| Objective:Hepatitis B is a common disease in China which is a serious threat to the lives and health of residents.Unfortunately,there is still lack of a very effective medicine for the treatment of hepatitis B currently.The ocean is an important part of the earth.Organisms living in marine may have a variety of anti-disease effect.This study attempted to evaluate the anti-HBV effect of 20 natural small molecules extracted from marine organisms in vitro.We hope to screen some substance which has an effect on anti-hepatitis B virus.Methods:20 marine small molecules in this study were provided from Guangxi Beibu Gulf marine research institute.These 20 compounds were as follows:1.In this study,HepG2.2.15 cell was used to evaluate the anti-HBV effect of marine small molecule in vitro.After HepG2.2.15 cells were attached to the wall,the secretion of HBsAg and HBeAg in the supernatant within 6 days was observated.If everything was normal,model was established successfully.The evaluation method of toxicity of marine natural active small molecules on HepG2.2.15 cells:Small molecule were configurated into 20mM,lOmM,5mM,2.5mM,1.25mM,0.625mM this 6 concentration gradient.Lamivudine(positive control)was configurated into 1 mM concentration.HepG 2.2.15 cells were seeded in 96 well plates with 1×107/L.After the cell were attached to the wall,then different concentration group and normal group in different drug containing medium respectively.2.After the cell were treated for 6 days,supernatant were blotted.Then 20?L of 5g/L MTT was added into plates.The plates were incubated at 37?for 4h and then shocked for lOmin.Then A490nm were detected.Finally,the cell inhibition rate and TC50 were calculated.3.Method for the determination of HBsAg and HBeAg in the supernatant of cell culture:The small molecule compounds H5,H10,H12 etc were dissolved by DMSO,then diluted by 1640 complete medium and prepared in 20mM,10mM,5mM,2.5mM,1.25mM 5 concentration gradient(The final concentration of DMSO is less than 0.1%).At the same time a negative control group(only with cell culture medium)and positive control group(lamivudine 1 mM).At the same time,a negative control group(only with cell culture medium)-and positive control group(lamivudine 1 mM)were set up.Each group set up 3 wells.After cell passage,the cells in good conditions at exponential phase were taken.The cell concentration was adjusted to 1×107/L and then inoculated in 96 well plates.Each hole was 200 uL.The plates were cultured in a Incubator for24h.After cell got adherent,the corresponding medium containing drugs were added to each drug group.Normal culture medium were added to negative control group.Supernatant at day 3 and day 6 after cells got adherent were collected respectively.The supernatant were kept at-20?.HBsAg and HBeAg supernatant at day 3 and day 6 were deteted by ELISA.After the color reaction appeared,A450nm were read by microplate reader.Then the antigen inhibition rate and 50%inhibition concentration(IC50)were got through the formula.4.Determination of HBV DNA in the supernatant of cell culture:According to the third experimental methods,supernatant at day 3 and day 6 were got.According to HBV DNA PCR quantitative determination kit,routine operation was taken.HBV DNA quantitative results were got and the inhibition rate of DNA and HBV IC50 were calculated.Results:1.After HepG2.2.15 cells appeared adherence,the secretion of HBsAg and HBeAg in the culture supernatant appeared stable growth within 6 days.There was no significant difference in cells inhibition between 0.625mM compounds group and negative control group.The difference in cytotoxicity 20mM compound group and 1mM lamivudine group was not significant.2.Inhibition of small molecule on the secretion of HBsAg and HBeAg:This study evaluated the anti-HBV activity of 20 marine natural small molecular co mpounds.Among them,when H5 concentrations was above 2.5mM,there was statistically significant differences in HBsAg and HBeAg in the supernatant on d3 and d6 between drug treatment group and negative control group(P<0.05).On d6,IC50 of H5 on HBsAg and HBeAg were 11.6mM and 12mM,and TI were 2.1 and 2 respectively.when H10 concentrations was above 2.5mM,there was statistically significant differences in HBsAg and HBeAg in the supernatant on d3 and d6 between drug treatment group and negative control group(P<0.05).On day6,IC50 of H10 on HBsAg and HBeAg were 9.9mM and 12mM,and TI were 2.5 and 2.1 respectively.When H12 concentrations was above 2.5mM,there was statistically significant differences in HBsAg and HBeAg in the supernatant on d3 and d6 between drug treatment group and negative control group(P<0.05).On d6,IC50 of H12 on HBsAg and HBeAg were 8.74mM and 9.7mM,and TI were 2.7 and 2.4 respectively.3.Inhibition of marine small molecules on HBV DNA:Among 20 compounds,when H5 concentrations was above 2.5mM,there was statistically significant differences in HBV DNA in the supernatant on d3 and d6 between drug treatment group and negative control group(P<0.05).On day6,IC50 of H5 on HBV DNA was 11.5,and TI was 2.1 respectively.When H10 concentrations was above 5mM,there was statistically significant differences in HBV DNA in the supernatant on d3 and d6 between drug treatment group and negative control group(P<0.05).On day6,IC50 of H5 on HBV DNA was 19.9 and TI was 2.5 respectively.When H12 concentrations was above 1.25mM,there was statistically significant differences in HBV DNA in the supernatant on d3 and d6 between drug treatment group and negative control group(P<0.05).On day6,IC50 of H5 on HBV DNA was 8.6 and TI was 2.73 respectively.Conclusions:1.The HepG2.2.15 cell line we established could stably secrete HBsAg and HBeAg and as time increased the secretion increased.2.The toxicity of all 20 Marine natural active small molecule substance to HepG2.2.15 cells is not obvious.3.Through the screening of 20 kinds of marine natural small molecules,it is found that H5,H10,H12 three compounds have anti-HBV activity.And with the increase of the concentration,their inhibitory action of HBsAg,HBeAg and anti-HBV DNA in HepG2.2.15 cell culture supernatant increase. |
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