| Schistosomiasis japonica is a serious,parasitic disease which is mainly present in Asia.The intermediate host of S.japonicum is Oncomelania hupensis.Cercariae emerge from the snail and enter the water.People infect with S.japonicum cercariae by water contact.Adult worms pair off and lay eggs.Eggs deposited in the tissue can result in chronic hepatic fibrosis.Nowadays,drug chemotherapy is the main control of schistosomiasis in endemic areas.Praziquantel has been recommended by the WHO as its efficacy against all Schistosoma species.Long-term,mass deployment of PZQ may serve to accelerate drug resistance or tolerance.S.mansoni and S.japonicum strains showing resistance or insensitivity to PZQ have been reported in laboratories and sporadic reports of reduced susceptibility have come from the field.Consequently,novel antischistosomal candidate should be procured in advance of PZQ inefficacy.DW-3-15(patent NO:ZL201110142538.2),a hybrid compound linked to the endoperoxide bridge of artemisinin at position 10 of PZQ,was developed in our laboratory.In the previous studies,DW-3-15 was shown to be effective against all developmental stages of S.japonicum in vivo.In the present study,synthesis of DW-3-15 was performed by a pharmaceutical company,and the schistosomicidal efficacy and stability of the compound were further confirmed.Moreover,proteomic analysis and polyclonal antibody preparation were used to evaluate the changes of protein level and transcription level in S.japonicum worms after treatment with DW-3-15.Part one.Lethal effect of praziquantel derivative DW-3-15 against Schistosoma japonicum in vitroObjective:To explore the lethal effect of DW-3-15 on different developmental stages of S.japonicum in vitro.Methods:1.The mice were individually infected transcutaneously with S.japonicum cercariae.Worms recovered from infected mice at 14 days(juvenile worms),21 days(juvenile worms),and 35 days(adult worms)post-infection were collected through perfusion of the hepatic portal system and mesenteric veins using citrated saline.Five adult worms or eight juvenile worms per well were incubated with 3mL DMEM,and adult worms separated by sex.The final concentrations of DW-3-15 were 50-100?mol/L.Adult and juvenile worms were exposed to the drugs for 24 h,rinsed three times with DMEM the next day,and subsequently cultured in drug-free DMEM for 72 h.The worms were observed at 24,48,72,and 96 h post-incubation and assigned a viability score.After 96 h of exposure to 100?mol/L DW-3-15 and 100?mol/L PZQ in vitro,samples of adult worms were fixed overnight and examined under scanning electron microscope.2.One pair of adult worms per well were incubated with 50-100?mol/L DW-3-15.The percentage pairing was examined at 1,6,12,and 24h.Moreover,eggs laid in the well were collected and counted after 24 and 96 h of incubation.Results:1.Higher concentrations of DW-3-15 resulted in significantly reduced viability in male,female,and juvenile stages at 24,48,72,and 96h(all P<0.001).The lethal effect of DW-3-15 was concentration-dependent,with DW-3-15 at 100?mol/L showing the most potent effect.From 24 h to 96 h periods,DW-3-15 reduced the viability scores of female and male by 91.1-97.8%and 93.3-96.3%,with the reductions in the viability scores of 14-day-old worm and 21-day-old worm by 91.7-94.4%and 95.4-99.1%,respectively.The viability scores of male and female worms incubated with 100?mol/L DW-3-15 were close to these in the PZQ group,while the viability scores of 14-day-old worm and 21-day-old worm incubated with DW-3-15 at 100?mol/L were significantly lower than these in the PZQ group(all P<0.05).Moreover,all adult worm pairs separated within 24 h of incubation with DW-3-15 at 50-100 ?mol/L.In contrast to the negative control,egg output of adult worm pairs cultured with DW-3-15 at 50-100 pmol/L for 24 or 96 h was significantly lower(P<0.001).2.After 96 h of DW-3-15 treatment(100 ?mol/L),females and males shortened with rod-type body shape showed a complete loss of movement and sucking capacity.Both male and female worms exposed to PZQ became curled,paralyzed,and