| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 95 === Part I
We previously developed a gene-gun-based in vivo screening system and reported the identification of shikonin as a potent suppressor of tumor necrosis factor TNF- gene expression. Here, we show that shikonin can selectively inhibit the expression of TNF- at the RNA splicing level. Treatment of lipopolysaccharide-stimulated primary monocytes and THP-1 cells with shikonin resulted in normal transcriptional induction of the TNF- gene, but unspliced pre-mRNA accumulated at the expense of functional mRNA. This effect occurred with noncytotoxic dosages of shikonin and was highly specific, because neither a housekeeping gene nor another inflammatory cytokine gene, IL-8, was affected under the same treatment. Moreover, administration of shikonin increased the expression of the IL-8 protein, which was accompanied by suppressed-activation of the dsRNA-activated protein kinase (PKR) pathway in THP-1 cells. Because PKR inactivation has been shown to downregulate the splicing process of TNF-RNA and interfere with translation, our findings suggest that shikonin may achieve differential modulation of TNF- and IL-8 protein expression through inactivation of the PKR pathway in THP-1 cells and also reveal that regulation of pre-mRNA splicing may constitute a promising target for future anti-inflammatory application.
Part II
To characterize possible specific immune-modulatory activities of three anti-inflammatory phytocompounds (emodin, shikonin, and cytopiloyne), and a putative medicinal herb extract from Echinacea purpurea, we conducted a focused DNA microarray study for selected immune-related genes, using the LPS-stimulated THP-1 monocytic cells. As a quick response (0.5 hour post incubation), cells treated with shikonin showed its significantly inhibitory effect on expression of several genes, including cytokines (TNF-α, IL-1 and IL4), chemotaxis and cell migration (CCL4 and CCL8), and inflammatory response (NFATC3 and PTGS2). Emodin, surprisingly, showed a quite similar early (~ 0.5 hours) response on most of these genes. In contrast, both cytopiloyne and BF/S+L/Ep showed little inhibitory effect on the expression of genes tested at early stage. In comparison, BF/S+L/Ep showed the inhibitory effect at the late stage (~12 hours post incubation) on expression of several genes, including cytokines (IL4), chemotaxis and cell migration (CDH1 and ITGA2), and inflammatory response (NFATC3, PTGS2, and GATA2). By Key node analysis specified with the TRANSPATH® database, the possible key regulators for emodin or shikonin were identified in E6-AP or Rad23A respectively, which primarily regulate the ubiquitin pathway. Our results also demonstrated a similar effect of BF/S+L/Ep and cytopiloyne on the kinetic gene expression pattern in LPS-induced THP-1 cells, and we identified the ERK1/2 activation pathway as the putative target of these two preparations by Key node analysis. Taken together, our results indicated that shikonin and emodin possessing strong and quick anti-inflammatory activity might interfere with the ubiquitin pathway, and suggested the presence of similar activity between cytopiloyne and BF/S+L/Ep. This study offers mechanistic insights and candidate molecular targets for the future development of these phytocompounds or fractionated plant preparations as a defined health supplement or herbal medicine.
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