青蒿琥酯体外抗弓形虫作用的实验研究

Anti-Toxoplasma Gondii effect of artesunate in vitro

目的:观察青蒿琥酯(Artesunate,Art)对体外培养的弓形虫感染模型的抑制作用。方法:采用表达绿色荧光蛋白的转基因弓形虫RH株(RH-GFP)感染小鼠腹腔巨噬细胞(Macrophage,Mφ),建立体外作用模型并分为对照组(0.9%氯化钠溶液阴性对照);Art组,药物浓度分别为1.5625、3.125、6.25、12.5、25、50、100和200μg/mL;阿奇霉素(Azithromycin,Azm)组(阳性对照),药物浓度分别为12.5、25、50、100、200、400、800和1600μg/mL;Art+Azm组,药物浓度分别为:Art1.5625+Azm12.5、Art3.125+Azm25、Art6.25+Azm50、Art12.5+Azm100、Art25+Azm200和Art50+Azm400μg/mL。各组孵育24 h后以流式细胞仪检测感染体系中虫体和细胞比例变化,以吉姆萨染色观察速殖子形态和结构变化。结果:与对照组相比,Art组弓形虫速殖子比例自3.125μg/mL起随着药物浓度的增加明显下降(P<0.01),50μg/mL时游离虫体比例低于0.3%,Mφ感染率低于1.6%。Azm组速殖子在400μg/mL时比例低于0.6%,Mφ感染率低于3.5%。Art+Azm组中Art12.5+Azm100μg/mL起速殖子比例低于0.04%,此时Mφ感染率低于1.8%。吉姆萨染色显示,各组随着药物浓度增加,弓形虫速殖子由香蕉状逐渐肿胀变形,细胞质内呈泡沫状,内部结构崩塌瓦解,直至细胞膜破裂内容物溢出,视野内仅残留固缩深染的胞核或碎片。结论:体外实验中,Art可显著杀伤弓形虫游离速殖子,降低宿主细胞感染率,该抗虫效果在一定范围内与浓度呈正相关。

Objective:To explore the inhibitory effect of artesunate(Art)on an in vitro cultured model of Toxoplasma Gondii infection.Methods:An in vitro infection model was established by infecting macrophages(Mφ)in the peritoneal cavity of mice with Toxoplasma Gondii RH strain expressing stable green fluorescent protein in the cytoplasm(RH-GFP).The experiment was divided into four groups:control group(0.9%sodiuom chloride solution),Art group(concentrations of 1.5625,3.125,6.25,12.5,25,50,100 and 200μg/mL),Azithromycin(Azm)group(positive control,concentrations of 12.5,25,50,100,200,400,800 and 1600μg/mL),Art+Azm group(concentrations of Art 1.5625+Azm 12.5,Art 3.125+Azm 25,Art 6.25+Azm 50,Art 12.5+Azm 100,Art 25+Azm 200 and Art 50+Azm 400μg/mL.After 24 hours of incubation,the change in proportion of parasites and cells was detected by flow cytometry,and the morphology and structural changes of tachyzoites were observed by Giemsa staining.Results:Compared with the control group,the proportion of tachyzoites in the Art group decreased significantly with the increase of concentration from 3.125μg/mL(P<0.01),the proportion of free parasites was less than 0.3%at 50μg/mL,and the infection rate of Mφwas less than 1.6%.In Azm group,the proportion of tachyzoites was less than 0.6%at 400μg/mL,and the infection rate of Mφwas less than 3.5%.In the Art+Azm group,the proportion of tachyzoites in the Art 12.5+Azm 100μg/mL group was less than 0.04%,and the infection rate of Mφwas less than 1.8%.Giemsa staining showed that with the increase of concentration in each group,the Toxoplasma Gondii tachyzoites gradually swelled and transformed from a banana shape to one with foamy cytoplasm.The internal structure collapsed and disintegrated until the cell membrane ruptured and contents spilled out,and only the solidified and deeply stained nuclei or fragments remained in the field of view.Conclusions:Art can significantly kill the free tachyzoites of Toxoplasma Gondii in vitro experiment and reduce the infection rate of host cells,and the anti-Toxoplasma Gondii effect is positively correlated with the concentration within a certain range.