| Schistosomiasis resistance is one of the focuses of schistosomiasis control work,and praziquantel(PZQ)resistance of S.mansoni has been reported.So far,praziquantel is still the only specific drug for the treatment of S.japonicum,and there is obviously a potential risk of resistance.Therefore,it is necessary to study new anti-schistosomiasis drugs that can replace praziquantel.However,praziquantel has been used in the treatment of schistosomiasis for more than 30 years,but its drug target and mechanism are still unclear.Previous studies have established a method to induce resistance of adult S.japonicum in mice with subtherapeutic dose of PZQ,and successfully induced resistance of PZQ to S.japonicum.Based on the previous work,this study intends to induce and collect PZQ-tolerant worms and detect their differentially expressed proteins by proteomics.It will provide scientific basis for further elucidating the mechanism of PZQ against S.japonicum and identifying potential targets,and also provide new strategies for the development of new anti-schistosomiasis drugs.Prat 1 Induction of PZQ-tolerant Schistosoma japonicumObjective:Induction of PZQ-tolerant S.japonicum lays an effective foundation for screening differentially expressed proteins between PZQ-tolerant and non-induced S.japonicum.Methods:60-80 single male cercariae of S.japonicum infected mice.After 3 weeks of infection,the mice were given PZQ subtherapeutic dose(ED50,25.98 mg/kg)for 30 days and stopped for 3 weeks.Then the mice were given PZQ therapeutic dose(200 mg/kg)for 5 days.After 2 weeks,the mice were killed and dissected by dislocation of cervical vertebra.Worms in 100%active state were cultured in DMEM low sugar culture medium preheated in advance.PZQ of different concentration was added.After 16 hours,the worms were washed with normal saline three times and then replaced with new culture medium.The worm vitality score was observed and recorded under the visual microscope every 24 hours to evaluate the drug sensitivity of PZQ-induced tolerant insects.Result:The critical lethal concentration of PZQ against non-induced S.japonicum in vitro(the lowest concentration of 90%activity reduction after 72 hours of drug treatment)was 1000 pmol/L.The decrease rate of activity of PZQ-tolerant worms was 66.7%,which was significantly different from that of non-induced worms(92.2%)(P<0.0001).The critical lethal concentration of PZQ against PZQ-tolerant worms in vitro was 4 times(4000?mol/L)that of non-induced S.japonicum.Conclusion:The susceptibility of S.japonicum induced by PZQ decreased by 3 times and showed obvious tolerance.Therefore,the induced PZQ-tolerant worms met the requirements of subsequent experiments.Part 2 Identification of functional proteins of Schistosoma japonicum PZQ tolerant worms by Western Blot and identification by mass spectrometryObjective:The differential expressed proteins of S.japonicum PZQ-tolerant and non-induced worms were identified by Western Blotting in rabbit sera infected with S.japonicum,which provided a new experimental basis for elucidating the mechanism and potential targets of PZQ.Mothods:Polyacrylamide gel electrophoresis(SDS-PAGE)was used to separate the total proteins from two species of worms.Western Blotting was used to identify the differentially expressed sites of functional proteins in PZQ tolerant and non induced worms of S.japonicum,and the bands of functional differentially expressed proteins identified in the gel were identified by liquid chromatography-mass spectrometry(LC-MS).The differentially expressed proteins of PZQ-tolerant and non-induced worms were identified.The functional properties of differentially expressed proteins were retrieved by online Uniprot.Result:Western Blotting identified three differentially expressed functional protein loci in PZQ-tolerant worms of S.japonicum.Gray scale analysis showed that there were two up-regulated loci,a loci(2.5±0.06 vs 1.00±0.00,P<0.0001)and b loci(1.60?0.03 vs 1.00±0.00,P<0.0001),and one down-regulated locus was b(0.70 ± 0.00 vs 1.00±0.00,P<0.0001).LC-MS was used to screen and identify functional protein loci of PZQ-tolerant worms.The results showed that there were five differentially expressed proteins.The differentially expressed proteins of PZQ-tolerant worms were mainly one membrane-related protein and three Ca2+ binding-related proteins(calpain B,calpain,leucine zipper containing EF-chiral transmembrane protein 1).Conclusion:PZQ can promote or inhibit the expression of some specific protein molecules of S.japonicum under sustained pressure.It was found that calpain B,calpain and leucine zippers in this study contain EF-chiral transmembrane protein 1,which are all Ca2+ binding related proteins,and participate in cell signal transduction process,suggesting that the anti-insect target of PZQ may be related to Ca2+ binding related proteins.Part 3 Analysis and identification of TMT differential proteins of Schistosoma.japonicum PZQ-tolerant wormsObjective:The differentially expressed proteins of S.japonicum PZQ-tolerant and non-induced worms were analyzed and identified.Similar functional or similar proteins were screened by comparing with the results of the second part.The mechanism and potential targets of PZQ were further elucidated by TMT proteomics.Method:Quantitative proteomic tandem mass tagging(TMT)technique was used to detect the total proteins of S.japonicum PZQ-tolerant and non induced worms.Quantitative analysis of proteins was carried out to screen differentially expressed proteins with significant changes in PZQ-tolerant worms(differentially increased by more than 1.3 times,differentially decreased by less than 1/1.3),and the differentially selected proteins were analyzed by bioinformatics.Result:31 protein molecules were differentially expressed in S.japonicum PZQ-tolerant worms by TMT-labeled quantitative proteomics analysis.6 protein molecules were up-regulated and 25 protein molecules were down-regulated.The ratio of four-transmembrane protein expression was 2.031,with the most significant difference.The main biological pathways for differentially expressed protein enrichment are ribosome and lysosome.Two ribosomal protein molecules are up-regulated and nine ribosomal protein molecules are down-regulated in the ribosomal pathway.Three protein molecules(four-transmembrane protein,V-type proton ATPase protein lipid subunit,SJCHGC01869 protein involved in the process of Neurol phospholipid)are up-regulated and four proteins(carboxypeptidase,hypothetical protein with cysteine-type peptidase activity,unknown protein with deoxyribonuclease II activity,SJCHGC04223 protein involved in protein demethylation)were up-regulated in lysosome pathwayConclusion:The results of TMT proteomics(quantitative detection)showed that the differential expression of four transmembrane proteins in PZQ-tolerant worms was the most significant,while LC-MS(qualitative detection)showed that there was one FAM62B protein differentially expressed,and one transmembrane protein(leucine zipper contains EF-chiral transmembrane protein 1).The results of both detection techniques suggest that the potential target proteins of PZQ against S.japonicum may be closely related to membrane-related proteins/transmembrane proteins and Ca2+binding-related proteins(calpain B,calpain).The exact target proteins need to be further studied.This study suggests that the target of PZQ is Tetraspanin.Proteins with Ca2+binding properties and involved in Ca2+ signal transduction process(calpain,calmodulin,leucine zipper containing EF-chiral transmembrane protein 1)and protein involved in translation process(ribosomal protein and translation initiation factor IF-2 non-taxonomic subunit)may be closely related to PZQ insect-resistant targets. |
PDF Full Text Request