穿心莲内酯干预铜绿假单胞菌BF形成及QS系统相关基因表达的实验研究

【作者】 陈思敏；

【导师】 曾南； 彭成；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 博士

【摘要】 目的1.观察穿心莲内酯对铜绿假单胞菌生物被膜(BF)形成的干预作用,并分析药物对BF的干预作用与细菌群体感应(QS)系统的相关性,在分子水平探讨穿心莲内酯的作用机制。2.观察穿心莲内酯提高左氧氟沙星抗BF阳性铜绿假单胞菌活性的作用,探讨穿心莲内酯作为抗菌增效剂的可行性。方法1.以铜绿假单胞菌PA01野生株为研究对象,首先采用电镜技术(扫描电镜SEM和透射电镜TEM)观察穿心莲内酯在300、30、3μg·mL-1剂量下对PA01所生成的BF形态学的影响；其次采用96孔板定量测定法,定量检测穿心莲内酯在300、250、200、150、100、50、25、15、10、5μg-mL-11O个剂量下,对72h内PA01形成BF厚度的影响；最后构建体外BF渗透模型,观察穿心莲内酯在300、30、3μg·mL-1剂量下,在构建的BF模型上对左氧氟沙星渗透性的影响。以此综合分析和评价穿心莲内酯对铜绿假单胞菌BF的干预作用。2.采用实时定量荧光PCR (Real-Time PCR)技术观察穿心莲内酯在150、75、37.5μg·mL-1剂量下对铜绿假单胞菌QS系统中相关基因lasR和rhlR,以及调节基因pvdQ的mRNA表达水平的影响,分析药物对BF的干扰是否与QS系统相关。3.采用传统的体外药物敏感性实验,观察穿心莲内酯与左氧氟沙星不同配比的22个样品对160株临床近期分离BF阳性铜绿假单胞菌的MIC值,并采用概率单位分析法,对受试药物效价强度进行拟合,以残差平方和和相关系数为主要判别,进行线性、对数性、Emax和Sigmod Emax的对比,评价不同配比受试药物的抗菌活性；以PA01构建小鼠铜绿假单胞菌腹腔感染模型,观察体外抗菌活性较强的13个配比受试药物的体内抗感染作用。4.采用体内炭粒廓清实验,以吞噬指数为指标观察穿心莲内酯对正常小鼠网状内皮细胞吞噬功能的影响；采用巨噬细胞体外培养实验,观察穿心莲内酯体外对巨噬细胞吞噬作用的影响。结果1.SEM结果显示,空白对照组PA01附着于玻璃载体表面在72h可形成分化成熟、较厚、结构不均匀并密布有孔状通道的BF,TEM镜下可见PAO1聚集生长,菌体边缘清晰,周围可见呈团块,形态似“塔状”或“蘑菇状”的不规则BF结构,未见明显的培养基染色颗粒。穿心莲内酯3μg·mL-1剂量下,PAOl产生的BF未能形成成熟的结构,BF明显稀薄且平整,可见附着的PAOl菌体,TEM镜下PAOl有一定的聚集,可见少量“蘑菇状”BF结构,但较小且不明显；在穿心莲内酯30、300μg·mL-1剂量下,PA01分散附着,均未见明显的BF结构,TEM镜下PA01多数分散生长,少见聚集,菌体边缘不清晰,周围有大量颗粒状物质。2.穿心莲内酯在300、250、200、150、100、50、25、20、10、5μg·mL-110个剂量下,72h内均可影响PA01BF的生长。穿心莲内酯在20μg·mL-1以上剂量下,12、24、36、48、60、72h时BF厚度明显变薄。3.BF渗透性实验显示,穿心莲内酯在300、30、3μg.mL-1剂量下均可以提高铜绿假单胞菌生物被膜模型对左氧氟沙星的渗透性,在模型构建后的2、4、6、8、10、24h的药物渗透量显著升高。4.实时定量荧光PCR测定结果显示,穿心莲内酯在150μg.mL-1浓度下增强lasR基因表达,30、3μg.mL-1剂量下则降低其表达；穿心莲内酯在300、30和3μg·mL-1的剂量下均可降低rh1R和pvdQ基因的表达；5.体外药物敏感性实验结果显示,在左氧氟沙星比穿心莲内酯在1：0.001～1：0.01范围内,药物的Emax和ED50虽然有一定的波动,但总体趋势上表现出Emax高于单用左氧氟沙星和ED50低于单用左氧氟沙星,表明穿心莲内酯在一定剂量范围内有提高左氧氟沙星抗菌活性的作用。体内细菌感染保护性实验结果显示,所观察的13组配方对铜绿假单胞菌小鼠感染模型的体内保护性作用均优于左氧氟沙星单用。体内与体外实验结果表明穿心莲内酯可提高巨噬细胞的吞噬功能。结论穿心莲内酯可干扰PAO1生物被膜BF的产生和成熟,降低BF厚度,增强喹诺酮类药物左氧氟沙星的渗透性；抑制铜绿假单胞菌群体感应系统中lasR、rslR基因和pvdQ基因nRNA的表达,可能是其干预BF形成的作用机制；穿心莲内酯提高体外铜绿假单胞菌对左氧氟沙星的敏感性及增强巨噬细胞吞噬能力,提示穿心莲内酯可以发挥抗菌药物增效作用。更多还原

