氨溴索对粘液型铜绿假单胞菌生物膜藻酸盐作用的体外研究

The effect of ambroxol on alginate of Pseudomonas aeruginosa biofilms in vitro

目的探讨氨溴索对铜绿假单胞菌临床分离株形成的生物膜(biofilm,BF)主要成分藻酸盐的干预作用,研究其对藻酸盐合成过程中起重要作用的基因表达和合成过程中限速酶活性的影响,以及其对藻酸盐降解的影响。方法建立铜绿假单胞菌临床分离株BF体外模型,培养7d后得到成熟BF。将BF内的细菌振荡下来后,用硫酸-苯酚法检测氨溴索对藻酸盐含量的影响;RT-PCR检测藻酸盐合成过程中重要基因algD、algU、algR和mucA的mRNA表达;分光光度计检测合成过程中限速酶——GDP-甘露糖脱氢酶(guanosine diphospho-D-mannosedehydrogenase,GMD)的活性,并检测藻酸盐的降解情况。结果在氨溴索3.75mg/ml作用下,藻酸盐含量(mg/g)由86.4024±0.8588下降到59.9199±0.5803(F=66.2,P<0.01);其合成重要基因algD、algU、algR和mucA的mRNA的表达分别由1.2994±0.0173、1.0488±0.0457、0.9888±0.0267和0.8731±0.0336变化为1.0253±0.0265、0.9594±0.0106、0.8536±0.0179和1.0770±0.0503(F=91.9,41.1,88.4和56.9,P均<0.05);其合成限速酶GMD活性由0.0989±0.0055下降到0.0558±0.0016(F=121.2,P<0.01);藻酸盐的降解量(△mg/g)由1.4122±0.0073变化为1.4175±0.0019(F=21.81,P>0.05)。1.875mg/ml氨溴索作用下,有同样的趋势但效应不如高浓度明显。结论氨溴索可以降低铜绿假单胞菌BF藻酸盐的含量,影响藻酸盐合成过程中重要基因algD、algU、algR和mucA的mRNA的表达,降低藻酸盐合成限速酶GMD活性,但对藻酸盐的降解无影响。

Objectives To explore the effect of ambroxol on alginate which was the primary component of biofilm （BF） induced by clinical isolate of Pseudomonas aeruginosa. Further to investigate the effect of ambroxol on the expression of important genes and the activity of key enzyme involved in synthesis of alginate,and the effect of ambroxol on the degra-dation of alginate. Methods Plate culture method was used to establish biofilm model of Pseudomonas aeruginosa in vitro. Mature BF formed after 7-day culture. Bacteria embedded in BF were isolated through vibration and collected for investigation. The effect of ambroxol on the contents of alginate was detected through Sulphuric acid-Oxybenzene Method. The mRNA expression of important genes involved in synthesis of alginate such as mlgD,algU,algR and mucA were detected by using RT-PCR;Activity of guanosine diphospho-D-mannose dehydrogenase（GMD） ,the key enzyme involved in synthesis of algi- nate,was detected with spectrophotometer;Degradation of alginate were determined by the difference between the contents of alginate right after isolation and those having been placed for 1 hour. Results After the treatment by ambroxol with the concentration of 3.75 mg/ml,the content of alginate （mg/g） was decreased from 86. 4024±0. 8588 to 59. 9199 ±0. 5803 （ F = 66.2, P 〈 0.01 ） ; the mRNA expression of algD, algU,algR and mucA were changed from 1. 2994 ±0.0173,1. 0488 ± 0.0457,0. 9888 ± 0.0267 and 0. 8731 ± 0. 0336 to 1. 0253 ± 0. 0265,0. 9594 ±0. 0106,0.8536 ±0. 0179 and 1. 0770 ± 0. 0503 respectively（ F = 91.9,41.1,88.4 and 56.9, P 〈 0.05 ）. The activity of GMD decreased from 0.0989 ± 0. 0055 to 0. 0558 ± 0.0016 （ F = 121.2, P 〈 0.01 ）. The degradation of alginate （△ mg/g） changed from 1.4122 ± 0. 0073 to 1.4175 ± 0. 0019 （ F = 21.8,P 〉 0.05 ）. The effect of ambroxol with the concentration of 1. 875 mg/ml had the same tendency as that of 3.75 mg/ml,not so obviously though. Conclusion Ambroxol can decrease the contents of alginate,influence the mRNA expression of algD,algU ,algR and mucA,and decrease the activity of GMD. But it had no effect on degradation of alginate.