双氢青蒿素与头孢呋辛对大肠杆菌的协同抗菌作用及机制研究

Synergistic antibacterial effect and mechanisms of dihydroartemisinin and cefuroxime incombination

该实验用双氢青蒿素(dihydroartemisinin,DHA)与头孢呋辛(cefuroxime,CFX)或氨苄青霉素联用,探讨其对大肠杆菌的协同抗菌作用及其机制。采用微量肉汤稀释法和2,3,5-氯化三苯基四氮唑(2,3,5-triphenyl-2H-tetrazolium chloride,TTC)法确定出DHA、CFX和氨苄青霉素的最低抑菌浓度(minimum inhibitory concentration,MIC)分别为300,25,25μmol·L^-1,头孢呋辛的最低杀菌浓度(minimum bactericidal concentration,MBC)为25μmol·L^-1,DHA的MBC>600μmol·L^-1;采用棋盘稀释确定出DHA与头孢呋辛或与氨苄青霉素联合的分级抑菌浓度指数(fractional inhibitory concentration index,FICI)分别为0.375和0.75,表现为协同作用或加和作用。从时效曲线可以得出两药联用后对大肠杆菌的作用明显强于DHA和CFX各自单用的,说明DHA联用后,可以减少CFX的使用量。继而研究了DHA与头孢呋辛协同抗菌的机制:聚丙烯凝胶电泳结果显示,两药联用的条带比各自单用的更浅,有的谱带甚至消失,说明两药联用后对总蛋白谱带具有明显的抑制作用;电导率法结果显示,两药联用后菌液的电导率值明显增高;碱性磷酸酶(alkaline phosphatase,AKP)检测显示,联用组碱性磷酸酶渗漏最为明显,提示菌体完整性可能受到破坏;扫描电子显微镜(scanning electron microscope,SEM)结果显示,两药联用后对大肠杆菌形态的破坏作用比单独用药组强;荧光定量PCR结果显示,两药联用能抑制大肠杆菌超氧压力反应相关基因(soxS)的表达。结果证明双氢青蒿素与头孢呋辛联用具有协同作用。

In this study,we explored the antibacterial effect and mechanism of dihydroartemisinin(DHA)combined with cefuroxime(CFX)or ampicillin against Escherichia coli.The minimum inhibitory concentration(MIC)of DHA,cefuroxime,and ampicillin against E.coli was 300,25,25μmol·L^-1,respectively,determined by broth microdilution method and 2,3,5-triphenyltetrazolium chloride(TTC)method.The minimum bactericidal concentration(MBC)was 25μmol·L^-1 for cefuroxime,above 600μmol·L^-1 for DHA.The fractional inhibitory concentration index(FICI)of DHA combined with cefuroxime or ampicillin was 0.375 and 0.75,respectively,determined by checkerboard microdilution assay,suggesting the synergistic effect or additive effect of the drug combination.Moreover,the time-effect curve showed that the antibacterial activity of DHA and CFX combination was much stronger than that of either of the drugs,suggesting that combination with DHA can decrease the CFX dosage.Then we studied the synergistic mechanism of DHA combined with cefuroxime and found that the combination of the two drugs had a significant inhibitory effect on the total protein bands,as shown by the results of polypropylene gel electrophoresis.The results of conductivity method and alkaline phosphatase test respectively showed that its conductivity value and alkaline phosphatase(AKP)leak were significantly higher than either of the drugs,suggesting that the integrity of bacteria may be damaged.The scanning electron microscope(SEM)results showed that the morphology of E.coli was destroyed most in the combination group.The quantitative fluorescence PCR technology showed that the combination of two drugs can inhibit the expression of superoxide stress gene soxS.In summary,the combination of dihydroartemisinin and cefuroxime has a synergistic antibacterial effect on E.coli.