Anti-viral mechanism of natural constituents from Artemisia morrisonensis against hepatitis B virus and molecular phylogenetic analysis of related Artemisia plant species

Autor: Shu-Heng Liu, 劉書亨
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
Artemisia morrisonensis （AM）is an endemic alpine species of Artemisia in Taiwan and is proposed to have wide range of therapeutic effects on liver related disease. However, the active constituent of A. morrisonensis against hepatitis B virus (HBV) is still remained unknown. The aims of this study were attempted to elucidate the anti-viral mechanism of natural occurring constituents against HBV from A. morrisonensis and the molecular phylogenetic analysis of Artemisia related species was also be conducted. Initially, the extracts of AM-AQ, AM-EA1, AM-EA2, AM-EA3, AM-EA4 and pure compound AM-EA22-3 were obtained from A. morrisonensis and tested their cytotoxicity on HepG2 2.2.15 cells during 9-day treatment. The cytotoxic effect was occurred during the treatment of HepG2 2.2.15 with AM-EA and EA3 whose dose was higher than 40 μg/ml, and with AM-EA2 higher than 10 μg/ml after 9-day treatment. The cytotoxic effect of AM-EA22-3 was only observed in the doses more than 160 μg/ml. However, there is no apparent cytotoxic effect on cells that with AM-AQ, AM-EA1, and AM-EA4 during treatment. On the other hand, the conditioned media from non-cytotoxic treatment of AM-EA2, AM-EA3, and AM-EA22-3 were collected to measure the secreted level of HBsAg and HBeAg. The results indicated that all of these constituents reduced the secretion of viral antigens (HBsAg and HBeAg) in a dose dependent manner. Furthermore, viral DNA of secredted viral particle was isolated from cell cultural media after non-cytotoxic treatment and detected with Real-Time PCR in order to determine the effect of AM constituents on replicative viral DNA level. The results indicated that all of these three extracts exhibit the inhibitory effect on HBV viral DNA replication. To examine the anti-viral mecanism of these three AM constituents (AM-EA2, AM-EA3, and pure compound AM-EA22-3), HBV viral promoter-luciferase reporter transfected Huh7 cell was used as model to test whether the treatment of these constituents have effect on viral gene promoters’ activity. The results indicated that the promoters correspond to HBV Core, X, and PreS were significantly up-regulated by AM-EA3 and AM-EA22-3. Furthermore, treatment of AM-EA2 specifically increases the HBV Core and PreS promoters’ activity. The activation of HBV viral mRNA levels was also detected in Northern blot during treatment. The previous study have shown that the increase of PreS relative to S promoters’ activity will lead to the overexpression of large surface protein and cause the intracellular accumulation of viral particle and induce endoplasmic reticular (ER) stress. To examine this possibility, HBV transfected Huh7 cell were treated with AM-EA2, AM-EA3 and AM-EA-22-3 and Western blot was conducted to analyze the expressed level of viral surface proteins (HBsAg) and ER stress induced GRP proteins. The results indicated that treatment of these two extracts and one pure compound significantly decreased the level of GRP78 protein, but slightly increase GRP94 portein level in Huh7 cells. However, the expression level of three froms of viral surface proteins (SHBs, MHBs and LHBs) in treated cells has no apparent change. In addition, the accumulation of viral surface antigen (HBsAg) was observed during treatment. These results suggest that the anti-hepatitis B virus mechanism of A. morrisonensis might mediate through the regulation of viral gene expression and induce ER stress and lead to the intracellular accumulation of viral particle. Taken together, the bioactive constituents of A. morrisonensis could serve as resources of therapeutic agent in the drug of development on virus-caused hepatitis. In the molecular phylogenetic analysis, four closely-related species of Artemisia and four Compositae species were chosen and the Internal Transcribed Spacer (ITS) sequences within these species were cloned and sequenced for phylogenetic analysis. The results revealed that A. oligocarpa and A. capillaries were co-evolved with A. morrisonensis, suggesting the possible naturally occurred constituents of these three Artemisia species may possess similar constituents against HBV.
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