目的 评估去甲二氢愈创木酸对外排泵系统MexCD-OprJ介导铜绿假单胞菌头孢他啶耐药性的影响并探讨其相关机制。方法 将0.5麦氏浓度的菌液稀释接种于96孔板内，棋盘稀释法加入去甲二氢愈创木酸和头孢他啶，同时设置不加药的阳性对照组、去甲二氢愈创木酸对照组、头孢他啶对照组，培养24 h后，酶标仪测定吸光度，记录各药物的最低抑菌浓度（MIC），并计算抑菌率和部分抑菌浓度指数（FIC）；取96孔板内的菌液涂布法接种细菌，培养24 h计数菌落数；实时荧光定量PCR（qRT-PCR）检测外排泵膜蛋白MexC、MexD、OprJ及nfxB基因的表达情况。结果 与单用头孢他啶或去甲二氢愈创木酸相比较，头孢他啶与去甲二氢愈创木酸联合应用能更明显抑制外排泵系统MexCD-OprJ介导的头孢他啶耐药的铜绿假单胞菌的生长（P<0.05），头孢他啶与去甲二氢愈创木酸的作用主要表现为协同或相加作用；联合用药后，头孢他啶与去甲二氢愈创木酸的MIC值均明显降低，其中，部分头孢他啶的MIC值与头孢他啶敏感的铜绿假单胞菌质控菌株的MIC值无明显差异。与单用头孢他啶相比较，头孢他啶与去甲二氢愈创木酸联合应用后细菌外排泵膜蛋白MexC、MexD和OprJ的基因表达明显降低（P<0.05），而nfxB的表达明显升高（P<0.05）。结论 去甲二氢愈创木酸能逆转外排泵系统MexCD-OprJ介导铜绿假单胞菌头孢他啶的耐药性，其机制与其能够下调该耐药菌外排泵膜蛋白MexC、MexD及OprJ的表达、上调上述3个蛋白的负向调节基因nfxB的表达有关。

Objective To evaluate the effect of nordihydroguaiaretic acid (NDGA) on ceftazidime resistance of Pseudomonas aeruginosa mediated by efflux pump system MexCD-OprJ and explore its mechanism. Methods The bacterial solution with a concentration of 0.5 mcburney was diluted and inoculated in a 96-well plate, and NDGA and ceftazidime were added by the checkerboard dilution method. At the same time, the untreated control group, NDGA control group and ceftazidime control group were set; After being cultured for 24 h, the absorbance was measured by an enzyme micro-plate reader, the minimum inhibitory concentration (MIC) of each drug was recorded and the bacteriostatic rate and fractional inhibitory concentration (FIC) index were calculated. Bacteria were inoculated with the bacterial liquid coating method in the 96-well plates, and the bacterial colony number was counted after 24 h of culture. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the gene expressions of efflux pump membrane protein MexC, MexD, OprJ and nfxB. Results Compared with ceftazidime or NDGA alone, combination of ceftazidime and NDGA significantly inhibited the growth of efflux pump system MexCD-OprJ-mediated ceftazidime-resistant P. aeruginosa (P<0.05); The pharmacological effects of ceftazidime and NDGA showed synergistic or additive effects; After combined administration, the MIC values of ceftazidime and NDGA were significantly decreased, and the MIC value of some ceftazidime had no significant difference from that of ceftazidime-sensitive P. aeruginosa; Compared with ceftazidime alone, the gene expressions of efflux pump membrane proteins MexC, MexD and OprJ were significantly decreased after combined application of ceftazidime and NDGA (P<0.05), while the expression of nfxB was significantly increased (P<0.05). Conclusion The mechanism of NDGA on ceftazidime resistance of P. aeruginosa mediated by efflux pump system MexCD-OprJ is related to its ability to down-regulate the gene expression of efflux pump membrane proteins MexC, MexD and OprJ, and up-regulate the gene expression of the negative regulatory gene nfxB of the above three proteins.

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