| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === Helioxanthin-145 (HE-145) is a lignin isolated from the heartwood of Taiwania cryptomerioides Hayata. Previous studies showed that HE-145 has good anti-hepatitis B virus (HBV) activity. HE-145 suppresses HBV RNA, viral protein expression, and HBV DNA replication. In order to find out the target of HE-145, a series of HE-145 analogs have been synthesized. After screening of HBsAg-inhibiting analogs, it was found that an analog 145-12 exhibiting good potency. In the present study, human hepatoma cells HepG2/A2 and HepES2 were used as models to investigate whether 145-12 has any anti-HBV effects like HE-145.
Both cell line HepG2/A2 and HepES2 are derived from HepG2 cells by transfecting HBV DNA. HepG2/A2 cells constitutively secret HBsAg and HBeAg into the culture medium. HepES2 cells continually produce ayw type HBV viral particles. In HepG2/A2 cells, 145-12 suppressed HBsAg and HBeAg secretion with EC50 = 0.02 ug/ml and 0.05 ug/ml. According to the results of reporter assays, 145-12 suppressed the activity of core promoter (CP), S1 promoter (S1P), S2 promoter (S2P), and enhancer I/X promoter complex (E/XP) involving enhancer I. However these suppressions were not observed in 293T cells, suggesting these effects are liver specific. In HepES2 cells, 145-12 not only suppressed the secretion of Dane particles (HBV virion), but also inhibited HBV core protein and RNA expressions. These results suggest that 145-12 has good anti-HBV activity that is similar to HE-145.
Activities of promoters and enhancer I are regulated by transcription factors. To study how HE-145 and 145-12 suppress the expressions of HBV. Transcription factors were overexpressed in HepG2/A2 cells to investigate the effects of these transcription factors on HBV inhibition. It was found that overexpression of PPARγ reversed HE-145 or 145-12-mediated suppressive effects on E/XP activity. In HepES2 cells, overexpression of PPARγ reversed HE-145 or 145-12-mediated suppressive effect on HBV core proteins and also reversed the suppressions on HBV 3.5 kb RNA and 2.4/2.1 kb RNA of these two compounds.
In the present study, I show 145-12 has good anti-HBV activity similar to HE-145. Moreover, I found that PPARγ is involved in HE-145 or 145-12-mediated HBV suppression, offering a clue that the target might be one of members in PPARγ pathway.
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