Correlation Between P-gp And MRP1 And Resistance To Praziquantel In Schistosoma Japonicum

Schistosomiasis is a major infectious disease which seriously damages human health and hampers economic development in developing countries, and over 200,000 deaths occur due to schistosomiasis per year in sub-Saharan Africa. In China, schistosomiasis is predominantly endemic in the marshland and lake regions along the middle and lower reaches of the Yangtze River and some mountainous regions of two provinces of Yunnan and Sichuan, and the disease is defined by the central government as one of the priorities for communicable disease control(HIV/AIDS, hepatitis B, tuberculosis and schistosomiasis).Since praziquantel was developed in 1970 s, it has been recommended the choice of drug for treatment of human schistosomiases due to high efficacy, low toxicity, easy administration, few adverse reactions and low cost. Currently, an estimated 30 million high-risk populations undergo praziquantel treatment worldwide per year. It is considered that drug resistance may occur following long-term extensive application. Experimental induction of praziquantel resistance in S. mansoni receives global attention. In addition, praziquantel-resistant S. japonicum isolates have been experimentally induced successfully. Exploring the mechanisms underlying resistance of S. japonicum to praziquantel is therefore urgently needed.P-glycoprotein(P-gp) and multidrug resistance-associated protein 1(MRP1) are two firstly identified ATP binding cassette(ABC) transporter, which induce active transportation of multiple biomolecules via cell membrane in an ATP-dependent manner, including amino acids, carbohydrates, lipids, inorganic ions, polysaccharides, metal ions, polypeptides and toxins. It is reported that mammalian tumor cells may reduce intracellular accumulation of antitumor agents through increasing P-gp and MRP1 expression to resist the antitumor activity, resulting in multiple drug resistance.It has been proved that the resistance of S. mansoni to praziquantel is associated with P-gpand MRP1 expression, and the up-regulation of P-gp and MRP1 expression is found to be involved in the development of drug resistance. However, there is no knowledge on the association of P-gp and MRP1 expression with praziquantel resistance in S. japnoicum till now. The purposes of this study were to identify P-gp and MRP1 genes in S. japnoicum, compare the difference of P-gp and MRP1 gene expression between praziquantel-resistant and-susceptible isolates of S. japonicum, and determine the alteration of P-gp and MRP1 gene expression following post-exposure to praziquantel at sub-lethal doses, so as to evaluate the correlation between praziquantel resistance and P-gp and MRP1 expression in S. japonicum. The results of the current study would provide basis for the extensive investigation of the mechanisms underlying the resistance to praziquantel in S. japonicum. This study includes the following four parts.Part I Amplification and identification of P-gp and MRP1 genes in adult worms of Schistosoma japonicumObjective: To identify P-gp and MRP1 genes in Schistosoma japonicum.Methods: The coding DNA sequences(CDSs) of SMDR2, a P-gp homologue, and Sm MRP1, a MRP1 homologue in S. mansoni were obtained through literature review, and the CDS of P-gp gene of in S. japonicum was captured using BLAST software in Gen Bank. In addition, the CDS of MRP1 gene in S. japonicum was captured using BLAST software in S. japonicum genome database. According to the sequence of P-gp and MRP1 genes, specific primers were designed using the software Primer Premier 6 and Oligo 7. Adult worms were captured from the portal vein and mesenteric vein of S. japonicum-infected mice. Total RNA was isolated from adult worms, and transcribed into c DNA. PCR amplification was performed, and the PCR product was checked with electrophoresis on 2% agarose gel. The amplification band was used for q PCR assay.Results: The homologous sequences of P-gp and MRP1 genes in S. japonicum were captured using BLAST software. Three pairs of specific primers were designed, including 18 S internal control. Electrophoresis showed 3 amplification products with respective sizes comparable to expected. The amplification efficiency was estimated to be 0.90, 1.04 and 0.96, revealed by the standard curve, which met the requirement of relative quantitative 2-ΔΔCT method.Conclusion: Genes encoding P-gp and MRP1 proteins are detected in the adult worms of S.japonicum.Part II Alteration of P-gp and MRP1 m RNA expression in adult worms of Schistosoma japonicum following exposure to praziquantelObjective: To evaluate the correlation between P-gp and MRP1 gene expression and praziquantel in adult worms of Schistosoma japonicum.Methods and Results:In vitro experiments: S. japonicum infection mouse model was established through cercarial placement on mouse abdomen. After 42 days of infection, mice were sacrificed with the cervical dislocation method. Mice were dissected, adult worms were recovered, and active adult worms without damages were incubated in RPMI 1640. Then, adult worms were treated with praziquantel at sub-lethal doses at 37°C containing 5% CO2. Following 0.4, 4 and 24 hours of treatment, adult worms from the experimental and control groups were collected. Total RNA was extracted from adult worms, and transcribed into c DNA. Real-time quantitative PCR(q RTPCR) assay was performed to detect the m RNA expression of P-gp and MRP1 genes.Results of in vitro experiments: q RT-PCR showed that the P-gp m RNA expression was 2.81 times greater in the adult worms treated with praziquantel for 0.5 h than in controls(P < 0.05), and 1.54 times higher in the adult worms exposed to praziquantel for 4 h than in controls(P > 0.05), while the P-gp m RNA expression recovered to baseline levels following exposure to praziquantel for 24 h, which did not differ from that in controls(P > 0.05). The results indicated that the P-gp m RNA expression appeared a transient incline in the adult worms treated with praziquantel in vitro, and then