The anti-HBV activity and its mechanism of Boehmeria nivea

• Main Authors: Huang Kai-Ling, 黃凱玲
• Other Authors: Lai Yiu-Kay
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/65530755282219726790

博士 === 國立清華大學 === 生物科技研究所 === 95 === Hepatitis B virus (HBV) has infected more than 400 million people worldwide. About 20% of patients infected with HBV may lead to chronic hepatitis, liver cirrhosis, and hepatocarcinoma. Chronic hepatitis type-B patients are clinically treated with interferon alpha (INF-��) and nucleoside analogue such as lamivudine (3TC), adefovir or entecavir, which are analogues of reverse-transcriptase inhibitors; however, low responsiveness and drug-resistant mutant are major problems in treatment with chronic hepatitis B. Boehmeria nivea (B. nivea) has been distributed and used therapeutically in China and Taiwan for diuretic, antipyretic, and hepatoprotective purposes. In this thesis, the Chinese herbal medicine, the root extract of B. nivea (BNE) was studied for its anti-human HBV effects and its mechanism of action mode. In HepG2 2.2.15 cells, HBeAg and particle-associated HBV DNA secretion into supernatant were significantly inhibited by BNE at a dose of 100 μg/mL, whereas the HBsAg was not inhibited. With different doses of BNE, the reduced HbeAg was correlated with the inhibition of HBV DNA. The anti-HBV effect of BNE was not caused by its cytotoxicity to cells or inhibition of viral DNA replication and RNA expression. In an in vivo animal experiment, BNE also showed a potential anti-HBV activity in HBV-viremia SCID mice model. BNE was effective to inhibit the production of HBsAg and HBV DNA, while virus-producing tumor growth was not affected. Although, both nucleocapsid formation and envelopment by viral surface proteins were not altered by BNE; however, viral core and large-surface proteins accompanied with their encapsidated viral DNA were observed to accumulate within the cells. The proper folding and assembly of viral surface proteins are mediated by host chaperones. It has been reported that viral large- but not small- and middle-surface protein is required for the translocation process in endoplasmic reticulum (ER) during HBV morphogenesis and an ER luminal chaperone 78 kDa glucose-regulated protein (GRP78) bound to preS1 domain of large-surface protein to assist in proper folding. For investigating the target site of BNE, ER-related chaperone proteins were assessed. The ER chaperones such as GRP78, 94 kDa glucose-regulated protein (GRP94) and calnexin were not induced by BNE, BNE specifically decreased the intracellular GRP78 protein amounts. The possible mechanism of blockage of virions secretion by reduced GRP78 might be resulted from (1) mis-folded large-surface protein leading to failure of assembly of virus particle (2) failure to induce the ER stress response by accumulated large-surface protein, which turns to activate the synthesis of middle- and small-surface proteins, GRP78 and GRP94 in assistance of virions envelopment. Furthermore, the antiviral ability of BNE was observed reversible and the HBV DNA was restored by thapsigargin. By combining BNE and 3TC, it revealed an apparently additive anti-HBV effect, suggesting that BNE exploited different anti-HBV action mode from nucleoside analogues could combine other drugs for chronic hepatitis B therapy. In conclusion, the anti-HBV ability of BNE was evaluated and validated in in vitro and in vivo assay systems, and BNE has a potential to be developed as an anti-HBV drug.