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目的探讨双氢青蒿素(dihydroartemisinin,DHA)对体外培养的蓝氏贾第鞭毛虫(Giardialamblia)滋养体蛋白质的损伤作用。方法用改良TYI-S-33培养基培养蓝氏贾第鞭毛虫滋养体;设立实验组(DHA组)和正常对照组(C组)。实验组用含双氢青蒿素的培养基(含LC50=200μg/mL的DHA)培养;对照组培养基不含药物。提取各组虫体总蛋白,经Brad-ford法进行蛋白质定量,样本经2D-电泳和硝酸银染色后,扫描检测凝胶电泳图蛋白点的数量和种类,获取蛋白质点匹配信息,挑选匹配性高的蛋白点,进行质谱(MALDI-TOF MS)分析。结果经双氢青蒿素作用后虫体总蛋白质含量(0.6993μg/mL)明显低于对照组(4.8241μg/mL),二者差异有显著性(P<0.001)。蛋白质二维电泳凝胶图分析结果显示,正常对照组(C组)蛋白点密集,约1253个点,布满整块凝胶,蛋白表达量高(蛋白点颜色深且清晰、明亮);与此相反,实验组(DHA组)蛋白点显著减少,DHA作用6h后约617个点,作用12h后约326个点,蛋白表达量明显降低(蛋白点颜色浅,不清晰)。其次,正常对照组与双氢青蒿素组的12kDa~30kDa、等电点pI3.0~7.6范围内蛋白点表达量存在明显差异,即实验组该范围内大多数蛋白点完全消失,45kD~60kDa的蛋白点仅剩6个点。质谱分析鉴定结果显示,在正常对照组胶图所选的80个匹配良好的蛋白点中,鉴定出6种骨架蛋白(14个点),而实验组(DHA组)蛋白胶图中相对应的蛋白点消失。结论双氢青蒿素对蓝氏贾第鞭毛虫滋养体的蛋白质具有明显的损伤作用,其中包括细胞骨架蛋白。
Objective To investigate the damaging effect of dihydroartemisinin (DHA) on the trophozoites of Giardia lamblia cultured in vitro. Methods The trophozoites of Giardia lamblia were cultured in modified TYI-S-33 medium. The experimental group (DHA group) and the normal control group (C group) were established. Experimental group with dihydroartemisinin medium (containing LC50 = 200μg / mL of DHA) culture; control group medium without drugs. The total protein was extracted from each group, and the protein was quantified by Bradford-D method. After the samples were stained with 2D-electrophoresis and silver nitrate, the number and type of protein spots in the gel electrophoresis map were scanned to obtain the matching information of protein spots. High protein spots were analyzed by mass spectrometry (MALDI-TOF MS). Results The total protein content (0.6993μg / mL) in the worm body after dihydroartemisinin treatment was significantly lower than that in the control group (4.8241μg / mL). The difference was significant (P <0.001). The two-dimensional electrophoresis gel analysis showed that the protein spots in the normal control group (C group) were densely packed with about 1253 spots, which covered the whole gel with high protein expression (the protein spots were dark and clear and bright) In contrast, the DHA group showed a significant reduction of protein spots, with about 617 points after 6 hours of DHA treatment and 326 points after 12 hours of treatment. The protein expression was significantly reduced (the protein spots were light and unclear in color). Second, there was a significant difference in the expression of protein spots between 12kDa ~ 30kDa and isoelectric point pI3.0 ~ 7.6 in the normal control group and the dihydroartemisinin group, that is, most of the protein spots in the experimental group completely disappeared in the range of 45kD ~ 60kDa protein spots only 6 points left. Mass spectrometry analysis showed that among the 80 well-matched protein spots selected by the control group, 6 kinds of scaffold proteins (14 spots) were identified, while in the experimental group (DHA group), the corresponding Protein spots disappear. CONCLUSION Dihydroartemisinin can significantly damage the protein of Giardia lamblia trophozoites, including cytoskeletal proteins.