Preliminary Study On The Effect And Mechanism Of Auranofin On Toxoplasma Gondii

Objective: To study the Mechanism of auranofin inhibiting the Proliferation of Toxoplasma gondii.Methods: The MB-231 cells infected with Toxoplasma gondii were divided into 3 groups with Auranofin(control,10μg/ml and 20μg/ml group).The Cell count method was used to observe proliferation of Toxoplasma gondii RH strain.Intracellular ROS levels were measured by Flow cytometry relying on the DHE for superoxide,Cal-biochem and CM-H2 DCFDA for H2O2.The transmission electron microscopy(TEM)was used to observe the ultrastructure of Toxoplasma gondii after treating by Auranofin.The level of LC3 in the brain of mice infected with toxoplasm was measured by immunofluorecence,immunohistochemical and Western blotting.The changes of CD4+and CD8+ T lymphocyte subsets were detected by flow cytometry.The changes of Toxoplasma gondii tachyzoites in the brain of mice were observed by HE staining.Results: The results of cell count showed that the number of Toxoplasma gondii tachyzoites in the experimental group(20.8±3.96×104/ml)was significantly lower than that in the control group (102.8±5.8×104/ml).The content of reactive oxygen species of test group was evidently higher than that of control group.Ultrastructural observations showed the internal structure of Toxoplasma gondii was destructed more serious(The body cell membrane rupture,cytoplasm and uniform change,the contents overflowed,mitochondria and rough endoplasmic reticulum disappeared,nuclear pyknosis,nuclear membrane rupture and cytoplasmic vacuolization appeared.In some severe cases,the disintegration of the body was homogeneous.The effect of 20μg/ml concentration group(group C)more obvious than that of 10μg/ml(group B)concentration group with Auranofin.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=0.000).The results(1208.55 ±580.07)of immunohistochemistry showed that the expression of LC3 protein was significantly higher than that before treatment(544.54 ±225.04)(p=0.000).The results of immunoblotting assay showed that the expression of LC3 protein was significantly higher(41767.39±40.83)than that before treatment(21800.33 ±783.61)(p = 0.001).The proliferation of Toxoplasma gondii Tachyzoites in the brain of mice was significantly less(218.8±21.17)than that before treatment(495.4 ±31.91)(p=0.000).The expression of CD4,CD8 and CD4/CD8 were increased in AURANOFIN group.Conclusions: AURANOFIN could not only kill Toxoplasma gondii directly in vitro,but also suppress Toxoplasma gondii proliferation by improve the effect of autophagy and inhibit the proliferation of Toxoplasma gondii by increasing the immune function of body.