RESUMO
Objective: To investigate the killing effect of hypericin on tachyzoites of Toxoplasma gondii RH strain in vitro. Methods: Normal saline ï¼group Aï¼ and different concentrations of hypericin (5 µg/ml, group B; 50 µg/ml, group C; 500 µg/ml, group Dï¼ were added to T. gondii tachyzoites in 24-well plateï¼1×10(6)/wellï¼. The tachyzoites were harvested after 2, 4 and 6 h, and underwent the following treatment: trypan blue staining to calculate the dyeing rate, Giemsa staining to observe the morphological and structural alterations of tachyzoites, and transmission electron microscopy to observe the ultrastructure of tachyzoites. In addition, flow cytometry was performed to calculate the survival rate of YFP-carrying Toxoplasma with the same treatment. Results: The trypan blue dyeing rate at 2 h after treatment in groups B, C and D wasï¼11.0±3.6ï¼%, ï¼25.0±6.3ï¼% andï¼40.0±2.7ï¼% respectively, with a significant difference of group D versus B and C ï¼P<0.01ï¼, and groups C and D versus group A ï¼»ï¼6.0±3.0ï¼%ï¼ï¼½. The dyeing rate at 4 h and 6 h in group D wasï¼97.0±2.0ï¼% and ï¼98.0±1.7ï¼%, respectively, both significantly higher than that of groups C ï¼»ï¼30.0±7.2ï¼%, ï¼42.7±5.5ï¼%ï¼½, B ï¼»ï¼20.0±3.0ï¼%, ï¼34.0±6.6ï¼%ï¼½ and A ï¼»ï¼10.0±1.0ï¼%, ï¼19.3±4.9ï¼%ï¼½ï¼P<0.01ï¼. Giemsa staining showed gradual end swelling and necrosis of tachyzoites with increased treatment duration and dosage. Transmission electron microscopy showed swelling of worm body, gap between cell membrane and matrix, increase and enlargement of vacuoles inside worm body, disruption of cell membrane, and dissolving of inner structures, with increased treatment duration. Flow cytometry showed significant difference of tachyzoite survival rate at 2, 4 and 6 h after hypericin treatment with that of the control groupï¼P<0.01ï¼. The survival rate of group C at 2 h after hypericin treatment wasï¼7.9±1.9ï¼%, significantly lower than that of groups B ï¼»ï¼38.1±5.5ï¼%ï¼½ and A ï¼»ï¼81.8±6.0ï¼%ï¼½ ï¼P<0.01ï¼. No tachyzoite was found to survive in group D at 2 h and in group C at 4 h. The survival rate of group B at 4 and 6 h after hypericin treatment wasï¼14.3±7.9ï¼% and ï¼1.4±1.8ï¼%, respectively, both significantly lower than that of group Aï¼»ï¼73.8±11.3ï¼% andï¼64.1±14.4ï¼%, respectivelyï¼½ ï¼P<0.01ï¼. Conclusion: Hypericin has a remarkable killing effect on T. gondii tachyzoites, and the efficacy positively correlates with the dose and treatment duration.