| Schistosomiasis(Schistosomiasis)is a tropical and subtropical zoonosis caused by Schistosomiasis infection,which is mainly prevalent in the Middle East,South America,Southeast Asia and sub-Saharan Africa.In China,only S.japonicum is endemic and transmitted.The spread of the disease depends on the emergence of cercariae from an intermediate host,oncomelania snails,which invade the human body through the skin and develop into juvenile worms.The juvenile worms eventually migrate to the hepatic hilar mesenteric vein system and develop into adults and lay eggs.The egg deposits in the liver tissue and forms a large number of egg granulomas.At the late stage,the egg granulomas are gradually replaced by fibrous tissue,resulting in liver fiber,which seriously affects the patient’s ability to work and quality of life.However,no effective schistosomal vaccine is available and chemotherapy is still the cornerstone for control and elimination of schistosomiasis.Since PZQ discovered,owning to its good tolerability,low cost,broad therapeutic profiles and single administration with high efficacy,has been recommended as first choice drug by the WHO for treatment all major forms of schistosomiasis.However,due to the repeated long-term and large-scale treatment of PZQ for schistosomiasis,the development of drug-resistance remains a great concern.It has been reported that PZQ resistant strains can be induced under laboratory conditions.Thereafter,emergency of resistance to PZQ for treatment of schistosomiasis in the clinical field has been reported.In addition,However,whilst PZQ is effective against the adult worms of Schistosoma spp,it displays poor efficacy against immature schistosomula and can not prevent reinfection.These factors may explain the low cure rates and rapid reinfection of residents in endemic areas,who are frequently infected with both juvenile and adult parasites.Therefore,it is imperative to develop new antischistosomal drugs that can alternative or complement PZQ.In previous study,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.It was shown to be effective against adult and juvemile wormsin vitro and in vivo by commercial synthesis.In addition,TMT(Tandem Mass Tags Labeling Proteome Quantification)analysis showed that the insecticidal mechanism of DW-3-15 may be related to the inhibition of SjHAT,and its target may be different from that of PZQ.Here,we investigated the antischistosomal effect of PZQ combined with DW-3-15 on different developmental stages of Schistosoma japonicum in vitro and in vivo,which would verify whether there was synergistic effect of the two compounds.In addition,the effects of the two compounds on the transcription and expression of adult and juvenile worms of SjHAT were discussed,and the potential targets and mechanisms of action of the two compounds against Schistosoma japonicum were further explored,so as to provide a scientific basis for exploring their combined chemotherapy as a potential novel antischistosomal method.Part one.Lethal effect of PZQ combined with its derivative DW-3-15 against Schistosoma japonicum in vitroObjective The aim of this part is to investigate the lethal effect of PZQ combined with DW-3-15 on different developmental stages of Schistosoma japonicum in vitro.Method The mice were individually infected transcutaneously with S.japonicum cercariae.S.japonicum worms recovered from infected mice at 14 days postinfection(juvenile worm)and at 35 days postinfection(adult worm)by perfusion of the hepatic portal system and mesenteric veins method,and were placed in 6-well plates containing complete DMEM medium.The control group was added with 0.1%DMSO;The final concentrations of PZQ and DW-3-15 in the single drug group were 25～100 μM;The combination groups included PDa(50 μM PZQ combined with 50 μM DW-3-15)and PDb(100 μM PZQ combined with 100 μM DW-3-15);The adult and juvenile worms were treated by the compound of these groups.All the S.japonicum worms were exposed to the chemicals for 16h,then cultured in chemical-free complete DMEM for 72 h.During the incubation period,the viability and survival rate of the worms were observed every day,and the degree of tissue death of the worms was observed by Propidium Iodide(PI)staining.After the culture,the synergistic index was calculated,and the ultrastructural features of the schistosome were