| Chronic hepatitis B is a kind of infective disease caused by HBV. One-third of population in the world was infected by HBV, half population live in high epidemic area, mainly in under developed area, such as Southeast Asia, China, Australia and Africa. In America, 200, 000 people become new carrier every year. The epidemiologic survey showed 5%-20% infected people become chronic carrier. The chance for these people to get hepatic cirrhosis or hepatic cellular cancer is higher than others. Chronic hepatitis B becomes a serious public health problem. People in the world pay more and more attention on this issue.Persistent viral infection and immune function disorder are the major reason of chronic hepatitis B. Therefore, clearance of virus infection is the major method to therapy the chronic hepatitis B. Currently, the drugs used to therapy chronic hepatitis B include immunomodulator and virus inhibitor. However, these drugs had some side effects and appeared drug resistance. For example, the response rate of INF-αis very low, the side effects is common and serious. For lamivudine, the YMDD mutation is very difficult to overcome.The drugs discussed above can inhibit and eliminate the HBV DNA in serum immediately, but the concentration of these drugs in liver is low, so it is a little difficult to eliminate the virus in liver and the virus can stay in liver for a long time. Therefore, it has already become a focus to develop and prepare anti-HBV drug with high effect and low price.Polyoxometalate is a kind of metal-oxygen clusters compound. Currently, the synthesis of ployoxometalate enter molecule phrase. The novel polyoxometalate with biology activity can be synthesized by molecule self-assembly method. The antivirus activity of polyoxometalate was studied since 1970's. Many researchers found the polyoxometalate with keggin structure can inhibit both RNA virus and DNA virus. In the recent years, the study of antivirus of polyoxometalate is focus on anit-HIV, influenza virus and SARS virus. Some researchers found the mechanism of antivirus of polyoxometalate is to prevent the viral adsorption and penetration, inhibit the activity of retroviridase. Nowadays, the study of antivirus of polyoxometalate becomes a focus in the world.We synthesized a kind of novel polyoxometalate based on HBV structure. The anti-HBV activity of the novel polyoxometalate was evaluated in this paper.1. Anit-HBV activity in vitro: HepG 2 2.2.15 cell lines were used in in vitro experiment. The cytotoxicity of PTW-6 on HepG 2 2.2.15 cell lines was detected by MTT assay. The result showed the IC50 is 1590.46μg/mL. The concentration of PTW-6 in other experiments was decided according the IC50. The highest concentration was 90μg/mL, which is no toxicity for cells. The effect of PTW-6 on HBsAg and HBeAg in supernatant was detected with ELISA assay. The results showed the inhibitory effect of PTW-6 on HBsAg and HBeAg increased with the concentration of PTW-6 increased. For HBeAg, the inhibitory rate is highest after 5 days treatment, the inhibitory rate is 90.6 % at the highest concentration. For HBsAg, the inhibitory rate is 95.1 % at the highest concentration. However, the inhibitory rate is very low in positive group. The significant difference was observed compared with positive control group(P<0.05). The inhibitory effect of PTW-6 on intracellular HBV mRNA and HBV DNA were detected by fluorescent quantitation PCR assay and southern blot assay, respectively. The results showed PTW-6 had inhibitory effect both on intracellular HBV mRNA and HBV DNA. The inhibitory effect of PTW-6 on extracellular HBV DNA was detected by dot blot assay. The result showed PTW-6 inhibited extracellular HBV DNA. The inhibitory effect is closed to that of positive control at 45μg/mL and 90μg/mL group.2. Anit-HBV activity in vivo: The ducks were used as animal model. The copy number of virus before and after treatment was analyzed by quantitation PCR. The results showed that the DHBV DNA decreased obviously after treatment with PTW-6, especially in high-dose group. Compared with negative control group, obvious significant difference was observed in high dose group(P<0.01), however, no significant difference was observed in middle and low dose group (P>0.05,P>0.05). 2 weeks after treatment, the copy number of DHBV DNA in PTW-6 groups kept stable, however, the copy number of DHBV DNA in ADV group is reversible. PTW-6 relieved the inflammation of liver. In high-dose, PTW-6 decreased the level of AST and ALT in serum.3. Toxicity of PTW-6: Kunming mice and wistar rats were used as animal model to evaluate the toxicity of PTW-6. The result of the acute toxicology of PTW-6 showed LD50 of PTW-6 is 2533.7 mg/kg, which prove PTW-6 is a compound with low toxicity. No observable toxicity can be found in 90 days experiment and special toxicity experiment. The effect of PTW-6 on mitochondria was detected by dot blot. No toxicity was observed. In a word, a novel polyoxometalate with anti-HBV activity was synthesized, which will improve the development of polyoxometalate on anti-DNA virus research.
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