Pharmacodynamic Evaluation Of Thymol Against Staphylococcus Aureus Pneumonia

Staphylococcus aureus?S.aureus?,gram-positive bacteria,which is a significant zoonotic pathogen that causes a variety of diseases in human and animals.It has been discovered that surface proteins,exocrine proteins and enzymes play an important role in S.aureus infections.Alpha-hemolysin?Hla?is an important exocrine toxin.Many researchs have demonstrated that Hla plays a vital role in S.aureus pneumonia,mastitis and keratitis in animal diseases models.Hla is considered a potential target to treat staphylococcal infections for drug discovery.Thymol exhibited a significant potency in inhibiting the hemolysis of rabbit red blood cells in S.aureus culture supernatant,when co-culture with S.aureus.In this thesis,there were four experiments to study the pharmacodynamics evaluation of thymol against S.aureus pneumonia in mice model.Firstly,the effects of thymol on the expression of S.aureus Hla and the protective effect of thymol on A549 cell injury induced by S.aureus Hla in lung cancer epithelial cells were studied in vitro and cells model.And then,the acute toxicity of thymol to mice was study before the protective effect of thymol on S.aureus pneumonia model in mice.The following studies were carried out in this thesis:1)The effects of thymol on the expression of S.aureus Hla in vitro were studied.In this experiemnt,we determined the minimal inhibitory concentration?MIC?of thymol against S.aureus and the growth curve of S.aureus cultured with different levels thymol.The hemolytic activity and the Hla content in S.aureus culture supernatant were detected by rabbit red cells and western blotting,respectively.The genes transcription information were performed by fluorescent quantitative reverse transcriptase PCR?RT-PCR?.2)To study the protective effect of thymol on cell injury induced by S.aureus Hla,the live/dead cells and LDH relaease were detected in the A549 cells co-culture with S.aureus.3)The acute toxicity of thymol in the mice were studied by intraperitoneally injected,and the LD₅₀ was obtained by the Bliss method.4)To study the protective effect of thymol on the damage of S.aureus pneumonia in mice,the protective trial and therapeutic trial were performed.Results:The MICs range of thymol to different strains of S.aureus was 128-256?g/mL.Thymol had no effect on the growth of S.aureus USA300 at the concentration of 0-32?g/mL.Thymol inhibited the hemolytic activities of bacteria supernatant in concentration-dependent manner.At the concentration of 32?g/mL,the hemolytic activity of USA300 culture supernatant was 34.8%compared with negative control group.We also foun the same appearances in the protein immunoblotting experiment.The Hla bands were significantly decreased when bacteria treated with high levels thymol.The transcriptional levels of hla and RNAIII also decreased in the USA300co-cultured with high concentration thymol.At the level of 32?g/mL,the transcriptional level of hla and RNAIII decreased to 5.5%and 10.5%,respectively,compared with the negative control group.In the co-culture system of S.aureus and A549 cells,the number of death cells significantly reduced in the high concentration of thymol.At the concentration of 32?g/mL,the release amount of LDH was significantly decreased?4.8%,p<0.01?.The mice were showed depressed,convulsions,and even death after intttraoeritoneal injection of thymol.Its clinical symptoms become more severe when the dose of thymol were increased.The intraperitoneal administration of thymol in mice was 608.246 mg/kg the median lethal dose(LD₅₀),and the 95%confidence interval was?500.149⁸07.214?mg/kg.Thymol can significantly reduce the mortality of mice when compared with the positive control group?only 8325-4 groups??p<0.05?.Pulmonary pathological changes in the drug delivery group can be found to be significantly reduced in ophthalmic lung and pathological section compared with the positive control group.Compared with the positive control group,the W/D value of the drug group was significantly lower than that in the positive control group?p<0.05?.The number of lung colonies in the drug group was significantly lower than that in the positive control group?p<0.01?,and the colony count was 1.1×10⁵ CFUs/mg and 6.8×10⁷CFUs/mg,respectively.Conclusion:Thymol has some effects against S.aureus in vitro.Thymol has no effect on the growth of S.aureus USA300 at a concentration of 0-32?g/mL.Thymol decreased the expression of hla by down regulating the transcriptional level of hla and RNAIII.The phenomenon of hemolysis decrease and concentration dependent was observed,negative correlated.Thymol has protective effect on A549 cell injury mediated by S.aureus Hla.Thymol is mildly toxic to intraperitoneal administration of mice.Thymol can reduce the mortality of S.aureus pneumonia in mice,reduce the lung pathological changes and W/D value,and reduce the colonization of S.aureus in lung tissue.