Effection And Mechanism Of Pulsatilla Saponin D On Schistosoma Japonicum And S.Mansoni

Part 1 Study on the effect of PSD against Schistosoma japonicumObjective:To observe schistosomicidal effects of（Pulsatilla saponin D,PSD）on different age and gender of Schistosoma japonicum in vivo.Methods:Divide the ICR mice which were infected with the cercariae of Schistosoma japonicum into two batches randomly,each batch has six groups and ten mice in each group.The first batch were given PSD 14d after infection,the second batch were given PSD 35d after infection.Three groups were given different doses of PSD by tail vein injection in both 14-18d batch and 35-39d batch.The drugs which were divided into 10mg/kg、20mg/kg and 40mg/kg were given twice a day.The mice in（Praziquantel,PZQ）group were given PZQ of the does of 300 mg/kg orally at once.The mice in artesunate（Artesunate,ART）group were given artesunate of the does of 300mg/kg orally at once.The mice in the control group didn’t receive treatment after infection.After 49 days,remove the mice’s eyes and collect the blood.After the mice were dissected,collect the worms,calculate the worm reduction rate and the female worm reduction rate.Use 4%potassium hydroxide to digest the liver and calculate the egg reduction rate.Results:The average number of worms in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The worm reduction rate in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The average number of female worms in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The female worm reduction rate in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The average number of eggs in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.Conclusion:Different doses of PSD（10mg/kg,20mg/kg,40mg/kg）have the killing effects on the Schistosoma japonicum of different developmental stages in vivo.In the same growth period,different doses of PSD have effects on mice with Schistosoma japonicum and high dose（40mg/kg）group has the most effective schistosomicidal effect.At the same dose condition,PSD has a better killing effect on35-day-old worms than 14-day-old worms.In the treatment groups of different doses of all,the female worm reduction rate is higher than the worm reduction rate.Compared with the untreated group,the number of females decreased obviously.Part 2 Study on the effect of PSD against Schistosoma mansoniObjective:To observe the schistosomicidal effects of PSD on different age and gender of Schistosoma mansoni in vivo.Methods:Divide the mice which were infected with the cercariae of Schistosoma mansoni into two batches randomly,each batch has four groups and ten mice in each group.The first batch were given PSD 14d after infection,the second batch were given PSD 42d after infection.The mice were given PSD of the single dose of 40mg/kg by tail vein injection in both 14-18d batch and 42-46d batch.The drug was given twice a day.The mice in PZQ group were given PZQ of the does of 300 mg/kg orally at once.The mice in ART group were given ART of the does of 300 mg/kg orally at once.The mice in the control group didn’t receive treatment after infection.After 56 days,remove the mice’s eyes and collect the blood.After the mice were dissected,collect the worms,calculate the worm reduction rate and the female worm reduction rate.Use 4%potassium hydroxide to digest the liver and calculate the egg reduction rate.Results:The average number of worms in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The worm reduction rate in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The average number of female worms in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The female worm reduction rate in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.The average number of eggs in PSD groups was different from the control groups significantly（*P<0.05,**P<0.01）.Conclusion:The single dose of PSD（40mg/kg）has killing effect on the Schistosoma mansoni of different developmental stages in vivo.PSD has a better killing effect on 42-day-old worms than 14-day-old worms.In all the treatment groups of the single dose（40mg/kg）,the female worm reduction rate is higher than the worm reduction rate.Compared with the untreated group,the number of females decreased obviously.PSD can significantly reduce the number of worms and female worms which are 42-day-old in the mice.Part 3 Preliminary study on the immune regulation and schistosomicidal effect of PSD on the mice infected with Schistosoma japonicum.Objective:Section the liver tissue to observe the effect of PSD on the hepatic tissue pathology of mice infected with Schistosoma japonicum in optical microscope.To detect the dynamic changes of related humoral immune factors,and then explore the effect of PSD on inflammatory reaction caused by schistosomiasis japonica.Also,to observe the morphological change of Schistosoma japonicum epiderm by scanning electron microscopy and study the mechanism of action of PSD on Schistosoma japonicum.Methods:Divide the ICR mice which were infected with the cercariae of Schistosoma japonicum into two batches randomly,each batch has six groups and ten mice in each group.The first batch were given PSD 14d after infection,the second batch were given PSD 35d after infection.Three groups were given different doses of PSD by tail vein injection in both 14-18d batch and 35-39d batch.The drugs which were divided into 10mg/kg,20mg/kg and 40mg/kg were given twice a day.Section the liver tissue to observe the effect of PSD on liver granuloma in optical microscope.Using sandwich ELISA method to determine IgG,TNF-alpha,IL-4 and IL-17 in serum.Pour 0.3%liquor sodii citratis into ICR mice which were infected with 30cercariae after 42d,obtaining sterile adult worms of Schistosoma japonicum.Culture adult worms of Schistosoma japonicum with the 4ml RPMI 1640 culture medium containing 10%fetal bovine serum,100 U/ml penicillin,100μg/ml streptomycin and80μl rabbit red blood cells.After 12 hours,replace different concentrations of PSD（3.75,7.5,15,30,60μg/ml）then culture adult worms of Schistosoma japonicum in the culture medium for 72 hours.Culture all the worms in the environment of 5%CO₂ and95%air at 37℃for 7 days.Change the culture medium every 3 days and observe the killing effect of PSD on the adult worms of Schistosoma japonicum in vitro.Culture the sterile adult worms of Schistosoma japonicum in the culture medium containing 60μg/ml PSD for 4 hours,and then place them in 10%glutaraldehyde buffer at 4℃for 4hours,and then in PBS（1%）at 4℃for 2 hours.After the worms were washed by PBS,dehydrate them in ethanol and dry them in liquid CO₂.Finally,scan the specimens with electron microscope to observe the influence of PSD on the epidermis of worm body.Results:The organizational structure of mice’s liver changed obviously which were infected with Schistosoma japonicum and didn’t receive treatment.The results showed the inflammatory infiltration and a large number of granuloma as morphological characteristics.Compared with the untreated group,the size of hepatic granulomatous inflammation treated by PSD were much smaller.Since the mice were treated by 20mg/kg PSD 14-18 days and 35-39 days after infection,the number of inflammatory cells in the liver was significantly reduced,the structure of hepatic lobule nearly back to normal tissue and most of the liver cells showed normal appearance.After determining the immune response of mice treated by PSD with the ELISA method,results showed that:compared with the infection group,20mg/kg PSD can effectively downregulate the expression of IgG,TNF-alpha,IL-4 and IL-17,which showed the best immune regulation effect.In addition,scanning electron microscope examination results showed that the epidermis of male worm body treated by PSD showing abnormal structure.Also,there were extensive edema,erosion and peeling between the nodule cortex area.Conclusion:20mg/kg and 10mg/kg of PSD can significantly reduce the inflammatory reaction of granuloma,inhibit fibrosis caused by Schistosoma japonicum egg and reduce damage of liver.Scanning electron microscope examination results showed that the surface damage of male worms was most severe in the 60μg/ml PSD treatment group.The damaged cortex fell off and exposed the subcutaneous tissue.These results show that PSD has the effect of anti schistosomiasis activity.