observed under scanning electron microscope(SEM).Results 1.In vitro,while for DW-3-15 and PZQ,the antischistosomal effect was both concentration-dependent,with PZQ or DW-3-15 at 100 μM demonstrating the most potent effect.After 72h,the viability reduction rate of adult worms exposed to PZQ or DW-3-15 were 67.8%～72.3%and 94.3%～94.4%,respectively.And the viability reduction rate of juvenile worms exposed to PZQ or DW-3-15 were 68.9%and 97.8%.The pronounced antischistosoamal effect were observed between PDa and PDb,and there was statistically significant difference in terms of viability compared to control gruop(P<0.0001).After 72 h,all the male and juvenile worms were killed by the higher concentration group(PDb),and the PDb resulted 99.0%reduction in viability of female worms.Exposure to combination of lower concentration group(PDa)resulted in 100%and 93.3%～94.4%reduction in viability of juvenile worms and adult worms,respectively.The antischistosomal effect of PDa on adult worms was similar to that of 100 μM DW-3-15 and better than that of 100 μM PZQ.The combination index(CI)of PZQ combined with DW-3-15 against juvenile,male and female worms was 0.28～0.57,0.27～0.63 and 0.53～0.65,respectively,indicating the combination of PZQ and DW-3-15 producing strong synergism.In addition,worms treated with combination of PZQ and DW-3-15 displayed brighter red fluorescence of juveniles and adult worms than worms incubated with single compound,showing the damage of worm tissue being more serious of combination groups.2.Morphological observation showed that when treated with 100 μM PZQ for 72 h,the adult and juvenile worms showed spastic paralysis,curled up in spiral shape and stiff posture,while when treated with 100 μM DW-3-15 for 72 h,the adult worms showed obvious swelling,became shorter and curled up,and the juvenile worms stiffened like rods or slightly curled up.When the two compound combined,the worms exposed to PDa and PDb showed stiff and slight swelling.Adult worms were U-shaped or O-shaped curled up,while juvenile worms were straight rod shaped or slightly curled up.The tegumental surface of juvenile worms showed vacuole changes,and complete loss of movement capacity.3.Compared to control and single drug groups,extensive tegumental damages of worms were induced by combination groups.The male worms showed severe tegumental damage in the form of extensive sloughing,with exposure of the subtegumental muscle layer,muscle injury and muscle dissolved.Meanwhile,all the subtegumental structures of the oral suckers were exposed,with hole-shaped erosions on the muscle layer,and the ventral sucker showed extensive swelling and collapse of the tegument,a loss of the spine,and complete fusion,with the formation of long irregular disorganized splits.Conclusion The combination of PZQ and DW-3-15 showed significant antischistosoma effect on adults and juvenile worms of S.japonicum in vitro,and the effect was better than that of single drug groups,showing synergistic effect.Part two.Antischistosomal activity of PZQ combined with its derivative DW-3-15 against Schistosoma japonicum in vivoObjective The aim of this part is to investigate the antischistosomal activity of PZQ combined with DW-3-15 on different developmental stages of S.japonicum in vivo and the influence on egg granuloma formation.Method The mice model were established by infected transcutaneously with S.japonicum cercariae.These mice were treated at 14 days postinfection(juvenile worm),21 days postinfection(both juvenile and adult worms infection)and 28 days postinfection(adult worm).The mice of control group were treated by 0.5%carboxymethyl cellulose sodium,and the mice of single drug group were treated by 100～400mg/kg PZQ or DW-3-15.The combination groups included the lower dose of PDc(100 mg/kg PZQ combined with 200 mg/kg DW-3-15)and the higher dose of PDd(200 mg/kg PZQ combined with 400 mg/kg DW-3-15).All the mice were treated for 5 consecutive day and killed at 21 days posttreatment.The worm and egg burden,combination index and the area of granulomas were calculated to evaluate the antischistosomal effect.Results 1.For PZQ or DW-3-15 monotherapy group,the reduction of total worm and egg burden showed dose-dependent.The highest worm and egg reduction induced by PZQ monotherapy group(96.9%and 85.2%,respectively)were higher than DW-3-15 monotherapy