摘要
目的通过测定结核分枝杆菌活性氧含量及过氧化氢酶活性,初步探讨吡法齐明抗耐药结核分枝杆菌的作用机制。方法以1、2、4、8倍于最小抑菌浓度(minimal inhibitory concentration,MIC)的氯法齐明和吡法齐明处理H37Rv标准株24h,应用荧光探针2',7'-二氯荧光黄双乙酸盐(DCFH-DA)活性氧检测试剂盒测定不同浓度的氯法齐明和吡法齐明处理前后结核分枝杆菌菌体内的活性氧含量水平变化。以过氧化氢酶活性检测试剂盒测定5株对氯法齐明和吡法齐明均耐药、3株对氯法齐明耐药但对吡法齐明敏感、1株对氯法齐明和吡法齐明均敏感(编号13385)的结核分枝杆菌及H37Rv标准株的过氧化氢酶活性,比较氯法齐明和吡法齐明耐药菌株与敏感菌株的过氧化氢酶活性的差异。采用SPSS 19.0软件进行数据的统计学分析,并对吡法齐明药物处理浓度与所测得的荧光强度值进行Spearman相关性分析和线性回归分析,以P<0.05为差异有统计学意义。结果活性氧含量水平测定结果显示,结核分枝杆菌在经不同浓度氯法齐明和吡法齐明处理24h后,各浓度药物处理样本的荧光强度值均值排序分别为1MIC(6668.5)>8MIC(2751.5)>2MIC(2572.0)>4MIC(2015.0),8MIC(2637.5)>4MIC(2297.0)>2MIC(2085.5)>1MIC(1896.0),菌体内积累的活性氧含量均高于无药物刺激的阴性对照样品(H37Rv标准株+DCFH-DA,878.5)至少2倍;且在各浓度吡法齐明处理的菌株中,菌株测定的荧光强度值与药物浓度间呈现明显的正相关(Spearman相关性分析,r=0.976,P<0.001),经线性回归方程检验结果显示回归模型有统计学意义(F=92.576,P=0.011)。菌株过氧化氢酶活性测定结果显示,氯法齐明耐药菌株的过氧化氢酶平均活性(0.87U/10^4cell)是其敏感菌株(0.48U/10^4cell)的近2倍;而吡法齐明耐药菌株的过氧化氢酶活性均值(0.88U/10^4cell)略高于其敏感菌株(0.71U/10^4cell),但与氯法齐明耐药菌株均值相近。结论氯法齐明和吡法齐明作用于结核分枝杆菌后均会引起菌体内活性氧含量的积累,两药可能有着相似的作用机制;过氧化氢酶活性增高可能与结核分枝杆菌对氯法齐明和吡法齐明耐药有关。
Objective To explore the mechanism of TBI-166 against drug-resistant Mycobacterium tuberculosis(MTB)by determining the reactive oxygen content and catalase activity of MTB.Methods H37Rv standard strains were treated with clofazimine(CFZ)or TBI-166 at 1,2,4,8 times the minimal inhibitory concentration(MIC)for 24 hours.The levels of reactive oxygen species(ROS)in bacteria were determined with fluorescent probe 2',7'-dichloro fluorescent yellow diacetate ROS detection kit,and the changes of ROS levels in bacteria were compared before and after the medication of CFZ or TBI-166 with different concentrations.The catalase activities of 5 strains resistant to both CFZ and TBI-166,3 strains resistant to CFZ but sensitive to TBI-166,1 strain(13385)sensitive to both CFZ and TBI-166,and H37Rv standard strain were determined by the catalase activity detection kit.The difference in catalase activity between drug-resistant strains and sensitive strains to both CFZ and TBI-166 was compared.SPSS 19.0 software was used for statistical analysis.Spearman correlation analysis and linear regression analysis were performed upon the concentrations of TBI-166 and the fluorescence values.The significance level was 0.05.Results The ROS results showed that after 24 h of treatments with different concentrations CFZ and TBI-166,the rank order of the fluorescence value of samples were 1 MIC(6668.5)>8 MIC(2751.5)>2 MIC(2572.0)>4 MIC(2015.0),and 8 MIC(2637.5)>4 MIC(2297.0)>2 MIC(2085.5)>1 MIC(1896.0),respectively.The amount of ROS accumulated in the bacteria was at least 2 times higher than that in the negative control samples without drug(H37Rv standard strain+DCFH-DA,878.5).Moreover,in the strains treated with different concentrations of TBI-166,the fluorescence values were positively correlated with the drug concentration(Spearman correlation analysis,r=0.976,P<0.001).The regression equation test results showed that the regression model was significant(F=92.576,P=0.011).The catalase activity results showed that the average catalase activity of the CFZ-resistant strains(0.87 U/10^4 cell)was nearly twice that of the CFZ-sensitive strains(0.48 U/10^4 cell).The average catalase activity of the TBI-166-resistant strains(0.88 U/10^4 cell)was slightly higher than that of the TBI-166-sensitive strains(0.71 U/10^4 cell),and was similar to that of CFZ-resistant strains.Conclusion Both CFZ and TBI-166 can induce the accumulation of intracellular ROS in MTB,and the two drugs may have similar mechanisms of action.Increased catalase activity may be associated with the drug resistance to CFZ and TBI-166 in MTB.
作者
张叶
李媛媛
徐建
陈曦
王彬
付雷
陆宇
ZHANG Ye;LI Yuan-yuan;XU Jian;CHEN Xi;WANG Bin;FU Lei;LU Yu(Department of Pharmacology,Beijing Key Laboratory of Drug Resistance Tuberculosis Research,Beijing Chest Hospital,Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)
出处
《中国防痨杂志》
CAS
CSCD
2019年第11期1160-1166,共7页
Chinese Journal of Antituberculosis
基金
国家自然科学基金(81973367)。
关键词
分枝杆菌
结核
抗药性
药理作用分子作用机制
药物评价
临床前
氯法齐明
吡法齐明
Mycobacterium tuberculosis
Drug resistance
Molecular mechanisms of pharmacological action
Drug evaluation
preclinical
Clofazimine
TBI-166
