| Summary: | 博士 === 國立陽明大學 === 傳統醫藥學研究所 === 94 === Abstract
It is generally accepted that drug-drug, cell-cell, drug-cell interactions are involved in host responses to medical drugs. The aims of this study are to test our hypothesis that liver responses to a herbal remedy could be demonstrated by gene expression profiling, and to elucidate the effects of Scutellaria baicalensis Georgi (SbG) extract and its constituents on macrophage-hepatocyte interaction in primary cultures.
The compositions of the ingredients in the remedy containing Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd (S/B remedy) were analyzed and quantified by high performance liquid chromatography. By using 70% partial hepatectomy in BALB/c mice as in vivo model, effects of high dose (50 mg/kg) and low dose (1 mg/kg) S/B remedy were evaluated by cDNA microarray, followed by RT-PCR and real-time PCR confirmation. Factors affecting proliferative activities of mouse hepatocytes were measured by DNA flow cytometry, BrdU incorporation assay as well as serum interleukin-6 (IL-6) and transforming growth factor ��1 (TGF-��1) levels. In in vitro, trans-well (for Kupffer cells) and/or conditioned medium (for RAW 264.7 cells) systems, MTT and trypan blue exclusion and BrdU incorporation were used to elucidate the proliferative effects of SbG on primary hepatocyte cultures. SbG-stimulated cytokines productions, antibody-neutralization studies and mechanistic studies on TGF-β1 gene expression were performed in RAW264.7 cell line. Besides, recombinant human TGF-β1 (r-human TGF-β1) was added to elucidate the mechanisms of SbG effects on cultured hepatocytes. The mechanisms of SbG-treated primary hepatocyte growth were assayed by immunostain for NF-�羠 activation and toll-like receptor 4 mutant mice.
Based on global gene expression profiles, the results showed that high dose of S/B remedy up-regulated the expression of immediate early genes and cell cycle related genes, whereas low dose of S/B remedy had opposite effects. The gene expression of immediate early genes and cell cycle genes was further verified by real-time RT-PCR. Besides, proliferative activities, in terms of synthetic phase fractions and G2/M phase fractions in vehicle, low dose, high dose group were 18.45 ± 2.56%, 14.65 ± 1.06%; 9.27 ± 0.85%, 7.8 ± 0.11% and 18.45 ± 2.56%, 14.65 ± 1.06%, respectively. The serum IL-6 level was enhanced in high dose treatment whereas in low dose treatment had opposite effects. On the other hand, serum TGF-��1 level was decreased in high dose treated group and was increased in low dose treated group. In the in vitro cell-cell interaction experiments, the results showed that SbG stimulated the proliferation of cultured hepatocytes, whereas SbG-treated macrophage-induced conditioned medium (SbG-RAW-CM) and SbG in trans-well significantly suppressed the proliferation of cultured hepatocytes. The level of TGF-β1 in SbG-RAW-CM was higher than that of TNF-α and IL-6. Antibody-neutralization studies revealed that TGF-β1 was the main anti-mitotic cytokine in SbG-RAW-CM. The proliferation effect of SbG on cultured hepatocytes was inhibited by exogenous administration of r-human TGF-β1. Besides, the effects of SbG on hepatocyte growth were related to NF-�羠 activation but not to the toll-like receptor 4 pathway.
We conclude that global gene expression profiling by genomics approach may predict host responses to medical herbs, and that understanding the mechanisms of herbal effects on Kupffer cell-hepatocytes interaction provides a new direction for pharmaceutics studies on human diseases.
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