Mechanisms Of Styela Plicata (Lesueur) On Anti-Hepatitis B Virus In Vitro

Styela plicata is an ascidian which belongs to class Ascidiacea, subphylum urochordata, phylum Chordates, there are about 2000 kinds of ascidians have been found in the nature and they distribute in tropical and subtropical seas. Recent years, studying on the chemical composition and biological activity of ascidian has been one of the most popular domains in marine drug researches. Many chemical compositions such as peptid, alkaloide, macrolide, terpenoid, and so on, have been found in ascidian. Most of them have strong biological activity, such as anti-tumor, anti-virus, antibiosisi, immumity modulation and biocatalysisi, etc. And mechanisms on anti-tumor and anti-virus were better studed. But studies and reports were less seen in civil,our experiment institution was the only department which developed the research of Ascidiacea anti Hepatitis B virus in civil.In the vast ocean, specially in South China sea, there are abundant Styela plicata ascidian resources. On study of marine drugs, our research team made the basic research about the anti-virus effect of ascidian, the results showed the alcohol extract of Styela plicata has a definite inhibitory effect on hepatitis B virus in vivo and vitro. Now we had separated two activated monomers which are polypeptide, and we also separated its biochronometer and its n-butanol extraction position, we set up a finger print of Styela plicata's validation section. This study aimed to explore the anti-HBV mechanisms of Styela plicata further.Section 1 The inhibition effects of Styela plicata on Hepatitis B virus DNAObjectiveThis study was carried out according to cytology in vitro, we identified the Styela plicata's pharmacodynamic through inhiting HBsAg and HBeAg in HepG 2.215 cells,then tested HBV DNA copies in supernatant of cell culture by PCR to research the impacts and inhibition of HBV DNA.We analyzed the results and revealed the mechanisms of Styela plicata(lesueur) on anti -Hepatitis B virus in vitro.Methods1 HepG2 2.2.15 cells were incubated at 37℃5% CO2 5 days with different concentrations of Styela plicata and Lamivudine. By using ELISA to test HBsAg and HBeAg in culture supernatants, and MTT colorimetry to assay cell reproductive activity.2 HepG2 2.2.15 cells were incubated at 37℃5% CO2 with different concentrations of Styela plicata and Lamivudine, collecting culture supernanant of 48h and 96h.Using PCR to test the copies of HBV DNA.Results1 Differents concentrations of Styela plicata compared with cell group,the results had significant statistical difference(P=0.000,P<0.05).When Styela plicata's concentrations was higher than 0.5 mg·ml-1,it had good inhibition effects on both HBsAg and HBeAg.And its inhibition effects were influenced by the concentrations of Styela plicata.The concentrations were higher,the inhibition effects were stronger.2 The quantitative test results of HBV DNA copies from the drug-treated supernatant shows that different concentrations of Styela plicata extract inhibit HBV DNA expression in cells. And the inhibition effect increased with concentration (P <0.001).The inhibition effect of the HBV DNA secretion increased with concentration. As the highest concentration(2 mg·ml-1), and it inhibit 44.13%,46.40% of HBV DNA secretion at 48,96 hours respectively. The different concentrations of lamivudine also inhibit HBV DNA replication in cells. And The inhibition increased with concentration also. The inhibition effects increase with concentrations. When the concentration get to 1 mg·ml-1, it inhibit 17.41%,18.37% of HBV DNA secretion at 48,96 hours respectively. Both Styela plicata and lamivudine had similar inhibition effects on HBV DNA secretion. Section 2 The influence of Styela plicata on Hepatitis B virus DNA pclymeraseObjectiveThis part researched the activity of HBV DNA pclymerase and explored the mechanisms of Hepatitis B virus replication inhibition by Styela plicata. Then identified how the Styela plicata inhibited HBV DNA replication.MethodsPuring HBV DNA drops in HepG2.215 cell,PCR was beginning at the best action conditions and adding 32P-dCTP,which had radioactivity, in substrates, acting with different concentrations of Styela plicata and Lamivudine. At last,testing the radiation quantity of 32P-dCTPctp in HBV DNA,the double strand of HBV DNA were copied and extended by DNA pclymerase,if the activity of pclymerase were impacted,the radioactivity of 32P-dCTP in double strand would reduce,so we could know the impacts of DNA pclymerase activity which caused by Styela plicata and LamivudingResultsWhen the concentration of Styela plicata and Lamivudine were 1 mg·ml-1,their radiation quantity of 32P-dCTP compared with blank group, respectively(P< 0.05, >0.001).And when Lamivudine's concentration was 1 mg·ml-1,its radiation quantity of 32P-dCTP compared with blank group had significant difference. They both had statistical significance. From the results ,we knew Styela plicata extract product and Lamivudine both had impacts to activity of HBV DNA pclymerase, and their impacts were similar.The results of this experiment identied that Styela plicata extract product inhibited the secretion of HBV DNA from impacting activity of HBV DNA pclymerase. And its inhibition effects of HBV DNA replication was as well as Lamivudine. Our experiments based on predecessor' researchs but we had improvements that we identified the mechamisms of Styela plicata anti HBV,and the results of this task remind that Styela plicata might be an effective anticiral medicine in treating chronic hepatitis B virus .All of our experiments provided tational theories to develop this new and efficacious anti-HBV marine drug.