several worms showed some signs of recovery.Contracted phenotype of adult worm exposed to DW-3-15 seemed stiff.21-day-old worm treated with 100?mol/L DW-3-15 presented loss of movement and opaque,while 14-day-old worm became shortened and vacuolar changes occurred in the body.Juvenile worms(14 days and 21 days)incubated with 100?mol/L PZQ moved normally.3.Females and males exposed to 100?mol/L DW-3-15 showed significant ultrastructural changes in the tegument.Blisters and hole-shaped erosions were observed,as well as destruction of crests and extensive sloughing of the tegument with exposure of the sub-tegument layer of muscle tissue and even damage to the muscle tissue layer.Females and males exposed to 100?mol/L PZQ showed various changes in the tegument,with destruction of crests,shallow peeling,and pit-shaped erosion.The changes were confined to the tegument surface of the worm,with no damage observed in the muscle tissue layer.Conclusion:DW-3-15 showed a pronounced lethal effect against both adult and juvenile S.japonicum worms in vitro,namely activity against adult worm similar to that of PZQ,and the effect against juvenile worm superior to that of PZQ.Part two.Antischistosomal activity of praziquantel derivative DW-3-15 against Schistosoma japonicum in vivoObjective:To investigate the antiparasitic effect of DW-3-15 on S.japonicum in vivo.Methods:1.The mice were individually infected with approximately 60 S.japonicum cercariae.DW-3-15 was administered at doses of 100-400 mg/kg per day,35 days after S.japonicum infection.The positive control received PZQ at 100-400 mg/kg per day.Worms were collected from all groups after five consecutive days of treatment.The study was conducted to evaluate the effect of DW-3-15 on worm burden,liver weight/body weight,spleen weight/body weight,egg burden,and diameter of granulomas.2.The mice were individually infected with approximately 60 cercariae.DW-3-15 was administered at a dose of 400 mg/kg per day,14 days after S.japonicum infection.The positive control received PZQ at 400 mg/kg per day.Worms were collected from all groups by perfusion after 21 days of treatment.Worm burden,liver weight/body weight,spleen weight/body weight,egg production,and diameter of granulomas were assessed.Results:1.In vivo the oral administration of DW-3-15 at doses of 100-400 mg/kg significantly reduced the total worm burden and number of eggs in the liver(all P<0.05).400 mg/kg DW-3-15 treatment led to a 64.2%reduction in total worm burden and a 86.2%reduction in eggs numbers.Treatment with PZQ at 400 mg/kg led to 98.8 and 93.5%reductions in total worm burden and egg numbers,respectively.These reductions were higher than those induced by the DW-3-15 treatment.In addition,200-400 mg/kg DW-3-15 treatment ameliorated lesions of liver and spleen,and resulted in the reductions in the liver weight/body weight and the spleen weight/body weight,respectively(all P<0.05).Histological analysis of the livers showed a marked reduction in the average granuloma diameter after treatment with 400 mg/kg DW-3-15(P<0.01).2.The administration of DW-3-15 resulted in significant reductions in total worm burden(77.0%)and eggs numbers(95.4%),respectively(all P<0.05).Treatment with PZQ at 400 mg/kg led to 17.4 and 14.9%reductions in total worm burden and egg numbers,respectively.These reductions were lower than those induced by the DW-3-15 treatment.There were statistically significant reductions in the liver weight/body weight,the spleen weight/body weight,and the average granuloma diameter in the groups treated with DW-3-15 than in the untreated control groups(all P<0.001).Conclusion:Our findings further confirm that DW-3-15 not only possesses antiparasitic activity,but also attenuates egg-induced hepatic granulomas and fibrosis in vivo,and can be developed as a potential promising schistosomicide.Part three.TMT based proteomic analysis of adult S.japonicum worms after treatment with DW-3-15 in vivoObjective:To gain insight into the mechanism of action for DW-3-15 against shistosomiasis japonica.Methods:The mice were individually