[In vitro observation on the apoptosis induced by H2O2 in protoscolex of Echinococcus granulosus].

Objective: To explore the apoptosis induced by hydrogen peroxide (H2O2) in protoscolex of Echinococcus granulosus.

Methods: Protoscoleces were cultured in vitro, and used for the experiment in 2 groups: RPMI 1640 medium and RPMI 1640 medium added with glutamine. They were then treated with different concentrations of H2O2 to induce apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) was employed to observe the apoptosis. Protein expression of caspase-1, caspase-3 and Fas was detected by SP immunohistochemical technique, stained with DAB restained with hematoxylin. A yellow or brown color nucleus revealed positive apoptosis cells in protoscolex, a brown reaction product in cytoplasm showed positive cells of caspase-1 and caspase-3, and brown cell membrane and cytoplasm revealed Fas product; otherwise it was judged as negative. According to the percentage of positive cells in a protoscolex, the expression level was divided as 4 grades. The percentage of less than 5% was regarded as "-", 5%-25% as "+", 26%-50% as "++", more than 50% as "+++". The experiments were repeated 2 times with controls.

Results: In RPMI 1640 group, positive TUNEL was found in the protoscolex induced by 1 mmol/L H2O2 inducing for 12 hours. Induced by 1 mmol/L H2O2 for 4 h, the "+-++" expression rate of caspase-1 and caspase-3 in the protoscoleces was 86.6% and 77.8%, and for 8 h, 86.6% and 80.0% respectively, a significant increase in comparison to the control (P<0.05). Induced by 5 mmol/L H2O2 for 4 hours, the "++-+++" expression rate of caspase-1 was 93.0%, and the "+-++" expression rate of caspase-3 was 89.5%; induced for 8 h, the "++-+++" expression rate of caspase-1 decreased to 53.2%, and the "+-++" expression rate of caspase-3 decreased to 48.4% and "-" expression rate increased to 46.8%. Under 5 mmol/L H2O2 for 4 h the expression rate of caspase-3 significantly decreased at 8 h (P<0.01). In the group of RPMI 1640 plus glutamine, induced by 5 mmol/L H2O2 for 8 h, the "++ -+++" expression rate of caspase-3 in protoscolex was 100% (P<0.01). However, in RPMI 1640 group, induced by 5 mmol/L H2O2 for 8 h, the "++-+++" expression rate of caspase-3 in protoscolex was 32.2% and 46.8% were negative. The Fas product with positive reaction in protoscolex was found in both control and induced groups: in RPMI 1640 group, under 5 mmol/L H2O2 induced for 4 h, "+-++" expression rate was 53.0% and control was 20.0%; and in the group of RPMI 1640 plus glutamine, under 5 mmol/L H2O2 induced for 8 h, "+-++" expression rate was 88.7% and control was 71.4%, increased in both groups after induction (P <0.05).

Conclusion: Apoptosis in the protoscolex of E. granulosus may be induced by H2O2.