Biological Effects Of Praziquantel Derivatives Against S. Japonicum And Screening And Identification Of Differentially Expressed Proteins Of Praziquantel Resistant Parasites

Part1Praziquantel derivatives DW-3-15、P96and P96isomers exhibit activityagainst both juvenile and adult Schistosoma japonicum in vitroObjective：To study the killing effect of Praziquantel derivatives and P96isomersupon juvenile and adult worms(female/male) of Schistosoma S.japonicum in vitro.Method: ICR mice were infected with S.japonicum single-sex cercariae,Aftersixteen days or six weeks of infection, worms were obtained by portal vein perfusionfrom mice，then cultivated in DMEM medium containing with different concentrationsof DW-3-15、P96、P96isomers, Artesunate、praziquantel, then observed the death andmotility reduce rate.Results:In vitro test, juvenile worms incubated in DMEM medium containingDW-3-15or P96at the concentration of50μmol/L，33.3%or25.0%worms weresurvived， the same concentration of Artesunate role, the survival viability was42.9%.When anti-adult worms, the female and male survival rates were20.0%and41.9%at50μmol/L DW-3-15; respectively,all the males died and33.3%femalesurvivors after50μmol/L P96; PZQ15μmol/L can kill88.9%male worms, while thefemale worms were still37.8%survived.P96-3anti-adult worms at the concentration of100μmol/L, the survival rate was60.0%, when anti-juvenile at the concentration of50μmol/L, the survival rate was27.3%. Conclusion: PZQ derivatives DW-3-15and P96exhibit activity againstschistosomula japonicum in vitro，both better than Artesunate，the killing effect of P96on juvenile was the best. While against adult S.japonicum, derivatives against malesbetter than females,males are more sensitive than females to the derivatives.The killingeffect of9kinds of P96isomers were not as well as P96in vitro.Part2PZQ derivatives DW-3-15/P96exhibit activity against S.japonicum invivo in different animal modelsObjective：To prove the killing effect of two kinds of PZQ derivatives uponjuvenile and adult worms from mice and rabbit in vivo.Method:ICR mice and rabbit， different times after infection with S. japonicum,was given an oral administration of a dose of200mg/kg PZQ derivatives、Art and PZQfor5consecutive days.21days later the mice were killed and the worm reduction rateswere calculated. Rabbits were given different dose of P96(150mg/kg,300mg/kg) in the2nd week or4th week after infection，one week later,the same dose was given,then therabbits were killed at the10th week after infection by cardiac puncture, worm and eggreduction rate were calculated.Results:Mice experiments:Compared with control group, the death and motilityreduce rate of juvenile and schistosomal were obviously increased after two PZQderivatives’ treatment (P<0.05). The reduction rates were64.0%,56.2%and69.1%、70.4%while DW-3-15and P96against the3rd or14th day schistosomula, TheArtesunate’s killing effect on juvenile(3d,14d) were66.2%、67.4%, significantly higherthan PZQ group (22.0%,18.0%).The Dw-3-15、P96and Artesunate’s killing effect onadult worms were lower than PZQ（95.7%）with the death rate of81.1%.、82.8%and51.1%，no effective difference among the groups.9kinds of P96isomers administered ata dose of200mg/kg showed that the worm reduction of P96-3was60%whenanti-Schistosomula(14d)in vivo，it was similar to P96(76.4%)，with the function to killSchistosomula。Rabbits Experiments:When treated with P96at a dose of150mg/kg at the14th or28th day after infection for two times,respectively,worm reduction were25.3%and65.2%, egg reduction rate were30.5%and84.0%;while the dose add up to300mg/kg at the same time, worm reduction rates were62.2%and91.7%,egg reduction rates were86.2%and97.7%, significantly higher than150mg/kg P96’s treatment groups.Conclusion: Mice experiments showed that DW-3-15exhibit activity against both3-day-old and14-day-old juvenile and35-day-old adult S.japonicum in vivo.TheDW-3-15and P96’s killing effect on juvenile was equivalent to Artesunate’s whilesignificantly better than PZQ. Its Anti-adult effect was opposite. P96-3which was oneof9P96isomers had a killing effect on14d schistosomula, the structure-activityrelationship was worth further research.Rabbits experiments showed that high-dose therapy is better than low-dosetreatment, increasing the therapeutic dose, reduction rate and egg reduction rateimproved and showed a dose-dependent manner;especially the reduction rate reached91.7%after treatment with P96on the28th day,nearly to PZQ, indicating that P96exhibit activity not only on juvenile but also on adult S.japonicum in vivo in differentanimal models, it has the potential application value to become a new antischistosomiasis drug.Part3Real-time PCR evaluate the killing effect of P96by detecting the serumDNAlevels