穿心莲内酯的协同抗菌作用及相关机制研究

Study on the synergistic antibacterial effect of andrographolide and the related mechanisms

目的:研究穿心莲内酯对左氧氟沙星抗铜绿假单胞菌的协同抗菌作用及相关机制研究。方法:用铜绿假单胞菌PAO1复制小鼠腹腔菌感染模型,考察穿心莲内酯对左氧氟沙星的协同抗菌作用;利用实时荧光定量PCR(Real-Time PCR)技术考察穿心莲内酯在100μg/ml、1μg/ml、0.01μg/ml剂量下对铜绿假单胞菌PAO1群体感应系统(QS)系统中lasR、rhlR和pvd Q基因mRNA在24h内表达量的影响,分析药物协同抗菌作用的机制;考察穿心莲内酯在100μg/ml、1μg/ml、0.01μg/ml剂量下对PAO1胞内总蛋白和己糖激酶(HK)活性的影响作用。结果:体内抗感染保护性实验显示,左氧氟沙星和穿心莲内酯配比中剂量组(1∶0.1587)和低剂量组(1∶0.0373),感染动物24h^72h死亡率明显降低,作用明显优于单用左氧氟沙星,提示其保护性作用明显加强;Real-Time PCR实验结果显示,穿心莲内酯不同剂量对PAO1 lasR、rhlR和pvd Q基因mRNA的表达均呈现相似的先抑制后加强再抑制的动态调控趋势;在各剂量穿心莲内酯影响下,PAO1胞内总蛋白量未见明显变化,但己糖激酶活力降低明显,各剂量组与空白组比较均存在明显差异。结论:穿心莲内酯对左氧氟沙星呈现体内的协同抗铜绿假单胞菌作用,而且对PAO1 QS系统相关基因存在规律性调控,同时这一调节作用可能与降低细菌胞内己糖激酶活性存在关联。

Objective： To investigate the synergistic antibacterial effect of andrographolide on levofloxacin against Pseudomonas aeruginosa and the related mechanism research. Methods： Pseudomonas aeruginosa PAO1 was used to copy mice peritoneal infection type to investigate synergistic antibacterial effect of andrographolide on levofloxacin. Real-time fluorescence quantitative PCR（ Real-Time PCR） method was used to investigate the impact of Andrographolide at 100μg/ml,1μg/ml,0. 01μg/ml（ high,medium,low） doses on the expression of mRNA of PAO1 lasR,rhlR and pvd Q gene within 24 h against Pseudomonas aeruginosa PAO1 QS system. The mechanism of drug synergistic antibacterial activity was analyzed. The effect of andrographolide at 100μg/ml,1μg/ml,0. 01μg/ml（ high,medium and low） on the activity of total protein and hexokinase（ HK） in PAO1 was investigated. Results： Protection experiments in vivo against infection showed that when Levofloxacin and andrographolide was the ratio of middle dose（ 1： 0. 1587） and low dose（ 1： 0. 0373）,the mortality of infected animals was significantly reduced in 24 h~ 72 h and there was a significant difference when compared with the model group（ P 0. 01 ~ 0. 05）,the role was significantly better than levofloxacin alone,which suggested that there was a strengthening trend of protective effect. The result of Real-Time PCR showed that different doses of andrographolide on expressions of mRNA of PAO1 lasR,rhlR and pvd Q gene all showed a similar regulation trend of inhibition,strengthening and re-inhibition. Under the influence of each dose of andrographolide,the total protein content of PAO1 did not significantly change,but the activity of hexokinase was obviously decreased,and there were significant differences between the two groups（ P 0. 01 ~ 0.05）. Conclusion： Andrographolide combined with levofloxacin presentes in vivo synergistic inhibition effect against Pseudomonas aeruginosa,and also exists a regularity regulation role on the PAO1 QS system-related genes. At the same time this regulation may be related to the reduction of intracellular hexokinase activity.