Supression Of Tyrosine Hydroxylase Expression In Schsitosoma Japonicum By RNA Interference

Schistosomiasis remains a serious public health problem world-wide, infecting more than 200 million people, and is endemic in 74 developing countries (Savioli,L,;2002). Schistosoma japonicum is epidemic in Asia. Schistosomasis is a kind of amphixenosis and human is the final host in the life cycle of schistosoma. The hazard of schistosomasis is main in the eggs, which are constantly produced by the adult worms in the portal vein and deposited in liver. The eggs can cause immunized inflammation and lead to the formation of granuloma and fibrosis in the liver. The granuloma and fibrosis in the liver can gradually develop to hepatic cirrhosis and endanger the life of infected person. Therefore more and more people start to study the parasite for the hazard after infecting schistosoma. So far, praziquantel is still the primary choice to treat schistosomasis. However this drug has evidently limitations for releasing many antigens after the death of worms and exists a series of side effects. So searching for new drugs or new treatment is still the urgent task to treat schistosomasis.TH, which is the firstly rate-limiting enzyme in the biosynthesis of CAs, catalyzes tyrosine to produce CAs such as dopamine, noradrenaline and adrenaline through a multistep enzymatic pathway for regulating the activity of muscle and the rate of oviposition in schistosoma. And Sj14-3-3 participates in the regulation of TH activity. People pay more attention to SjTH and Sj14-3-3 for these characteristics. RNA interference (RNAi) is the phenomenon to inhibit the highly conservative and specificedly homologous mRNA through inducing by dsRNA (double-stranded RNA). RNAi can provide the fast and high-efficiency method of inhibiting the expression of specific gene to study its function in the organism. The technique had been used to study nemathelminth and other livings contained, such as Schistosoma mansoni. However there are rarely used in the Schistosoma japonicum by RNAi technique.We choose SjTH and Sj14-3-3 as the target genes to synthesize dsRNA by transcription in vitro for the following RNAi in schistosoma japonicum. According to the results of SjTH and Sj14-3-3 in mRNA level and the level of SjTH protein, we can illuminate dsRNA can function in the cercaria of schistosoma japonicum more effectively.1. Decreased levels of mRNA in long dsRNA-treated parasitesThe level of SjTH mRNA did not decreased in parasites treated with SjTH dsRNA as compared with the control group which non-treated with anything after soaking 24h, while the mRNA levels in parasites treated with SjTH dsRNA were decreased by 58% and 85% after soaking 72h and 96h respectively (P<0.05).The level of Sj14-3-3 mRNA did not decreased in parasites treated with Sj14-3-3 dsRNA compared with the control group which was non-treated with anything after soaking 24h. While the mRNA levels in parasites treated with Sj14-3-3 dsRNA were decreased by 62% and 81% after soaking 72h and 96h respectively (P<0.05).2. Decreased levels of SjTH mRNA in long Sj14-3-3 dsRNA-treated parasitesThe level of SjTH mRNA did not decreased in parasites treated with Sj14-3-3 dsRNA compared with the control group which non-treated with anything after soaking 24h and 76h. Nevertheless the SjTH mRNA levels were decreased by 49% after the parasites soaking 96h (P<0.05).3. The infection of cercaria after inhibition by SjTH dsRNA and Sj14-3-3 dsRNA 24 mice were allocated to four groups including negative control, positive control and other two groups treated with dsRNA with 6 mice per group. Nagetive control was comparised by normal mice. Positive control was mice treated with normal cercaria. The mice of other two groups were infected by cercaria which were treated with SjTH dsRNA and Sj14-3-3 dsRNA. The pathological sections of the livers in the mice after infecting cercaria with 9 weeks, were observed by microscope. And it can be found that the positive group has obvious granuloma and fibrosis, while granuloma and fibrosis of SjTH dsRNA treated group and Sj14-3-3 dsRNA treated group are very light in the liver compared with control group. The activity of the mice in these two groups is very fine compared with the positive group.In conclusion, SjTH dsRNA and Sj14-3-3 dsRNA can work effectively to knockdown the expression of target genes in schistosoma japonicum. And the capability of infecting for the cercaria after treating with long dsRNA is very weak. These results prove that the target gene-SjTH plays important role in the life of schistosoma japonicum and may be the door to search the method of schistosomasis through studying SjTH by RNA interference.