金诺芬通过促进自噬对弓形虫增殖的影响

Effects of Auranofin on the proliferation of Toxoplasma gondii by promoting autophagy

摘要:

目的：金诺芬对弓形虫速殖子的作用及对弓形虫感染小鼠的自噬初步探讨。方法：透射电子显微镜观察用药前后刚地弓形虫超微结构的变化；采用免疫荧光、免疫组化和免疫印迹法检测感染有弓形虫的脑组织中自噬蛋白LC3的变化。HE染色观察金诺芬作用前后小鼠脑部的弓形虫速殖子变化。结果：透射电镜显示10 ?滋g/mL组（B组）的弓形虫速殖子内部结构被破坏，虫体细胞膜破裂，细胞质均质样改变，20 ?滋g/mL组（C组）的虫体内容物溢出，线粒体及粗面内质网消失，核固缩，核膜断裂，细胞质出现许多空泡，严重者虫体崩解呈均质样改变。免疫荧光结果显示实验组（77.49±15.68）的LC3蛋白的表达量较对照组（53.81±15.24）明显增加（P=0.001）。免疫组化结果显示用药后（1 208.55±580.07）的LC3蛋白表达量比对照组（544.54±225.04）明显增高（P=0.000）。免疫印迹法检测结果显示用药后（41 738.52±0.00）的LC3蛋白表达量比对照组（25 383.88±607.34）明显增高（P=0.000）。用药后（218.80±21.17）寄生在小鼠脑部的弓形虫速殖子增殖数量明显少于对照组（495.40±31.91）（P=0.000）。结论：金诺芬在体外有破坏弓形虫速殖子的作用，同时金诺芬可以提高弓形虫感染小鼠的自噬水平从而抑制弓形虫增殖。

Abstract:

Objective：To study the effect of Auranofin on Toxoplasma gondii tachyzoites and the autophagy in mice infected with Tox－oplasma gondii. Methods：The changes of Toxoplasma gondii ultrastructure were observed before and after treatment by transmission electron microscopy. The changes of autophagy protein LC3 in brain tissue with Toxoplasma gondii were detected by immunofluores－cence，immunohistochemistry and immunoblotting. The changes of Toxoplasma gondii tachyzoites in the brain of mice were ob－served by HE staining. Results：The transmission electron microscopy showed that in 10 ?滋g/mL group（group B），the internal struc－ture of Toxoplasma tachyzoite was destroyed，the cell membrane of the worm was ruptured，and the cytoplasm structure changed，while in 20 ?滋g/mL group（group C），the insect body content overflowed，the mitochondria and rough endoplasmic reticulum disappeared，karyopyknosis was shown，nuclear membrane was ruptured，many vacuoles presented in the cytoplasm，and in more serious situation，the insect body collapsed and presented a homogenous change. The results of immunofluorescence showed that the expression of LC3 protein in the experimental group（77.49±15.68） was significantly higher than that in the control group（53.81±15.24）（P=0.001）. The results of immunohistochemistry showed that the expression of LC3 protein was significantly increased after treatment（1 208.55±580.07 vs. 544.54±225.04）（P=0.000）. The results of immunoblotting assay showed that after treatment，the expression of LC3 protein was significantly higher（41 738.52±0.00） than that in the control group（25 383.88±607.34）（P=0.000）. After treatment，the proliferation of Toxoplasma gondii Tachyzoites in the brain of mice was significantly less（218.80±21.17） than that in the control group（495.40±31.91）（P=0.000）. Conclusions：Auranofin can destroy Toxo－plasma gondii tachyzoites in vitro，and meanwhile promote the autophagy of mice infected with Toxoplasma gondii to inhibit the proliferation of Toxoplasma gondii.