Some Biological Functions Of Thioredoxin Glutathione Reductase In The Development Of Schistosoma Japonicum

Schistosomiasis is a major highly prevalent and serious zoonosis parasite disease with over one million people infected and another 600 million living in areas at risk of infection, which is caused by schistosome. In addition to infecting people, Schistosome can infect over 40 species of wild and domestic animals which have varying impacts on human infection. The current control strategy of schistosomiasis is praziquantel treatment. However, chemotherapy does not prevent re-infection, and some isolates of Schistosoma mansoni are resistant to high doses of praziquantel have been found. Therefore, the development of a safe and effective vaccine target is necessary for reducing the morbidity of schistosomiasis.As schistosome live in the veins of the final hosts, redox enzyme is required for them to resist reactive oxygen species (ROS), which are released from host effector cells and themselves metabolism. Thioredoxin glutathione reductase (TGR) is a multifunctional enzyme, which replaces the function of TrxR、GR and Grx in schistosome. In this study, we investigated the immuneprotection, enzymatic activity and gene function of rSjTGR. We also analysed the inhibition effect of auranofin in Schistosoma japonicum (S. japonicum), which may contribute to understanding the role of SjTGR in the development of schistosome. 1、The immune protection of recombinant TGR against S. japonicum challengeTo evaluate the immunoprotection effects of rSjTGR, we carried out two experiments in BALB/c mice. After immunization of BALB/c mice with rSjTGR, reductions of 33.50% and 36.51%(P<0.01) were obtained in the adult worms burden, reductions of 33.73%and 43.44%(P<0.01) in the number of eggs in the liver compared with the control groups. The levels of specific IgGl, and IgG 2a antibodies and IFN-y were significantly higher (P< 0.01) in the group vaccinated with rSjTGR than the control groups, as detected by enzyme linked immunosorbent assay (ELISA). rSjTGR induced a mixed Thl/Th2 response in which Thl was dominant. The study suggested that rSjTGR induced partial immunoprotection against S. japonicum in BALB/c mice.2^ The analysis of enzyme and antioxidant activities of recombinant S. japonicum TGRThe enzymatic activity of rSjTGR is combined the characteristic activities of TrxR, GR and Grx. The results showed that the specific activities were 1.877±0.169 U/mg with DTNB,0.8±0.01 U/mg with GSSG, and 51.286±4.62 U/mg in a Grx assay coupled to HED. In addition, the kinetic analysis found that the optimal temperature and pH of SjTGR were 25 ℃ and 7, respectively. We also found the antioxidant effect of rSjTGR was base on ROS not H2O2. This study proved the enzyme activity of the recombinant protein SjTGR.3-. The effects on development of S. japonicum by RNA interference on TGRWe silenced rSjTGR expression using 4 small (si) RNA (si RNA S1, S2, S3 and S4). The expression of SjTGR at both the transcript and protein levels were partially inhibited by 200 nM S2 siRNA. In vitro, after knocking-down the expression of SjTGR, reductions of 23.16% and 36.19% were obtained in the adult worms burden compared with the control groups. We also found that the enzyme activity of TrxR was decreased in the S2 siRNA-treated group. The results of this preliminary study indicated that SjTGR may play an important role in the development of S. japonicum.4^ The analysis of TGR as drug target of auranofinThe aim of this study was to identify the inhibition of SjTGR with auranofin (AF). The results showed that the inhibitory effect of 12.5 μM AF on rSjTGR was as high as 64.29%. But inhibiting effect did not rise accordingly with the increased concentration of AF. We also found that schistosomula were susceptible to AF. In vitro, all the worms were dead after 3 hours treatment with 10 μM AF. This phenomenon may be relative to the environment and stress when the schistosomula encountered in the final host. The enzyme activity was reduced 85% with DTNB,69% with GSSG,58% with HED after 1 hour treatment with 10 μM AF. In vivo, after injection of BALB/c mice with AF, reduction of 39.18% was obtained in the adult worm burden. In summary, AF is an efficient inhibitor of SjTGR.