group(60.0%and 78.0%,respectively).Reductions of worm and egg burden induced by PZQ monotherapy group(43.3%and 83.2%,respectively)against juvenile were lower than DW-3-15 monotherapy group(70.3%and 84.7%,respectively).For the mice harboring both juvenile and adult worms,the administration of DW-3-15 resulted the higher reductions of total worm burden and lower reductions of number of eggs than that of PZQ.The highest worm burden and eggs burden reduction of DW-3-15 monotherapy group were both 74.5%,while those of PZQ monotherapy group were 64.1%and 79.4%,respectively.2.For the combination groups,the reduction of total worm and egg burden also presented dose-dependent.For the mice infected with 14-day-old,21-day-old and 28-day-old worms,treatment with the higher dose group resulted in a 83.8%,83.0%and 97.3%in total worm burden,respectively.Additionally,the antischistosomal effect of PDc on 14-day-old juvenile worms and 21-day-old multiple-stage worms were similar to 400 mg/kg DW-3-15,and better than that of 200 mg/kg PZQ.For the mice infected 28-day-old adult worms,the antischistosomal activity of PDc were similar to 200 mg/kg PZQ,and better than that of 400 mg/kg DW-3-15.These results indicated that combination compounds achieved the similar or even enhanced efficacy at lower doses.The effect of combination compound in egg burden was also better than that of single drug.Egg reductions of PDd against juvenile,multiple-stage and adult worms were 99.6%,94.1%and 87.3%,When the dose was halved,the egg reduction rates were 96.5%,86%and 81.4%in PDc group.Meawhile,the CI of combination groups against juvenile worms and multiple-stage and adult worms were 0.49～0.63,0.59～0.94 and 0.43～0.46,respectively,indicating the combination of PZQ and DW-3-15 producing synergistic effect.3.The histopathological features of granulomas showed that the pronounced reductions in S.japonicum-associated hepatic granuloma size were observed in the mice infected with juvenile,multiple-stage and adult worms treated with combination groups.The area of egg granuloma of low-dose and high-dose combination groups significantly different from that of the control group against(P<0.01;P<0.001).The area of liver granuloma was significantly reduced and the inflammatory cell infiltration was reduced after combination therapy,which was better than that of single drug group.Conclusion In vivo,PZQ combined with DW-3-15 had pronounced effect against 14-day-old juvenile worms,21-day-old mixed adults with juvenile worms and 28-day-old adults worms,showing synergistic effect.The combination cmpounds not only reduces the worm and egg burden,but also attenuates egg-induced hepatic granulomas and fibrosis,and can be developed as a potential promising method of schistosomiasis treatment.Part three.DW-3-15 affects the transcription and expression of SjHATObjective This part is to investigate the effects of different concentrations of DW-3-15 on SjHAT transcription and expression of adult and juvenile worms of Schistosoma japonicum in vivo and in vitro,and to provide a basis for exploring the antischistosomal mechanism of DW-3-15.Methods 1.In vitro,S.japonicum worms recovered from infected mice at 14 days postinfection(juvenile worm)and at 35 days postinfection(adult worm)by perfusion of the hepatic portal system and mesenteric veins method.The worms were exposed to 25～100 μM PZQ or 25～100 μM DW-3-15 for 16 h.The control group was added with 0.1%DMSO.After 16 h,these worms were cultured in chemical-free complete DMEM for 72 h.After 72 h,the worms samples of each groups were collected and used to extract the RNA and total protein.Real-time quantitative PCR and western blotting were used to detect the transcription and expression levels of SjHAT in each groups.2.In vivo,The mice were treated at 14 days postinfection and 35 days postinfection(adult worm).The mice were treated by 100～400mg/kg PZQ or DW-3-15,and the control group were treated by 0.5%carboxymethyl cellulose sodium.All the mice were treated for 2 consecutive day and killed at 2 days posttreatment.The worms samples of each groups were collected by perfusion of the hepatic portal system and mesenteric veins method.The worms samples of each groups were used to extract the RNA and total protein.Real-time quantitative PCR and western blotting were used to detect the transcription and