黄连解毒汤干预铜绿假单胞菌密度感应系统调控毒力因子和生物膜的作用初探

Inhibitory Effects of Huanglian Jiedu Decoction on Pseudomonas Aeruginosa Quorum Sensing System Regulating Virulence Factors and Biofilm

目的:探索黄连解毒汤铜对绿假单胞菌生长与毒力因子的干预作用。方法:将铜绿假单胞菌分别与不同浓度梯度的黄连解毒汤、黄连、黄柏、黄芩、栀子共同培养,观察对细菌生长、毒力因子、生物膜的影响。倍比稀释法观察黄连解毒汤对细菌生长的影响。刚果红弹性蛋白降解试验检测弹性蛋白酶活性,氯仿盐酸法测定绿脓菌素分泌量,结晶紫染色法观察生物被膜起始量,荧光定量PCR检测细菌密度感应(QS)基因转录水平。结果:黄连解毒汤最低抑菌浓度(MIC)为12.5 mg·ml^-1,黄连、黄芩、黄柏均为25 mg·ml^-1,栀子为50 mg·ml^-1。黄连解毒汤和黄连抑制绿脓菌素最低浓度均为6.25 mg·ml^-1,黄芩与黄柏为12.5 mg·ml^-1,栀子为50 mg·ml^-1。黄连解毒汤抑制弹性蛋白酶最低浓度为6.25 mg·ml^-1,黄连与黄柏均为12.5 mg·ml^-1,黄芩和栀子均为25 mg·ml^-1。黄连解毒汤抑制生物被膜的最低浓度为6.25 mg·ml^-1,黄连和黄芩均为12.5 mg·ml^-1,黄柏和栀子均为25mg·ml^-1。在低于最小抑菌浓度梯度之下,6.25 mg·ml^-1黄连解毒汤和12.5 mg·ml^-1黄连抑制las B、las I、lasR、rhl I、rhlR基因转录,12.5 mg·ml^-1黄岑和25.0 mg·ml^-1栀子抑制las B和las I基因转录,12.5 mg·ml^-1黄柏抑制las B和rhl I基因转录。结论:黄连解毒汤4种组分通过协同作用对细菌生长和密度感应系统调控的毒力因子发挥抑制作用。

Objective:To investigate the inhibitory effects of Huanglian Jiedu decoction(HLJDD)on the growth and virulence factors of Pseudomonas aeruginosa(P.aeruginosa).Methods:P.aeruginosa PAO1 strain was co-cultured with HLJDD or aqueous extract of following 4 herbs:Rhizoma coptidis(RC),Radix scutellariae(RS),Cortex phellodendri(CP)or Fructus gardeniae jasminoidis(FGJ).The minimum inhibitory concentrations(MICs)of HLJDD and the 4 herbs against PAO1 strain were determined by doubledilution analysis.The elastase expression was detected by Congo red degradation test,the secretion of pyocyanin was measured by ultraviolet spectroscopy,the biofilm formation was determined by crystal-violet staining,and the transcription level of quorum sensing(QS)gene was detected by real-time PCR.Results:HLJDD displayed the lowest MIC at 12.5 mg·ml^-1 when compared with MICs of 25 mg·ml^-1 for RC,RS and CP,and 50 mg·ml^-1 for FGJ.For the pyocyanin inhibition,the lowest concentration was 6.25 mg·ml^-1 for HLJDD and RC,12.5 mg·ml^-1 for RS and CP,and 50 mg·ml^-1 for FGJ.For the elastase expression inhibition,the lowest concentration was 6.25 mg·ml^-1 for HLJDD,12.5 mg·ml^-1 for RC and CP,and 50 mg·ml^-1 for RS and FGJ.For the biofilm formation,the lowest inhibitory concentration was 6.25 mg·ml^-1 for HLJDD,12.5 mg·ml^-1 for RC and RS,and 25 mg·ml^-1 for CP and FGJ.At the sub-MICs,6.25 mg·ml^-1 HLJJD and 12.5 mg·ml^-1 RC reduced the transcription of las B,las I,lasR,rhl I and rhlR;12.5 mg·ml^-1 RC and 25.0 mg·ml^-1 FGJ reduced the transcription of las B and las I;12.5 mg·ml^-1 CP reduced the transcription of las B and rhl I.Conclusion:HLJJD displays the synergism of its four constituents against the growth of P aeruginosa and the QS-dependent virulence factors.