of rabbits infected with schistosoma japonicumObjective:To resreach the dynamic changes of serum DNA levels of rabbits thatinfected with S. japonicum and treated with P96,valuated its insecticidal effect.Results:Rabbits infected with200cercariae of Schistosoma japonicum were orallyadministered with P96(150mg/kg and300mg/kg) and PZQ (150mg/kg) at14d,28dfollowing infection respectively. The highest concentration of DNA in two therapeuticdoses of P96after treatment were267.8,299.5(copy number), were higher than PZQgroup (249.5)(copy number), suggesting that P96was better than PZQ on the effect ofkilling schistosomula;However， the highest concentration of DNA were495.7、1049.5、1222.4respectively when the infected rabbits were treated with P96(150mg/kg、300mg/kg) and PZQ（150mg/kg）in the fourth week，the results showed that the killingeffect of P96was similar to PZQ when the dosage of300mg/kg.Different doses of P96can kill the worms,when treated adult worms showedthat the DNA in high-dose group(1049.5) was significantly higher than low-dosegroup(495.7);when treated schistosomula,although no significantly difference between the two groups,the DNA detected in high-dose group was still higer than low-dosegroup.The results indicates that P96’s killing effect has an apparent dose-dependenteffect.Conclusion:Real-time PCR for detecting serum DNA of rabbits infected withschistosome can evaluated the effect of drugs,further evidence of the P96insecticidaleffect in vivo,and the results showed that P96was better than PZQ while killingschistosomula and P96has an obvious dose-dependent effect.Its killing effect wasconsistent with PZQ at the dosage of300mg/kg.P96has the potential value as newanti--schistosomiasis candidate drug.Part4Induce resistance of S.japonicum with administration of PZQ-pressureand proteomics analysis of differentially expressed proteinsObjective:To screen and identify the differentially expressed proteins betweeninduced worms (the infected mice were treated intragastrically with ED50of PZQ)anduninduced worms;To clarify the mechanism of PZQ, further explore new vaccinecandidates and drug therapeutic targets and provide experimental evidence.Method:PZQ ED50was used to administrate mice that were infected byS.japonicum via intragastric for consecutive30days,21days later,given PZQ at the doseof200mg/kg for continuously five days,to observe the sensitivity of induced wormsagainst PZQ and PZQ derivatives.Collect the induced worms and normal worms, thetotal proteins were determined by2D-DIGE,then the diffentially expressed proteinswere identified by MALDI-TOF-MS and NCBI and combine with bioinformatics toanalyse the function of the proteins.Results:The induced worms were significantly decreased in sensitivity to PZQ,thesurvival rates were87.5%、82.0%、77.3%and75.6%respectively at the concentration of14μmol/L、28μmol/L、56μmol/L�'�112μmol/L，had no obviously difference comparedwith control group.When the concentration of PZQ was add up to112μmol/L(8times),75.56%worms were still survived. The induced worms showed significanttolerance to PZQ.In addition,the induced worms decreased in sensitivity to PZQderivatives DW-3-15and P96,the survival rates were95.83%and86.67%respectivelyat the concentration of15μmol/L and25μmol/L,while increase the concentration to fourtimes of the uninduced worms critical lethal concentration,the survival rates were10% and20%,significantly diffence with PZQ group （survival rate was75.56%at112μmol/L）(p<0.05).The tolerance of induced worms to PZQ derivatives was lowerthan PZQ,suggesting that the targets of PZQ derivatives and PZQ are different.34proteins spots were isolated by2D-DIGE and MALDI-TOF-MS,30differentially proteins were considered suitable for further study by MASCOT andNCBI,including12proteins up-regulated and18proteins down-regulated in the inducedgroup compared with the normal group These proteins respectively ascribed tocytoskeleton-associated protein, glucose and energy metabolism enzymes, stressproteins and thioredoxin peroxidase enzymes.Conclusion:The induced worms decreased in sensitivity to PZQ significantly，showed obviously tolerance.The tolerance of induced worms to PZQ derivatives waslower than PZQ,suggesting that the targets of PZQ derivatives and PZQ may bedifferent. Proteomic analysis showed there are partial differential expression proteinsbetween induced worms and uninduced worms.Up or down regulation of these diffentialproteins indicating that PZQ promote or inhibit the expression of some specificgenes.Screening and anlysis of the diffential expression proteins between inducedworms and uninduced worms,may help us clarify the machanism of PZQ,， whileproviding a scientific basis for exploring new vaccine candidate antigens and targets fordrug therapy.