expression levels of SjHAT in each groups.Results In vitro,the results of real-time quantitative PCR showed that the SjHAT transcription levels of male,female and juvenile worms were decreased with the increase of drug dose after treatment with different concentrations of DW-3-15 and PZQ,and showed a significant down-regulated(P<0.0001).The transcription level of SjHAT treated with 50 μM,75 μM and 100 μM DW-3-15 was significantly lower than that treated with the same concentration of PZQ(P<0.0001).The same changes of the SjHAT expression levels of male worms,female worms,and juvenile worms were observed between DW-3-15 and PZQ groups.Except for 25 μM PZQ to up-regulate the expression of SjHAT in Juvenile,the expression levels of SjHAT in all the other DW-3-15 and PZQ-administered groups were down-regulated,with the decrease at 100μM being most obvious.The expression levels of SjHAT in female worms and juvenile were significantly different from those in the normal control group(P<0.0001).In vivo,in 100～400mg/kg DW-3-15 treatment groups,the SjHAT transcription levels of male worms increased first and then decreased,and the transcription was up-regulated at 100 mg/kg dose(P<0.001)and down-regulated at 400mg/kg dose(P<0.05).SjHAT transcription level of females in 100～400 mg/kg DW-3-15 groups was significantly down-regulated(P<0.05;P<0.0001)and had a dose-dependent effect.The transcription level of SjHAT decreased with the increase of the dose of DW-3-15,but still showed a significantly up-regulated(P<0.05;P<0.0001).In the 100～400mg/kg PZQ treatment groups,transcriptional levels of SjHAT in males were higher than those in untreated control group(P<0.001;P<0.0001)and decreased with the increase of drug dose.The transc ription level of females SjHAT treated by PZQ decreased at first and then increased,and the transcription level of female SjHAT decreased significantly at the dose of 100 mg/kg and 200 mg/kg compared with the untreated control group(P<0.0001).When the dose increased to 400 mg/kg,the transcription level of female SjHAT increased,but was still lower than that of the untreated control group(P<0.01).The transcription level of SjHAT decreased with the increase of DW-3-15 dose,and showed a significantly up-regulated(P<0.05;P<0.0001).On the other hand,the transcription level of SjHAT in PZQ group increased with the increase of drug dose.The SjHAT transcription levels of juvenile in the 100 mg/kg PZQ treatment group were lower than those of the untreated control group(P<0.05),while they were significantly higher than the control group at the dose of 200 mg/kg and 400 mg/kg(P<0.001,P<0.0001).In terms of protein expression,different doses of DW-3-15 could down-regulate the expression of SjHAT in both males and females,with that of 400 mg/kg being most obvious(P<0.0001).In the PZQ groups,the expression levels of SjHAT in male worms decreased with the increase of the PZQ dose,which were significantly higher than those of the control group(P<0.0001);the expression levels of SjHAT in female worms were lower than those of the control group,while there was no dose-dependent effect.In addition,the expression levels of SjHAT in juvenile increased with the increase of DW-3-15 dose,and the SjHAT of worms was significantly up-regulated at the dose of 200 mg/kg and 400 mg/kg(P<0.05;P<0.001).In 100～400mg/kg PZQ treatment groups,the expression levels of SjHAT protein of juvenile worms gradually decreased with the increase of PZQ dose,presenting the significant up-regulated expression(P<0.0001).Conclusion The results revealed that both DW-3-15 and PZQ could affect the transcription and expression levels of SjHAT of adult and juvenile worms,and the two compounds had different effects on the transcription and expression,which further indicated that there might be differences in the targets and mechanisms against S.japonicum between the two compounds in vivo and in vitro.Meanwhile,this study also provided a new experimental basis for elucidating the antischistosomal mechanism of DW-3-15 and PZQ.Part four.Impact of DW-3-15 on histone acetylation of Schistosoma japonicumObjective The aim of present study is to examine the effect of DW-3-15 on histone H3 and H4 acetylation in adults and juvenile of S.japonicum in vivo and in vitro,and to provides the experimental data for further explore the antischistosomal mechanism