returned to baseline levels. The MRP1 m RNA expression was 2.14 times greater in the adult worms treated with praziquantel for 0.5 h than in controls(P < 0.05), and 2.69 times higher in the adult worms exposed to praziquantel for 4 h than in controls(P < 0.05); however, the MRP1 m RNA expression was 0.46-fold in the adult worms following exposure to praziquantel for 24 h than in controls(P < 0.05). The results indicated that the MRP1 m RNA expression appeared a transient incline in the adult worms treated with praziquantel in vitro, followed by a decrease tendency.In vivo experiments: S. japonicum infection mouse model was established through cercarial placement on mouse abdomen. All infected mice were assigned into 0.5 h treatment, 4 h treatment and 24 h treatment groups, while infected but untreated mice served as controls, of 3 mice in each group. Mice in the treatment groups were administered orally with praziquantel ata single dose of 75 mg/kg 40 days post-infection, and all animals were sacrificed with the cervical dislocation method 0.5, 4 and 24 h post-treatment. Mice were dissected, and adult worms were recovered. Then, total RNA was extracted from adult worms, and transcribed into c DNA. q RT-PCR assay was performed to detect the m RNA expression of P-gp and MRP1 genes. Results of in vivo experiments: q RT-PCR showed that the P-gp m RNA expression was 2.45 times greater in the adult worms treated with praziquantel for 0.5 h than in controls(P < 0.05), and 2.50 times higher in the adult worms exposed to praziquantel for 4 h than in controls(P < 0.05), while the P-gp m RNA expression recovered to baseline levels following exposure to praziquantel for 24 h, which did not differ from that in controls(P > 0.05). The results indicated that the P-gp m RNA expression appeared a transient incline in the adult worms treated with praziquantel in vitro, and then returned to baseline levels. The MRP1 m RNA expression was 1.81 times greater in the adult worms treated with praziquantel for 0.5 h than in controls(P < 0.05), and 1.95 times higher in the adult worms exposed to praziquantel for 4 h than in controls(P < 0.05); however, the MRP1 m RNA expression in the adult worms returned to baseline levels following exposure to praziquantel for 24 h(P < 0.05). The results indicated that the MRP1 m RNA expression appeared a transient incline in the adult worms treated with praziquantel in vivo, followed by a decrease tendency to baseline levels.Conclusion: Following exposure to praziquantel at a sub-lethal dose, the P-gp and MRP1 gene expression appeared a transient incline in the adult worms of S. japonicum.Part III Difference of P-gp and MRP1 mRNA expression in various development stages between praziquantel-susceptible and-resistant isolates of Schistosoma japonicumObjective: To compare the difference of P-gp and MRP1 mRNA expression between praziquantel-susceptible and-resistant isolates of Schistosoma japonicum.Methods: Oncomelania snails infected with praziquantel-susceptible and-resistant isolates of S. japonicum were placed in a beaker, and cercariae were released under light. Cercariae were collected using a series of centrifugations, including centrifugation at 6000 r/min for 3 min, at 4000 r/min for 5 min and at 10000 r/min for 3 min. Each mouse was infected with 40 cercariae to establish animal models of praziquantel-resistant and-susceptible S. japonicum infections. All mice were sacrificed 42 days post-infection, and adult worms were collected from the portal vein of mice. Mouse liver was collected, cut into pieces, homogenated, and filtered. S.japonicum eggs were harvested, and miracidia were collected. Total RNA was extracted from adult worms, cercariae and miracidia of praziquantel-resistant and-susceptible S. japonicum isolates, and transcribed into c DNA. The m RNA expression of P-gp and MRP1 genes was determined using q RT-PCR assay.Results: The P-gp m RNA expression was 2.21, 3.65 and 2.83 times higher in the adult worms, miracidia and cercariae of praziquantel-susceptible S. japonicum isolate than in those of praziquantel-resistant S. japonicum isolate(all P values < 0.05), while the MRP1 m RNA expression was 5.68, 1.36 and 1.50 times higher in the adult worms, miracidia and cercariae of praziquantel-susceptible S. japonicum isolate than in those of praziquantel-resistant S. japonicum isolate(all P values < 0.05)Conclusion: There was significant difference in the P-gp and MRP1 gene expression between praziquantel-susceptible and-resistant isolates of S. japonicum, and the expression of both Pgp and MRP1 genes was greater in adult, miracidia and cercariae of praziquantel-resistant isolate than in those of praziquantel-susceptible isolate of S. japonicum.Part IV Effect of P-gp and MRP1 inhibitors on egg production by adult worms of Schistosoma japonicumObjective: To evaluate the effect of P-gp and MRP1 inhibitors on egg production by adult womrs of Schistosoma japonicum, so as to investigate the correlation between P-gp and MRP1 expression and S. japonicum egg production.Methods: S. japonicum infection mouse model was established through cercarial placement on mouse abdomen. After 42 days of infection, mice were sacrificed with the cervical dislocation method. Mice were dissected, adult worms were recovered, and active adult worms without damages were incubated in RPMI 1640. Then, adult worms were treated with P-gp inhibitor R(+) Verapamil and MRP1 inhibitor MK-571 at 37°C containing 5% CO2. Following 48 h treatment, ee production was counted under an inverted microscope.Results: The egg production was 1.6, 2.3 and 3.6 times greater in the adult worms of S. japonicum following exposure to R(+)-Verapamil at doses of 0.5, 1 and 2 μM than in controls, while t The egg production was 1.6, 4.3 and 4.7 times greater in the adult worms of S. japonicum following exposure to MK-571 at doses of 5, 25 and 50 μM than in controls,Conclusion: Both P-gp and MRP1 inhibitors reduceegg production of S. japonicum in a dosedependent manner, suggesting that P-gp and MRP1 are involved in egg production of S.japonicum.