| Objective:(1) To selecte S .japonicum of decreased PZQ sensitivity and observe ultrastructural alterations of worms under drug pressure.(2) Investigate four kinds of Diazepam derivatives(B3,B30,B3-O2 and B3-2O2) and three kinds of 5-HT receptor/ dopamine antagonists(risperidone,olanzapine,quetiapine fumarate) activity on anti-schistosoma.Methods:(1) Infection of experimental animals: S. japomicum cercariae released from laboratory infected snails, were kindly provided by Institute of Parasitic Diseases of Jiangsu province. Mice were infected with 60±2 S. japonicum cercariae each, through the shaved abdominal skin(single-sex infection).(2) Collect adult worms: All groups of animals were sacrificed by cervical dislocation after the final treatment. In mice, worms were collected by the perfusion technique. Only male worms (usually 5-6) were distributed in duplicate tissue culture dishes (3.0 cm) in DMEM supplemented with 20% newborn calf serum. Dishes were kept at 37 ?C in an atmosphere of 5% CO2 in air.(3) Motility criteria: Motility was scored using the following criteria: 3 (moving whole body); 2 (moving only parts of body); 1 (immobile but not dead, unstained with vital dye); and 0 (immobile, stained with vital dye).(4) A batch of mice were infected with cercariae and six weeks later mice were randomly divided into seven groups (usually 10 mice per group) each of which was treated with subcurative doses of PZQ. Treatments were repeated for 5,10,20,30,60,90 days (1 time/per day),10 days (1 time/2 days) and mice were perfused three weeks after the initial treatment.(5) 3 weeks and 6 weeks after infection of S. japonicum, the mice were treated intragastrically with subcurative doses of PZQ for 30 consecutive days. The male adult worms were obtained by perfusion of mice three weeks after the initial treatment, then these worms were subjected in vitro in the medium with different PZQ concentrations. After incubation overnight (about 16 hours later), the worms were washed and resuspended in drug-free medium and observed for the subsequent 5 days.(6) Mice, 6 weeks after infection with S. japonicum, was induced with subcurative doses of PZQ for 30 consecutive days. The selected-worms were treated with 200 mg/kg PZQ repeatedly for 5 consecutive days. The survived worms were cultured in the medium with different PZQ concentrations and were observed for the subsequent 5 days.(7) In vitro test, adult male worms kept in medium were exposed overnight to four kinds of Diazepam derivatives(B3,B30,B3-O2 and B3-2O2) and 5-HT receptor/ dopamine antagonist(risperidone,olanzapine,quetiapine fumarate) with different concentrations. After that, worms were washed and resuspended in drug-free medium, then were observed during the following 5 days.(8) Preparation of specimen for scanning electron microscopy analysis: Worms were rinsed 3 times with physiological saline and fixed in 2.5% glutaraldehyde(pH 7.4) at 4°C for 24 hours, then worms were washed with phosphate( 0.2 mol/L, pH 7.4) two times and post-fixed in 1% osmium tetroxide for 1 hour. After that, the worms were dehydrated through a graded series of tert-butyl alcohol and stored at -20°C overnight. After drying, the worms were mounted on aluminium stubs and coated with platinum and paladium in an ion-sputtering apparatus, IB-5 (EIKO) for 10min. Then the worms were examined and photographed in a Hitachi scanning electron microscope S-570 (EDAX Japan).Results:(1) Seven groups of mice were treated intragastrically 6 weeks after infected with S. japonicum by the subcurative doses of PZQ in different days. After selecting for 30 consecutive days, the surviving worms'sensitivity to PZQ was significantly decreased. These findings showed that the treatment with subcurative doses for 30 consecutive days was the suitable time to select worms of sensitivity decreased. (2) The mice infected with S. japonicum 3 weeks and 6 weeks later were treated intragastrically with subcurative doses of PZQ for 30 consecutive days. Both of the surviving worms had the lower sensitivity to PZQ than the original unselected worms, the parasite sensitivity decreased more obvious when mice were treated in 3 weeks after infection. The damages of ultrastructure in the tegument and gynecophoral canal in the selected-worms were slighter compared with the worms from untreated mice by SEM.(3) While the selected-worms were repeatedly treated with 200 mg/kg PZQ for 5 consecutive days, the survived worms cultured in the medium with different PZQ concentrations were still alive completely. The damages of ultrastructure in the tegument and gynecophoral canal in the repeated treatment selected-worms were slighter compared with the worms from the mice after incubating to drug pressure for 30 days.(4) In vitro test, adult worms incubated in DMEM medium containing B3,B30 at concentrations of 50μmol/L were all killed; Worms cultured in risperidone have low survival rates.Conclusion:(1) After exposure to subcurative doses of PZQ for 30 consecutive days, the sensitivity of the survived worms in different stages to PZQ decreased, especially 3-week-old worms were strikingly refractory to the drug, which provide a foundation for designing respective clinical trials in humans.(2) Diazepam derivatives, B3 and B30 on S. Japonicum have showed its effect of killing worm; while 5-HT / dopamine receptor antagonist risperidone has a little effect on worms. These results would be helpful for further development of new broad-spectrum anthelminthics. |
PDF Full Text Request