(3.03±0.08),(2.70±0.09),(2.10±0.10),(2.20±0.10)(P<0.05).同时毒力因子外毒素A、弹性蛋白酶、鼠李糖脂、碱性蛋白酶的表达量也较培养基对照组明显减少,分别由(15220.16±988.89),(81.38±5.44),(289.73±6.72),(5011±108.78)减少至(3481.59±599.64),(29.76±3.84),(101.74±3.32),(1757±57.21)(P<0.05).经氨溴索1.875g/L干预后具有同样趋势,但效应不如高浓度明显(P<0.05).结论:氨溴索可以降低BFQS系统调控的毒力因子编码基因以及各自对应的毒力因子的表达.

AIM： To study the effect of ambroxol on the virulence factors of quorum-sensing （QS） system in pseudomonas aeruginosa biofilm（BF） and on the expression of the genes associated with virulence factors in an established biofilm model in vitro. METHOD： Mature pseudomonas aeruginosa BF was formed after 7 days＇ culture on plate. After the treatment of ambroxol at different concentrations, quantitative real-time RT-PCR was used to analyze the expression of virulence factors coding genes including toxA, rhlA, lasB and aprA, and ELISA was used to quantify the exotoxin A and elastase. Orcin-sulphuric acid method was used to quantify rhamnolipid and Folin-phenol colorimerry was utilized to quantify the activity of alkaline protease. RESULTS： After the treatment of ambroxol at the concentration of 3. 750 g/L, the mRNA expressions of toxA, rhlA, lasB and aprA were down-regulated（ P 〈 0.05 ） and the contents of exotoxin A, elastase, rhamnolipid and alkaline protease decreased respectively（P 〈 0. 05）. Ambroxol at the concentration of 1. 875 g/L displayed the same tendency, though less obvious （ P 〈 0.05 ）. CONCLUSION： Ambroxol down-regulates the virulence factors coding genes and the virulence factors of Quorum-sensing system in pseudomonas aeruginosa biofilm.