| 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. |
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