Brief Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jul 21, 2012; 18(27): 3617-3622
Published online Jul 21, 2012. doi: 10.3748/wjg.v18.i27.3617
Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment
Hong Shi, Lu Lu, Ning-Ping Zhang, Shun-Cai Zhang, Xi-Zhong Shen
Hong Shi, Ning-Ping Zhang, Shun-Cai Zhang, Xi-Zhong Shen, Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China
Lu Lu, Department of Gastroenterology and Hepatology, Shanghai Second People’s Hospital, Shanghai 200011, China
Author contributions: Shi H designed the research and wrote the paper; Shi H and Lu L performed the research; Zhang NP analyzed the data; Zhang SC and Shen XZ contributed to the design and critically revised the manuscript.
Supported by Zhongshan Youth Foundation of Fudan University, China, No. 257
Correspondence to: Hong Shi, MD, Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. shihongcn2000@yahoo.com.cn
Telephone: +86-21-64041990 Fax: +86-21-64038472
Received: January 30, 2012
Revised: April 18, 2012
Accepted: April 20, 2012
Published online: July 21, 2012
Abstract

AIM: To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following lamivudine treatment of HepG2.2.15 cells.

METHODS: HepG2.2.15 cells were treated with 2 μmol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (cytokine group), or treated with 2 μmol/L lamivudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 × 105 HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circular DNA that may have remained. Both HBV covalently closed circular DNA (cccDNA) and HBV DNA were examined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantified with enzyme-linked immunosorbent assay. Cell viability was measured with the cell counting kit-8 assay.

RESULTS: Compared to lamivudine alone (22.63% ± 0.12%), both sequential (51.50% ± 0.17%, P = 0.034) and cytokine treatment (49.66% ± 0.06%, P = 0.041) showed a stronger inhibition of HBV cccDNA; the difference between the sequential and cytokine groups was not statistically significant (51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88). The sequential group showed less inhibition of HBV DNA replication than the lamivudine group (67.47% ± 0.02% vs 82.48% ± 0.05%, P = 0.014); the difference between the sequential and cytokine groups was not statistically significant (67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071). The levels of HBsAg and HBeAg were significantly decreased in the sequential treatment group compared to the other groups [HBsAg: 3.48 ± 0.04 (control), 3.09 ± 0.08 (lamivudine), 2.55 ± 0.13 (cytokine), 2.32 ± 0.08 (sequential), P = 0.042 for each between-group comparison; HBeAg: 3.48 ± 0.01 (control), 3.08 ± 0.08 (lamivudine), 2.57 ± 0.15 (cytokine), 2.34 ± 0.12 (sequential), P = 0.048 for each between-group comparison]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells (58.03% ± 8.03% vs 100%, P = 0.000). Lamivudine pretreatment significantly reduced IFN-γ + TNF-α-mediated toxicity of HepG2.2.15 cells [85.82% ± 5.43% (sequential) vs 58.03% ± 8.03% (cytokine), P = 0.002].

CONCLUSION: Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-γ and TNF-α by decreasing the viral load.

Keywords: Hepatitis B virus; Covalently closed circular DNA; Interferon-γ; Tumor necrosis factor-α; Lamivudine