青蒿琥酯抗耐多药结核分枝杆菌的作用活性及机制研究  

Activity and mechanism of artesunate against multidrug resistant Mycobacterium tuberculosis

作　　者：喻容 周星 马小华[2] 石国民[2] 向延根[2] YU Rong;ZHOU Xing;MA Xiaohua;SHI Guomin;XIANG Yangen(Department of Clinical Laboratory,the Second Affiliated Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410005,China;Department of Clinical Laboratory,the Changsha Central Hospital,Changsha,Hunan 410004,China)

机构地区：[1]湖南中医药大学第二附属医院医学检验中心,湖南长沙410005 [2]长沙市中心医院检验科,湖南长沙410004

出　　处：《热带医学杂志》2024年第6期793-800,共8页Journal of Tropical Medicine

基　　金：湖南省卫健委科研项目(202111000097);湖南省自然科学基金医卫行业联合基金项目(2024JJ9454)。

摘　　要：目的探讨青蒿琥酯(ASN)单独或联合抗结核药物利福平(RFP)和异烟肼(INH)对耐多药结核分枝杆菌(MDR-TB)的抗菌活性及其作用机制。方法选择2021年1月-2022年5月经长沙市中心医院确认保存的MDRTB 20例,进行体外药敏试验。根据不同药物处理将其分为无药对照组、青蒿琥酯组、利福平组、异烟肼组、青蒿琥酯联合利福平组、青蒿琥酯联合异烟肼组,检测其最小抑菌浓度(MIC)。采用基因芯片法检测青蒿琥酯对MDRTB耐药基因突变位点分析,实时荧光定量PCR检测青蒿琥酯改善抗结核药物对MDR-TB影响与外排泵关系,扫描电镜和透射电镜观察青蒿琥酯处理前后细菌细胞的改变情况。结果20株耐药菌株经鉴定均为MDR-TB,耐药基因分别为rpoB531C-T突变、rpoB526C-T突变和rpoB516A-T突变,katG315G-A突变和katG315G-C突变;其中,10株为rpoB531C-T突变、katG315G-A突变;6株为rpoB531C-T突变、katG315G-C突变;4株为rpoB516A-T突变、katG315G-A突变。随机挑选3、8号菌株,MDR-TB耐药位点均为rpoB531C-T突变和KatG315G-C突变,经青蒿琥酯处理24 h前后,rpoB531C-T位点突变和KatG315G-C位点突变均未发生改变。青蒿琥酯单独作用MDR-TB时,MIC较高;青蒿琥酯与利福平、异烟肼联合作用时,MIC值明显降低,差异均有统计学意义(P均<0.001)。20株MDR-TB菌株中,仅在青蒿琥酯组中有2株能够显著抑制外排泵基因efpA、RV1250 mRNA表达,差异均有统计学意义(t=6.635、16.520,P均<0.05);其余菌株对efpA、RV1250 mRNA表达差异均无统计学意义(P均>0.05)。扫描电镜结果显示,与对照组比较,加入青蒿琥酯后,菌体干枯皱缩;透射电镜结果显示,青蒿琥酯处理后,出现脂质脱落,菌体死亡。结论青蒿琥酯单独作用对MDR-TB未显示出抗结核活性,但联合抗结核药时,可改善抗结核药物对MDR-TB的药物敏感性;药物外排泵efpA、RV1250基因可能不是青蒿琥酯发挥主要机制;青蒿琥酯可破坏结核分枝杆�Objective To explore the antibacterial activity of artesunate alone or in combination with anti-tuberculosis drugs rifampicin and isoniazid against multidrug resistant Mycobacterium tuberculosis(MDR-TB),and the mechanism of artesunate combined with anti-tuberculosis drugs on MDR-TB.Methods A total of 20 cases of confirmed multidrug resistant Mycobacterium tuberculosis strains stored in the Changsha Central Hospital from January 2021 to May 2022 were selected for drug sensitivity test in vitro.According to the different drug treatment,they were divided into drug free control group,artesunate group,rifampin group,isoniazid group,artesunate combined with rifampin group,and artesunate combined with isoniazid group,and their minimum inhibitory concentrations(MIC)were measured.Gene chip method was used to detect the mutation site analysis of MDR-TB resistance gene of artesunate;real-time fluorescence quantitative PCR was used to detect the relationship between artesunate improving the effect of anti-tuberculosis drugs on MDR-TB and efflux pump,and scanning electron microscopy and transmission electron microscopy were used to observe the changes of bacterial cells before and after drug treatment.Results The 20 drug-resistant strains were all identified as MDR-TB,and the resistance genes were rpoB531C-T mutation,rpoB526C-T mutation and rpoB516A-T mutation,katG315G-A mutation and katG315G-C mutation;among them,10 strains were rpoB531C-T mutation and katG315G-A mutation;6 strains were rpoB531C-T mutation and katG315G-C mutation;4 strains were rpoB516A-T mutation and katG315G-A mutation.Strains No.3 and 8were randomly selected.Their MDR-TB resistance sites were rpoB531C-T mutation and KatG315G-C mutation.After treatment with artesunate for 24 hours,rpoB531C-T site mutation and KatG315G-C site mutation were detected.None had been changed.When MDR-TB was treated by artesunate alone,the MIC was higher;when treated with artesunate combined with rifampicin and isoniazid,the MIC value was significantly lower,the differences were

关 键 词：青蒿琥酯 抗结核药物 耐多药结核分枝杆菌 电镜 最小抑菌浓度 

分 类 号：R563.8[医药卫生—呼吸系统]