Inhibitory Effect Of Astragaloside IV On Human Hepatitis B Virus Proliferation In Vitro

Chronic hepatitis B virus (HBV) infection is one of the major public health problems in the world. It is demonstrated by epidemiology data that more than 2 billion people have been infected with HBV and chronic HBV infection affects about 400 million people. Chronic HBV infection could cause cirrhosis, liver failure, and hepatocellular carcinoma (HCC). It is estimated that more than 500,000 people die annually due to cirrhosis and/ or hepatocellular carcinoma. China is the highly infected area in which 10 percent populations are infected with HBV and the healths as well as economic problems caused by HBV infection are big problems troubled people. Current options to treat chronic HBV infection are restricted to the use of interferon alfa (IFN-α) and nucleoside analogues such as lamivudine or adefovir, but these treatments have some drawbacks, including the impossibility to completely eliminate HBV from infected individuals, the spread of multi-drug resistance, insufficient therapeutic durability and frequent side effects associated with HBV as well as expensive drug price. It is therefore important to develop a new strategy to treat HBV patients. Scores of active components extracted from traditional Chinese medicines had been screened by us. Several active components had been demonstrated to have significantly antiviral effects against HBV such as artemisinin, matrine and liposome-encapsulated matrine, costunolide and ellagic acid extracting from Phyllanthus urinaria.Radix astragali is the dried root of perennial herbs, Astragalus membranaceus(Fisch.)Bunge, and Astragalus mongholicus Bunge, of the family leguminosae. Its chemical components include polysaccharides, flavonoids, saponins, numerous amino acids, trace elements, and various other components. Besides conventional usage, many antiviral activities of Radix astragali have been reported in the scientific literature. Astragaloside IV is a major bioactive constituent of the root of Radix astragali, which has been reported to have comprehensive pharmacological action, including anti-inflammatory, anti-hypertensive, and anti-diabetic. In the present work, it has been proved that astragaloside IV has antiviral effects against coxsackievirus B3 by upregulating expression of IFN-gamma mRNA. However, the effect of astragaloside IV for inhibition of HBV replication has not been examined. In the present study, we observed the effects of Astragaloside IV on HBV replication in vitro. The results might provide some useful data and idea for the further study of Astragaloside IV and for our native new medicine development.Experiment 1 HepG2.2.15 cell assaysHBsAg and HBeAg assays Objective: To study secretion of HBsAg and HBeAg in the collected culture supernatant. Methods: The HepG2.2.15 cells were cultured, and then set the ELISA kits as the positive control group, culture solution as the negative control group and collected culture supernatant from HepG2.2.15 cells at day10, 20, 30 as experiment group. The secreting effects of extracellular HBsAg and HBeAg in the collected culture supernatant of each well of cells were detected by enzyme-linked immunosorbent assay (ELISA). The optical density (A) was determined with the ELISA reader at 450nm. Result:HepG 2.2.15 cell stably secreted HBsAg and HBeAg in a time-dependent way. Conclusion: In the study, HepG2.2.15 cell line could be used as good model system in vitro.Experiments 21 Cytotoxicity assays Objective: To investigate the cytotoxic activity of astragaloside IV on HepG2.2.15 cells in vitro. Methods: Primarily the HepG2.2.15 cells which is stably transfected with HBV DNA were cultured with astragaloside IV in a series of concentrations, lamivudine at final concentrations of 10.0 mg/L, 5.0 mg/L and 1.0 mg/L, used as positive control, respectively. The same volume of culture medium was added to the control wells. HepG2.2.15 cells were observed and their viability was assessed by MTT (methyl thiazolyl tetrazolium) assay at 9 days. Results: After 9 days of treatment with 100mg/L and 50 mg/L of astragaloside IV, the toxicity on HepG2.2.15 cells was 50.6% and 35.1%, respectively. The 50% cytotoxic concentration (TC50) of astragaloside IV was about 97.4 mg/L. When HepG2.2.15 cells were treated with 10.0 mg/L～1.0 mg/L concentrations of lamivudine for 9 days, the toxicity on cells was 10.5%～6.9%. Conclusion: The cytotoxic activity of astragaloside IV was limited. In this study, no apparent cytotoxicity was observed for lamivudine.2 HBsAg and HBeAg evaluation Objective: To study secretion of HBsAg and HBeAg in the collected culture supernatant. Methods: The inhibitive effects of extracellular HBsAg and HBeAg in the collected culture supernatant of each well of cells were detected by ELISA. The optical density was determined with the ELISA reader at 450nm. Result:Astragaloside IV produced dose-dependent inhibition of HBsAg and HBeAg excreted by HepG2.2.15 cells. The 50% inhibitory concentration (IC50) of HBsAg and HBeAg was 53.5 mg/L and 59.4 mg/L at 6 days, respectively. When administrated for 9 days, astragaloside IV produced dose-dependent inhibition of HBsAg and HBeAg excreted by HepG2.2.15 cells, with a 50% inhibitory concentration (IC50) of 22.1 mg/L and 26.2 mg/L, respectively, and selectivity index (TI) was 4.4 and 3.7, respectively. When drug concentrations of lamivudine are 10 mg/L～1.0 mg/L, inhibitive rates of HBsAg and HBeAg were 25.3%～3.5% and 39.6%～3.6% at 6 days, respectively. At 9 days of treatment, lamivudine inhibited HBsAg and HBeAg to 35.2%～2.7% and 43%～4.2%, respectively. Conclusion: Astragaloside IV and lamivudine had inhibitive effects on expression of HBsAg and HBeAg secreted by HepG2.2.15 cells in a time-dose dependently.3 HBV DNA assay Objective: To assay extracellular HBV DNA in the collected culture supernatant. Methods: HBV DNA was extracted from culture medium and examined by real-time PCR based on the TaqMan technology. Result:Astragaloside IV had obviously effects on HBV DNA replication in vitro. IC50 of HBV DNA was 39.9 mg/L at 6 days and 13.2 mg/L at 9 days, respectively. Astragaloside IV's TI was approximately 7.4. In this study, lamivudine could inhibit the extracellular HBV DNA levels too. When HepG2.2.15 cells were treated with 10 mg/L～1.0 mg/L concentrations of lamivudine for 6 days, the extracellular HBV DNA levels were 36.4%～3.4% of the control levels, respectively. After an additional 9 days, the levels of extracellular HBV DNA increased to 49.5%～5.8% of the control levels. Conclusion: Astragaloside IV and lamivudine had inhibitive effects on proliferation of HBV DNA in a time-dose dependently.