infected transcutaneously with approximately 60 S.japonicum cercariae.DW-3-15 was administered at a dose of 400 mg/kg per day,35 days after S.japonicum infection.Females and males were collected from all groups by perfusion after five consecutive days of treatment,and the total proteins of worms were extracted.TMT-coupled LC-MS/MS was firstly used to investigate the proteome obtained from females and males after treatment with DW-3-15.By aid of database searching,bioinformatics information of differentially expressed proteins of females and males were analyzed.Results:After treatment with DW-3-15,407 differential proteins were identified in females.187 proteins upregulated and 220 proteins downregulated were mainly associated with translation,ribosome,and protein processing in the endoplasmic reticulum.After treatment with DW-3-15,41 differential proteins were identified in males.30 proteins upregulated and 11 proteins downregulated were mainly associated with calcium ion binding and lysosome processing.Conclusion:This study helps elucidate the mechanism of action for DW-3-15 against S.japonicum,and provides new insights into the potential drug targets for schistosomiasis.Part four.Preparation of polyclonal antibody of Schistosoma japonicum histone acetyltransferase(SjHAT)and the effect of DW-3-15 on SjHAT in adult worm in vivoObjective:To express recombinant SjHAT and prepare anti-SjHAT polyclonal antibody and explore the effect of DW-3-15 on SjHAT in adult worm in vivo.Methods:1.After optimizing the codon,the gene sequence of SjHAT was synthesized chemically and cloned into a vector petB2M.The recombinant plasmid petB2M-SjHAT was transformed into E.coli Rosetta(DE3)for expression.Highly purified rSjHAT protein was obtained from the form of inclusion body.2.New Zealand rabbits were immunized with the rSjHAT protein mixed with an adjuvant.The titer of anti-SjHAT polyclonal antibodies and rSjHAT-specific IgG antibodies in serum were evaluated by ELISA.3.Western blotting and real-time PCR analyzed expression levels of SjHAT in female and male worms after treatment with 400mg/kg DW-3-15 in vivo.Results:1.Recombinant plasmid petB2M-SjHAT was successfully constructed and highly expressed in E.coli Rosetta(DE3).The titer of anti-SjHAT polyclonal antibody was over 105,and rSjHAT induced high level of specific IgG antibodies in vaccinated rabbits.2.Western blotting analysis showed that anti-SjHAT polyclonal antibody(approximately 50kDa)probed with the total proteins extracted from female and male worms,respectively.The expression level of SjHAT in female worms was higher than that of males in the control group(P<0.05).After treatment with DW-3-15,the expression level of SjHAT in female worm was significantly decreased(P<0.05).QPCR analysis showed that the transcription level of SjHAT in female worm was higher than that of male worm in the control group,and DW-3-15 treatment significantly reduced the transcription level of SjHAT in female worm(all P<0.05).Conclusion:Our preliminary findings provide a basis for the development of SjHAT as a potential target of DW-3-15 against S.japonicum.In summary,DW-3-15,a commercialized PZQ derivative,exerted schistosomicidal effect on adult and juvenile S.japonicum worms.Notably,DW-3-15 induced clear damage to the tegument of adult worm similar to PZQ,and ameliorated egg-induced granuloma and fibrosis in the liver.After treatment with DW-3-15,TMT quantitative proteomics identified differential protein profiles of female and male worms,respectively.The anti-SjHAT polyclonal antibody was successfully prepared,and the transcription level and the protein level analysis suggested that SjHAT can be developed as a potential target of DW-3-15 against S.japonicum.The present work strongly encourages the development of DW-3-15 as a promising candidate drug for treating schistosomiasis japonica,and preliminary explores the mechanism of action for DW-3-15 against S.japonicum,and provides valuable information for screening potential drug targets for schistosomiasis. |
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