不同抗疟药物对红内期恶性疟原虫3D7线粒体呼吸的影响

Effects of antimalarial drugs on mitochondrial respiration in erythrocytic stages of Plasmodium falciparum 3D7

目的:利用Seahorse XFe96分析仪对6大类别共计13种国际一线抗疟药物作用于红内期恶性疟原虫3D7(Plasmodium falciparum 3D7)线粒体电子传递链(electron transport chain,ETC)进行评价。方法:采用三天抑制法和SYBR Green I荧光分析法对体外药物作用于P.falciparum 3D7的抗疟活性进行评价,采用磁珠分选技术对P.falciparum 3D7虫株进行分离纯化,采用Seahorse XF分析系统的线粒体耗氧率OCR值(Oxygen Consumption Rate)对P.falciparum 3D7线粒体不同时刻的生物能进行表征,进而考察不同抗疟药物对红内期恶性疟原虫线粒体有氧呼吸的影响。结果:流式检测结果显示,成功富集到虫期较为一致的滋养体期疟原虫。体外抗疟活性评价结果显示,除双胍类抗疟药物氯胍(proguanil,Pro)外,其余12种抗疟药物对P.falciparum 3D7的药效均为nmol/L级别。线粒体有氧呼吸考察的结果显示,双氢青蒿素(dihydroartemisinin,DHA)和氯喹(chloroquine,CQ)的5种浓度剂量(0.4、1、5、10、50×IC 50)对P.falciparum 3D7线粒体有氧呼吸均无明显影响。13种常用抗疟药物在5×IC 50浓度剂量下,青蒿素(artemisinin,ART)、蒿乙醚(arteether,ARE)和蒿甲醚(artemether,ARM)均能显著提高线粒体最大呼吸;奎宁(quinine,QN)和Pro仅对P.falciparum 3D7线粒体ETC的氧化磷酸化具有解偶联作用,未完全破坏线粒体ETC的功能;阿托伐醌(atovaquone,Ato)对P.falciparum 3D7线粒体有氧呼吸有明显的抑制作用,显著降低线粒体最大呼吸和质子的泄漏,完全破坏了线粒体ETC的功能;其余抗疟药物对P.falciparum 3D7线粒体有氧呼吸均无明显影响。结论:13种国际一线抗疟药物中大多数抗疟药物的靶标并非是疟原虫线粒体有氧呼吸的ETC通路。

AIM:The Seahorse XFe96 analyzer was used to evaluate the effects of thirteen types of international first-line antimalarial drugs in six categories on the mitochondrial electron transport chain(ETC)of Plasmodium falciparum 3D7(P.falciparum 3D7).METHODS:The antimalarial activity of in vitro drugs acting on P.falciparum 3D7 was evaluated using the three-day inhibition method and SYBR Green I fluorescence analysis method.MACS technology was used to separate and purify P.falciparum 3D7.The mitochondrial oxygen consumption rate(OCR)of Seahorse XF analysis system was used to characterize the bioenergy of P.falciparum 3D7 mitochondria at different times to investigate the effects of antimalarial drugs on mitochondrial aerobic respiration of Plasmodium falciparum.RESULTS:The results of flow cytometry showed that the Plasmodium of trophozoite stages was enriched successfully.The results of in vitro antimalarial activity evaluation showed that,except for the antimalarial drug proguanil(Pro),the other twelve antimalarial drugs were all of the nmol/L level against P.falciparum 3D7.The results of the mitochondrial aerobic respiration showed that the five concentrations of dihydroartemisinin(DHA)and chloroquine(CQ)(0.4,1,5,10,50×IC 50)on P.falciparum 3D7 mitochondria aerobic respiration had no significant effect.At the concentration of 5×IC 50 for thirteen antimalarial drugs,Artemisinin(ART),arteether(ARE)and artemether(ARM)can significantly increase mitochondrial maximum respiration,Quinine(QN)and Pro only had a decoupling effect on the oxidative phosphorylation of mitochondrial ETC of P.falciparum 3D7,which did not completely destroy the function of mitochondrial ETC.Atovaquone(Ato)had a significant inhibitory effect on P.falciparum 3D7 mitochondrial aerobic respiration,significantly decrease mitochondrial maximum respiration and proton leak,completely destroying the function of mitochondrial ETC,and the remaining ten antimalarial drugs had no significant effect on P.falciparum 3D7 mitochondrial aerobic respiration.CONCLUSION:The target of most antimalarial drugs in thirteen antimalarial drugs is not the ETC pathway of Plasmodium mitochondrial aerobic respiration.