【Abstract】 Objective1. Investigate the intervention effect that andrographolide against Pseudomonas aeruginosa biofilm(BF), analyze the correlation of drug to BF forming and bacterial quorum sensing(QS) system, and discuss the mechanism of andrographolide on a molecular level.2. Investigate the function of andrographolide improving the activity of levofloxacin anti-positive BF Pseudomonas aeruginosa. Discuss andrographolide’s feasibility as antibacterial synergist.Methods1. Using PAO1wild strains of Pseudomonas aeruginosa as the research object, firstly, we adopt electron microscopy(scanning electron microscopy SEM and transmission electron microscopy TEM), observe the differences in comparative morphology between BF generated by PAO1and blank group BF in the dose of andrographolide300,30,3μg·mL-1; secondly, we adopt96-well-plate quantitative measurement, making quantitative detection of the influence that andrographolide has on BF’s thickness generated by PAO1within72h in the dose of300,250,200,150,100,50,25,15,10,5μg·mL-1, and compare it with the blank control group; finally, build the BF penetration model in vitro, and investigate the influence that BF model structured by andrographolide has on levofloxacin permeability in the dose of300,30,3μg·mL-1.On theaccount, comprehensively analyze and evaluate the interventional effect that andrographolide has on Pseudomonas aeruginosa BF.2. Adopting the technology of quantitative PCR(Real-time PCR), we investigate the influence that andrographolide has on the related genes lasR and rh1R in the aeruginosa QS system and adjusting the mRNA expression levels of regulatory gene pvdQ in the dose of150,75,37.5μg·mL-1by the relative quantification method, and analyze that whether the interference that drug has on BF is related to the QS system.3. Using traditional experiments about the drug sensitivity in vitro, we investigate the influence that levofloxacin matching22andrographolide of different proportions has on MIC of160BF-positive Pseudomonas aeruginosa with clinical separation. Using probit analysis, we fit the potency the drug to be tested. Using the residual sum of squares and correlation coefficients as the main discrinant, we compare linear, logarithmic, Emax with Sigmod Emax and evaluate the antibacterial activity of different test drug. At the same time, we build mice’s Pseudomonas aeruginosa abdominal infection model with PAO1, and verify the protective effect of13drugs to be tested with better antibacterial activity, making24h,48h,72h animal mortality as indicators.4. Using the experiments about carbon particle clearance in vivo, we investigate the influence that andrographolide has on the phagocytosis of normal mice reticuloendothelial cells with the phagocytic index as an indicator. Using the experiments of macrophages cultivated in vitro, we directly observe the influence that andrographolide has on macrophage phagocytosis.Results1. The results of SEM test show that the blank control group PAO1form mature, thicker and uneven structured BF with dense porous channel when it has been attached to the glass surface for72h. Under TEM microscopy, we can see that POA1is growing in colonies. Bacterial edges are sharp, and clumps formed like "tower" and "mushroom-shaped" irregular BF structure can be seen around. There’s no significant medium staining particles. In the andrograholide dose of3μg·mL-1, BF generated by PAO1fails to form a mature structure and is obviously thin and smooth compared with the blank control group. And we can see attached PAO1thallus gathering under TEM microscope and a small amount of smaller and unapparent "mushroom-shaped" BF structure. Under the high dose(30,300μg· mL-1), POAls are attached dispersedly in andrographolide. And they all show no obvious BF structure. Most POA1grow dispersedly under TEM microscope, gathering is rare. The edge of the bacteria is not clear, and there is a large number of particular matter surrounded.2. With96-well plates, we determine that andrograopholide can influence POA1BF’s growth within72h in the10doses. In the dose of20μg·mL-1, andrographolide’s BF thickness and blank control group becomes significantly thinner. There is a statistically significant difference(P<0.01); When andrographolide is under the dose of10μg· mL-1and5μg· mL-1, the late growth of BF has no statistically difference compared with blank control group.3. BF permeability experiments show that andrographolide can improve the permeability that the Pseudomonas aeruginosa biofilm model against levofloxacin on the dose of300,30,3μg·mL-1, and the drug penetration shows a significant difference compared with the blank control group respectively in2,4,6,8,10and24h after building the model.4. The results of using the Real-Time PCR show that andrographolide can enhance gene expression on the dose of150μg·mL-1’for the lasR gene, while can reduce expression on the dose of30,3μg· mL-1; For rh1R and pvdQ, andrographolide can both reduce gene expression on the dose of300,30and3μg·mL-1.5. In vitro drug sensitivity results showed that in levofloxacin than andrographolide in1:0.001-1:0.01range, drug Emax and ED50although there are certain fluctuations, but the overall trend showed a higher Emax and lower ED50of levofloxacin levofloxacin alone.The results of drug in vitro sensitivity experiments suggest andrographolide can improve the antibacterial activity of levofloxacin role in dose within a certain range.6. The results of bacterial infection in vivo protective experiment show that the influences which the examined13formulas have on the protective effect of Pseudomonas mice infection model in vivo are all bigger than using levofloxacin only. The results of protective experiment in vivo have no significant dose relation with those of drug in vitro drug sensitivity experiment.ConclusionAndrographolide can interfere with the generation and maturation of the POA1biofilm BF, reduce the thickness of BF and enhance the penetration against quinolones levofloxacin; andrographolide can inhibit the expression of lasR, rslR gene and pvdQ gene mRNA in the Psedomonas aeruginosa quorum sensing (QS) system, suggesting that androgapholide may play a role in inhibiting the formation of BF through the regulation bavterial QS system; andrographolide can significantly improve the sensitivity that Pseudomonas aerugionasa to levofloxacin,and has a trend on enhancing macrophage phagocytosis, suggesting that andrographode can play a synergistic effect of antimicrobial drugs.更多还原