目的 評估去甲二氫愈創(chuàng)木酸對外排泵系統(tǒng)MexCD-OprJ介導(dǎo)銅綠假單胞菌頭孢他啶耐藥性的影響并探討其相關(guān)機(jī)制。方法 將0.5麥?zhǔn)蠞舛鹊木合♂尳臃N于96孔板內(nèi)，棋盤稀釋法加入去甲二氫愈創(chuàng)木酸和頭孢他啶，同時(shí)設(shè)置不加藥的陽性對照組、去甲二氫愈創(chuàng)木酸對照組、頭孢他啶對照組，培養(yǎng)24 h后，酶標(biāo)儀測定吸光度，記錄各藥物的最低抑菌濃度（MIC），并計(jì)算抑菌率和部分抑菌濃度指數(shù)（FIC）；取96孔板內(nèi)的菌液涂布法接種細(xì)菌，培養(yǎng)24 h計(jì)數(shù)菌落數(shù)；實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測外排泵膜蛋白MexC、MexD、OprJ及nfxB基因的表達(dá)情況。結(jié)果 與單用頭孢他啶或去甲二氫愈創(chuàng)木酸相比較，頭孢他啶與去甲二氫愈創(chuàng)木酸聯(lián)合應(yīng)用能更明顯抑制外排泵系統(tǒng)MexCD-OprJ介導(dǎo)的頭孢他啶耐藥的銅綠假單胞菌的生長（P<0.05），頭孢他啶與去甲二氫愈創(chuàng)木酸的作用主要表現(xiàn)為協(xié)同或相加作用；聯(lián)合用藥后，頭孢他啶與去甲二氫愈創(chuàng)木酸的MIC值均明顯降低，其中，部分頭孢他啶的MIC值與頭孢他啶敏感的銅綠假單胞菌質(zhì)控菌株的MIC值無明顯差異。與單用頭孢他啶相比較，頭孢他啶與去甲二氫愈創(chuàng)木酸聯(lián)合應(yīng)用后細(xì)菌外排泵膜蛋白MexC、MexD和OprJ的基因表達(dá)明顯降低（P<0.05），而nfxB的表達(dá)明顯升高（P<0.05）。結(jié)論 去甲二氫愈創(chuàng)木酸能逆轉(zhuǎn)外排泵系統(tǒng)MexCD-OprJ介導(dǎo)銅綠假單胞菌頭孢他啶的耐藥性，其機(jī)制與其能夠下調(diào)該耐藥菌外排泵膜蛋白MexC、MexD及OprJ的表達(dá)、上調(diào)上述3個(gè)蛋白的負(fù)向調(diào)節(jié)基因nfxB的表達(dá)有關(guān)。

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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