Screening Of Anti-Toxoplasma Gondii Drugs From Three Spider Toxins And Preliminary Study Their Mechanism

[Objectives]To investigate the effect of three kinds of China-specific spiders (Ornithoctonus huwena, Chilobrachys jingzhao and Ornithoctonus hainana) toxins against Toxoplasma gondii(T.gondii) in vitro and in vivo experiments, and to study their therapeutic mechanism.[Methods]1. Cytotoxicity assay The cytotoxicity of the toxins from Ornithoctonus huwena, Chilobrachys jingzhao and Ornithoctonus hainana was assessed by using MTT assay. The median toxic concentrations of three kinds of spider toxins in Hela cells (50% toxic concentration, TC50) were calculated.2. In vitro experiments2.1. Treatment before infectionFive different concentrations of three kinds of spider toxins were performed in a 96-U-well plate using two-fold serial dilutions. T. gondii tachyzoites were pretreated for 1h with these solutions at different concentrations or with medium alone and then incubated with cultured cells for 24 h. The inhibition of spider toxins to T. gondii tachyzoites invasion and intracellular replication was examined.2.2. Treatment after infectionAfter 3h of incubation with tachyzoites, the cells were treated with solutions at different concentrations previously described for 24h, and then were examined by the same procedures used for the first set of experiments.2.3. Time-effect relationshipAfter Hela cells were infected with T. gondii tachyzoites, these cells were incubated with spider toxins of the same concentration. The inhibition of T. gondii tachyzoites invasion to these cells was examined at different treatment times.3. In vivo experimentsTwo hours after the mice were infected with T. gondii tachyzoites by intraperitoneal inoculation, three kinds of spider toxin were also taken into the body of these mice intraperitoneally, with once per day for 7 days. Daily diet, activity, incidence and death time and mortality of the mice were recorded.[Results]1. MTT assay shows that the descending order of cytotoxicity of three kinds of spider toxins are Chilobrachys jingzhao toxins (JZTX)(TCso= 32.7μg/ml), Ornithoctonus huwena toxins(HWTX) (TC50= 41.2μg/ml) and Ornithoctonus hainana toxins (HNTX) (TC50= 109μg/ml).2. When the concentration of JZTX is 32.7μg/ml and 4.1μg/ml, the infection rates of T. gondii tachyzoites to cultured cells at the condition of pretreated T. gondii are 14.3% and 25.7%, respectively; the infection rates of T. gondii tachyzoites to cultured cells are 11.9% and 25.5%, respectively, when T. gondii is treated after cultured cells are infected, and both treatment conditions are lower than the control group(P<0.05); Moreover, under treatment conditions above, when the concentration of JZTX is 32.7μg/ml, the mean numbers of tachyzoite per parasitophorous vacuole are lower than those of the negative control group (P<0.05). When the concentration of HWTX are 41.2μg/ml and 10.3μg/ml, the infection rates of T. gondii tachyzoites to cultured cells at the condition of pretreated T. gondii are 16.4% and 23.9%, respectively; the infection rates of T. gondii tachyzoites to cultured cells are 15% and 23.9%, respectively, when T. gondii is treated after cultured cells are infected. In addition, compared with the control group, the infection rates of T. gondii tachyzoites of HNTX to cultured cells show no significant difference at the same conditions previously described. 3. When cultured cells are treated with spider toxins for 24 h, the infection rates of T. gondii to these cells are 40.8%(the negative control group),20.2% (JZTX group) and 20.5%(HWTX group), respectively. For 48 h, the infection rates of T. gondii to these cells are 85%(the control group),13%(JZTX group) and 36.3%(HWTX group), respectively.4. In vivo experiments indicated that the average survival days of the mice treated with JZTX (longer than 10.5 d) and with HWTX (longer than 14 d) were both longer than those of untreated group infected with T.gondii tachyzoite (P>0.05and P<0.05, respectively).[Conclusion]In vitro experiments demonstrate that both JZTX and HWTX are able to inhibit T.gondii tachyzoites invasion to the tested cells, and the inhibition effect correlates positively with the concentration of spider toxins. In vivo experiments show that the survival days of the infected mice treated with HWTX are significantly increased, and that HNTX have no significant therapeutic effect on infected hela cells.