| Objective: To evaluate whether PZQ derivatives P96 and DW-3-15 can be potentially novel drugs for treatment of schistosomiasis, we examined biological effects of P96 and DW-3-15 against PZQ-resistant adult worms of S. japonicum in vitro.Method: S. japonicum infections in mice were given subcurative(effective dose required to kill 50% of the worms, ED50) PZQ orally on the 21 st day after infection, for 30 consecutive days. Then with 21 days interval mice were treated again with curative(200 mg/kg) PZQ for 5 successive days. Two weeks post-treatment, worms were cultivated in DMEM medium containing different concentrations of PZQ, P96 and DW-3-15. The number of the live worms and the motility reduce rate were recorded.Results: We observed that the critical lethal concentration(the lowest dose required to make 72 h vitality reduction reach to 90%) of PZQ and P96 against adult worms induced was eight and four times respectively as high as uninducible worms, while the critical lethal concentration of DW-3-15 against PZQ-resistant adult worms was the same as that of the uninducible.Conclusion: The data revealed that the worms induced by PZQ were less susceptible to PZQ, which existed a significant difference from P96 and DW-3-15(P<0.05). And particularly PZQ-resistant worms showed no cross resistance to DW-3-15. These results indicate that the targets of P96 and DW-3-15 against S. japonicum may be different from PZQ. P96 and DW-3-15 will perhaps be potentially novel drugs for treatment of schistosomiasis.Objective: To test whether Ahnak of S. japonicum is related to the action of praziquantel, we subjected adult worms in vitro to Ahnak antibody(Ab) and PZQ, then to observe biological effect of PZQ against S. japonicum.Method: The worms of S. japonicum from mice were obtained by perfusion of portal vein, and cultivated in DMEM medium containing different concentrations of Ahnak Ab for different times. After that, PZQ was added and the worms were incubated overnight(16 h). Then the worms were washed, resuspended in fresh medium without drug and observed during the following 72 h.Results: When the concentration of Ahnak Ab was 1.2 μg/ml or 1.8 μg/ml, the worms pre-exposed to Ahnak Ab for 1 h, Ahnak Ab conferred an incomplete but significant inhibition of the schistosomicidal effects of PZQ and the 72 h worm survival both reached up to 80% and the vitality reduction is 71.1% and 64.4%, respectively. Other concentrations of Ahnak Ab and times of pre-exposure tested gave insignificant inhibition.Conclusion: As shown in the results, when the concentration of Ahnak Ab was 1.2 μg/ml, incubation for 1 h, Ahnak Ab emerged the most significant antagonism to schistosomicidal effects of PZQ. These results indicate that Ahnak might serve as an important target for the action of PZQ and are consistent with the hypothesis that calcium channels are involved in the mechanism of effect of PZQ. The function of Ahnak of S. japonicum deserves further study.Objective: Analyzing differentially expressed proteins of PZQ-resistant adult worms of S. japonicum induced so as to further explore the mechanism of schistosomicidal effects of PZQ.Method: PZQ-resistant adult worms and worms uninduced of S. japonicum were collected to extract the total protein. Differentially expressed proteins between the two groups were screened and identified by two-dimensional polyacrylamide gel electrophoresis(2-D PAGE) and liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results: There were 31 proteins up-regulated and 6 proteins down-regulated in the PZQ-resistant group compared with the uninduced. In addition, the variation tendency of 3 proteins is unidentified. These differentially expressed proteins respectively ascribed to cellular structure and motion-associated proteins(9), stress reaction related proteins(4), metabolic enzymes(7), regulatory proteins for the structure and translation of the protein(5), ion regulatory proteins(3) and some function-unknown proteins(12).Conclusion: The protein expression level of S. japonicum could change markedly under the continuous pressure of PZQ, suggesting that PZQ can promote or inhibit the expression of specific genes of S. japonicum. These differentially expressed proteins may be involved in the schistosomicidal effects of PZQ. Part 4 Transcript Analysis of PZQ-resistant Adult Worms of S.Objective:The relative expression of m RNA in PZQ-resistant adult worms and normal adult worms of S. japonicum was measured to analyze the differences of transcriptional levels between the two kinds of worms. And connecting with the differences in the levels of protein, we make a comprehensive analysis so as to explore mechanisms of PZQ-resistance.Method: Collect PZQ-resistant adult worms and normal adult worms of S. japonicum respectively and extract the total m RNA using TRIzol method, to obtain c DNA by reverse transcription. According to the accessions of the proteins differentially expressed, search the corresponding gene sequences in the NCBI, design the specific primers and detect c DNA using real-time fluorescent quantitative PCR technology.Results: The results revealed that the expression levels of m RNA of Ribosomal Protein/Large subunit and Calponin in PZQ-resistant adult worms were both lower compared with normal adult worms, the m RNA expression levels of Histone H2 A and HSP70 were both higher; while the m RNA expression levels of Globin-3, Triosephosphate isomerase, Eukaryotic translation elongation factor 1 alpha 2 and Myosin were not changed.Conclusion: It is suggested that the transcriptional level of PZQ-resistant adult worms under continuous PZQ pressure can change, and the variation tendency between transcription level and protein level is not the same. |
PDF Full Text Request