| Hepatitis B virus (HBV) persistent infection can cause chronic hepatitis,which often leads to liver cirrhosis and even hepatocellular carcinoma. Approximately 7.18% of the population in our country is hepatitis B surface antigen carrier, HBV chronic infection affect people's healthy severely, lakhs of people in china die each year from hepatitis B-related complications such as liver cirrhosis, hepatocellular carcinoma and fulminant hepatitis.The pathogenesis of chronic hepatitis is multiplicity, HBV specific cytotoxic T lymphocyte(CTL) are typically weak and rarely detectable in peripheral blood in chronic HBV infection. It is well known T Helper subpopulations are classified two types T Helper cell 1(Th1) and T Helper cell 2(Th2) according to the production of cytokines, Th1 principally induces cell-mediated immunity and play a crucial role in the protection from intracellular pathogens,including a number of viruses, while Th2-derived cytokines can assist the B cell to encourage antibody production and promote humoral immunity. Th1 cell play an important role in the CTL function maintenance. In immunopathogenesis of chronic hepatitis B, it has been suggested that a preferential shift towards a Th1-polarized phenotype could be associated with a successful control of HBV infection by promoting CTL function, while an early commitment towards the Th2 synthesis plays a significant role in HBV persistant infection. Andrographolide is a diterpene lactones compound isolated from the leaves of andrographis paniculata, extensive researchs of the last few decades have revealed it is useful as an anti-inflammatory, anti-viral, anticancer, antibacterial and immunomodulatory agent. The research shows andrographolide exerts immunomodulation by inducing IFN-γ, IFN-αand TNF-αsynthesis from PBMCs of healthy adult. It is unknown whether andrographolide treatment triggers the change of immune response to patients with chronic hepatitis B and its HBV inhibitory activity. The present study has two parts. in the first part of the study,PBMCs from patients with chronic hepatitis B were cultured with andrographolide, the expressions of IFN-y mRNA, IL-4 mRNA and IL-lOmRNA from PBMCs were detected by real-time PCR. in the second part, The different concentrations Andrographolide were cultured with HepG2.2.15 cells, the replication of HBV DNA in HepG2.2.15 cells were detected by real-time PCR to investigate its anti-HBV activity in vitro.Obejective: To investigate in vitro effect of andrographolide on the expressions of the Thl cytokine (IFN-y mRNA) and the Th2 cytokines (IL-4 mRNA, IL-10 mRNA) from PBMCs of patients with chronic hepatitis B.Method:Venous blood samples from patients with chronic hepatitis B and healthy volunteers were collected into heparinized tubes, PBMCs were obtained by centrifugation of blood over Ficoll solution,The cells were adjusted to the concentration of 4×106/mL, cells toxicity test, time course study and drug dose study were primarily assessed. PBMCs from patients with chronic hepatitis B were cultured with 10mg/L andrographolide diluted with Dimethyl Sulfoxide (experimental group) and 0.1% Dimethyl Sulfoxide (control group). The cells were cultured for 16 hours in cell incubator and then harvested. RNA was extracted from PBMCs and reversed transcription complementary DNA. The expressions of IFN-y mRNA, IL-4 mRNA and IL-10 mRNA from PBMCs were detected by real-time PCR.The cytokines expressions of PBMCs from patients with chronic hepatitis B and healthy volunteers were compared and we contrasted the difference of cytokines expressions between experimental group and control group. The results were analyzed using the Wilcoxon rank test.Results:The expression of IL-4 mRNA from patients with chronic hepatitis B was higher than healthy volunteers and the expressions of IFN-y mRNA and IL-10 mRNA had no obviously differences. PBMCs from patients with chronic hepatitis B were stimulated with 10 mg/l or 0.1%DMSO for 16 hours, The expression of IFN-y mRNA from PBMCs cultured with andrographolide was higher than from PBMCs cultured with 0.1% DMSO (Z=-2.78,P=0.005), and the expressions of IL-4 mRNA and IL-10 mRNA from PBMCs cultured with andrographolide were lower than PBMCs cultured with 0.1% DMSO (Z=-3.82,P<0.001). The proportion of IFN-γmRNA and IL-4 mRNA from PBMCs with andrographolide was higher than from PBMCs cultured with 0.1% DMSO (Z=-3.82, P<0.001)Conclusion:Andrographolide can modulate the expressions of IFN-γmRNA, IL-4 mRNA and IL-10 mRNA from PBMCs of patients with chronic hepatitis B and alter the balance of Th1/Th2 in vitro.Objective:To investigate the anti-hepatitis B virus activity of andrographolide by detecting the intracellular of HBV DNA level in HepG2.2.15 cells.Method:HepG2.2.15 cells were recruited and cultured in DMEM medium. The cells were adjusted to the concentration of 5×105/mL by DMEM medium and put into the 12-round-bottom-well plate. The experiental groups were cultured with different concentrations andrographolide 0mg/L,5mg/L, 10mg/L and the control groups were cultured with different concentrations adefoviur 0umol/L,25umol/L and 50umol/L for senven days, each parallel concentration were double-pored and the culture solution includind identical concentration medicine was changed every other day. Examine the HepG2.2.15 cells viability and extract the virus core particle HBV DNA. The intracellular HBV DNA level of experiental groups and control groups were determined by quantitative Real-Time PCR and compared the HBV DNA levels among groups cultured with different concentrations medicine.The results were analyzed with t test.Results:The intracellular HBV DNA level had no significant difference stimulated by different concentrations andrographolide (P>0.05), while the replication of HBV DNA was decreased in the control groups (P<0.05).Conclusion:No significant differences were observed on the duplicate levels of the intracellular HBV DNA among groups treated with different concentrations andrographolide, the study shows andrographolide had no inhibitory activity on HBV DNA replication. |
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