of DW-3-15 related to histone.Method 1.In vitro,S.japonicum worms recovered from infected mice at 14 days postinfection and at 35 days postinfection by perfusion of the hepatic portal system and mesenteric veins method.All the worms were exposed to 100 μM PZQ and DW-3-15 for 16 h,and then cultured in chemical-free complete DMEM for 72 h.The worm samples were collected to detect the acetylation levels of histone H3 and H4 by western blotting.2.The mice were treated by 400 mg/kg DW-3-15 and 400 mg/kg PZQ at 14 days postinfection and 35 days postinfection,while the mice of control group were treated by 0.5%carboxymethyl cellulose sodium.All the mice were treated for 2 consecutive day and killed at 2 days posttreatment.The worms samples were recovered by perfusion of the hepatic portal system and mesenteric veins method,and the the acetylation levels of histone H3 and H4 of these worms were tested by western blotting.Results 1.In vitro,both DW-3-15 and PZQ induced the significantly decreased of H4 acetylation of males and females(P<0.001;P<0.0001),and no significant change were detected in the level of H3 acetylation in males.The female worms exposed to DW-3-15 demonstrated a significant up-regulation in H3 acetylation(P<0.01),while the H3 acetylation level of females in PZQ group was significantly down-regulated(P<0.05).In the same condition,the decrease in the levels of acetylated H3 or H4 were observed in juvenile worms incubation with DW-3-15 or PZQ.The levels of H3 acetylation of juvenile worms in DW-3-15 group were not statistically significant compared with the control group(P>0.05),but it was significantly different in other groups compared with the normal control group(P<0.001;P<0.0001).2.In vivo,a significant down-regulated were tested in H4 acetylation of males and females of DW-3-15 groups(P<0.01;P<0.0001),while the H3 acetylation showed an opposite trend(P>0.05).The expression of H4 acetylation in both males and females of PZQ group was significantly down-regulated(P<0.01;P<0.0001).While the H3 acetylation level of females was significantly up-regulated in PZQ group(P<0.0001),and that of males was significantly down-regulated(P<0.01).In addition,DW-3-15 induced the up-regulation of H3 and H4 acetylation level of 14-day-old juvenile worms(P<0.01;P<0.001),while PZQ decreased the H3 acetylation level(P<0.001)and increased the H4 acetylation level of juvenile worms(P<0.001).Conclusion DW-3-15 and PZQ caused an influence of H3 and H4 acetylation in adult and juvenile worms,and more particularly of H4.Combined with the results of the third part,it was inferred that the the down-regulation of SjHAT induced by compounds may directly inhibit H4 acetylation in adults and exert the insecticidal effect.However,the changes of H4 acetylation levels were not consistent between the two compounds,suggesting that there might be differences in the targets and mechanisms of the two compounds against adult and juvenile worms.This part provided the new experimental data to further clarify the anti schistosomal mechanism of DW-3-15 and PZQ related to histone acetylation.In conclusion,we confirmed that the combination of PZQ with DW-3-15 produces synergistic effects and observed the antischistosomal activity against both adult and juvenile worms of S.japonicum in vitro and in vivo.Meanwhile,the PZQ combined with DW-3-15 could also ameliorate schistosomiasis hepatic pathology.These effect of combination groups is better than that of single drug groups.In vitro and in vivo,real-time quantitative PCR and western blotting were used to analyze the dynamic changes of transcription and expression of SjHAT of schistosome after treatment with different concentrations of DW-3-15 and PZQ.The results showed that the two compounds had different effects on the transcription and expression level of SjHAT in different developmental stages of S.japonicum,and could affect the levels of H3 and H4 acetylation.It is further suggested that SjHAT may be the potential target of DW-3-15 and PZQ against schistosome,and there may be differences in the mechanism of action between the two compound on SjHAT.These data also provides a scientific basis for further clariting the target and mechanism of schistosomicide,developing a new clinical treatment regimen for schistosomiasis. |
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