| Summary: | 碩士 === 中山醫學大學 === 營養學研究所 === 99 === In SARS-epidemic period, people thought that Isatidis Radix has an immuno-promotive effect, so Isatidis Radix is used as an adjuvant therapy for SARS. Indigofera suffruticosa Mill (ISM) is one of germplasm plants of Isatidis Radix. Because the leaves and stems of ISM could extract blue dye, Chinese people name ISM Ran-Bu-Qing. Our previous studies found that ISM extracts could inhibit lipopolysaccharides -induced inflammatory events in mouse RAW264.7 macrophages. However, the influence of ISM extracts on pi-glutathione S-transferase (GSTP) expression is not clear. We analyzed total amount of phenolic and flavonoids in ISM water extract (ISMWE) and ISM ethanol extract (ISMEE). Each gram of dry ISMWE and ISMEE contained 53.22±2.71 mg and 53.35±3.70 mg gallic acid equivalents, and 0.25±0.05 mg and 2.06±0.37 mg quercetin equivalents, respectively. Syringaldehydr is the major component of ISMWE and ISMEE analyzed by high pressure liquid chromatography (HPLC) and syringaldehyde could induce GSTP protein in Clone 9 liver cells. Clone 9 liver cells treated ISMWE (250, 500 or 1000 μg/ml) and ISMEE (250 or 500μg/ml) increased the pool of glutathione (p＜0.05). Immunoblotting analysis revealed that ISMWE and ISMEE increased GSTP protein levels in Clone 9 liver cells (p＜0.05). Additionally, ISMWE and ISMEE increased NAD(P)H quinone oxidoreductase 1 (NQO1) protein levels (p＜0.05). Real-time PCR analysis revealed that ISMWE and ISMEE increased GSTP mRNA expression (p＜0.05). Treated cells with 1000 μg/ml ISMWE or 500 μg/ml ISMEE significantly increased the phosphorylated extracellular signal-regulated kinase (ERK) protein levels (p＜0.05), but did not change the level of phosphorylated p38 and JNK. Clone 9 liver cells treated 20μM PD98059 (ERK inhibitor) could inhibit ISMEE-induced GSTP protein levels. Electrophoretic mobility shift assay (EMSA) revealed that 1000 μg/ml ISMWE or 500 μg/ml ISMEE increased binding affinity of nuclear protein to GSTP enhancer I (GPE I) DNA elements. Transient transfected with different GSTP promoter length plasmids (pTA-GSTP-Luc (-2713), pTA-GSTP-/--Luc (-2604), pTA-GSTP-/--Luc (-2375) or pTA-GPE I-Luc (-2713 to -2605)）showed that ISM induced luciferase activity when GPE I is included (p＜0.05). In addition, EMSA data showed that ISMEE also increased AP-1 binding to phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE). In summary, our data showed that ISM could induce GSTP expression.
|
|---|
