米托蒽醌的抗锥虫活性及其作用机制

The activity and mechanism of Mitoxantrone against trypanosomes

目的分析米托蒽醌体外抗锥虫的作用机制并考察其体内抗锥虫活性。方法通过倍比稀释合并alamar Blue染色测定米托蒽醌体外抗血流型布氏锥虫427（T.brucei BF427）的量-效和时-效曲线,并与抗锥虫药物喷他脒和美拉胂醇进行比较;通过DAPI染色、细胞周期表型分析考察米托蒽醌的抗锥虫作用机制;采用血虫症小鼠考察米托蒽醌的体内抗锥虫活性,米托蒽醌经腹腔给药,生理盐水为对照组;每天取血计数血虫密度;用药7d后停药,计算小鼠存活率。结果量-效曲线测得米托蒽醌对体外培养锥虫（起始密度为1×10^6个/mL）24h MIC为9.7μM;时-效曲线显示米托蒽醌和喷他脒都可显著抑制锥虫生长、持续降低锥虫密度;而经美拉胂醇处理后的锥虫密度在12 h内仍缓慢增长、随后出现明显降低;因此,在MIC作用浓度下米托蒽醌的体外抗锥虫活性好于美拉胂醇、与喷他脒相似。对照组小鼠的血虫密度迅速增加,感染6d时全部死亡;米托蒽醌明显抑制小鼠血虫增长,停药后2d小鼠出现死亡,最长存活14d,存活时间明显延长。DAPI染色和细胞周期表型分析显示锥虫核内（N）基因组DNA的复制受到抑制、而动基体（K）DNA的复制不受影响,表现为1N2K细胞型的比例明显上升,由10.8%上升至17.5%,2N2K细胞型的比例明显下降,由6.3%下降至3.0%。结论米托蒽醌具有体内抗锥虫活性,抑制锥虫核内基因组DNA的复制并使其产生损伤是作用机制之一。

Objective To explore the anti- trypanosome mechanism of Motoxantrone in vitro and evaluate its anti-trypanosome activity in vivo. Methods The dose-response and time-response of Mitoxantrone against Trypanosoma bruceiBF427 in vitro were determined by a series of double-diluted drug combined with alamar Blue staining. The anti-trypanosomeactivity of mitoxantrone was compared to that of Pentamidine and Melarsoprol. The mechanism of Mitoxantrone on anti-trypanosome activity was analyzed by fluorescence microscope after DAPI staining and cell cycle phenotypic analysis. In vivo,anti-trypanosome activity was estimated by treating the T. brucei BF427-infected mice with Mitoxantrone via intraperitonealinjection. Control mice were given the same volume of saline. The blood parasites were counted each day. Drug discontinuationafter 7 days and survival rate was calculated. Results The dose-response curve showed that the 24 h MIC of Mitoxantrone tocultivated T.brucei（initial cell density 1×10^6/ mL） was 9.7 μM.The time-response of the drugs against BF427 showed thatMitoxantrone and Pentamidine could inhibit the parasites significantly and reduce the cell density after administration. Incontrast,Melarsoprol caused the cell density reduction after treatment for 12 hours. These results indicated that Mitoxantroneshowed stronger anti- trypanosomal activities than Melarsoprol and similar to Pentamiwhen given at MIC in vitro. All theinfected mice in control group showed a big increase of the blood parasite density and died after 6 days of post-infection.Compared to the control mice,the infected mice with Mitoxantrone treatment showed much less blood parasites and began todie in two days after drug withdrawal,and the maximal survival time was 14 days of post-infection,prolonged significantly.DAPI staining and cell cycle phenotypic analysis showed that the replication of nuclear（N） DNA of the parasite was inhibited,and the replication of kinetoplast（K） DNA was not affected,which showed that the 1N2 K cell number increased from 10.8% to17.5%,and the 2N2 K cell number declined from 6.3% to 3%. Conclusion Mitoxantrone has strong anti- trypanosomeactivity in vivo,and the potential mechanism is related to the genomic DNA replication inhibition and DNA damage.