Study On The Mechanism Of Celastrol Combined With Polymyxin B Against MRSA

Objectives: To study the synergistic antibacterial ability and mechanism of celastrol and polymyxin B,and analyze whether celastrol could be used as an effective auxiliary compound to enhance the antibacterial effect of polymyxin B,so as to achieve the attenuation and synergistic effect of polymyxin B,and found new drugs and treatment methods to address the growing serious problem of bacterial drug resistance.Methods: 1.Evaluate the antibacterial activity of celastrol by various in vitro experiments,such as using the micro broth dilution method to determine the minimum inhibitory concentration of celastrol and polymyxin B against pathogenic bacteria,or using crystal violet staining method to determine the inhibitory effect of celastrol on the biofilm formation ability of pathogenic bacteria;2.Evaluate the in vitro antibacterial effect of the combined use of celastrol and polymyxin B through different drug synergistic experiments,such as using the checkerboard dilution method to determine the inhibitory concentration index of the combination of celastrol and polymyxin B,or using the micro-broth dilution method to determine the growth curve of combined celastrol and polymyxin B,and using a persistence bacteria model to detect the killing effect of the combination of the two on the persistent bacteria,and finally,using the electron microscope to observe the combination of the two on the bacterial morphology;3.To study the mechanism of action of celastrol combined with polymyxin B on Staphylococcus aureus through strand-specific differential transcriptome sequencing;4.To study the in vivo antibacterial activity of celastrol combined with polymyxin B by establishing a granulocytopenia mouse thigh infection model.Results: 1.We found that celastrol had inhibitory activity against MRSA,with a MIC and MBC concentration at 2 μg/m L and 16 μg/m L,respectively,and the inhibition rate of celastrol on MRSA biofilm formation reached 70% at 1μg/m L.2.Celastrol and polymyxin B had synergistic antibacterial effect,and the synergistic antibacterial index was 0.375～0.5.Celastrol could enhance the inhibitory effect of polymyxin B on MRSA and reduced its MIC by 8 times;in addition,the combination of the two could effectively kill persistent MRSA bacteria that could not be killed by the two alone;furthermore,the combination of celastrol and polymyxin B can cause serious deformation and shrinkage of the cell morphology of MRSA,accompanied by the destruction of the integrity of the bacterial wall and membrane and the phenomenon of bacterial death.3.Transcriptome results showed that the combined treatment of MRSA with celastrol and polymyxin B could significantly down-regulate the expression of Agr B,Sar R,Rpi R and other genes.GO functional annotation analysis found that these differential genes were mainly enriched in the membrane components,cytoplasm and cell membrane in molecular composition.The GO functions of the down-regulated genes were mainly enriched in the categories of biosynthesis and metabolism of tetraterpenoids and the biosynthesis and metabolism of carotenoids.The KEGG pathways in the down-regulated genes were mainly enriched in the categories of the carotenoid biosynthesis,fatty acid degradation,valine,leucine and isoleucine biosynthesis.4.The results of animal experiments showed that the combined treatment of celastrol and polymyxin B significantly reduced the area of abscess in the infected thigh of mice compared with the model group,and the amount of bacteria carried in the infected thigh also decreased.Conclusions: 1.Celastrol had excellent antibacterial effect on MRSA,with MIC and MBC values of 2 μg/m L and 16 μg/m L,respectively,which could significantly inhibit the formation of MRSA biofilm.2.Celastrol was an effective auxiliary compound,which could enhance the antibacterial activity of polymyxin B against MRSA and reduced its MIC by 8times;in addition,the combination of celastrol and polymyxin B could not only effectively kill the persistent bacteria formed by MRSA but also destroyed the cell morphology of MRSA,leading to its death.3.Transcriptome data revealed that the mechanism of action of celastrol and polymyxin B might be to block the process of QS(Quorum sensing)by targeting the Agr QS system gene Agr B,thereby reducing intercellular communication and signal transduction;furthermore,the secretion of virulence factors such as Sar R and PSM could also affect the biosynthesis and metabolism of carotenoids in MRSA.4.The combination application of celastrol and polymyxin B could effectively alleviate the symptoms of MRSA infection in animals.