染布青萃出物對大鼠Clone 9 肝細胞pi屬麩胱甘肽轉移酶表現之影響

在SARS流行期間，因板藍根具有提高免疫力增強抵抗力的保健功效，認為板藍根可做為一種輔助治療SARS之方法。染布青為中藥材板藍根的基原植物之一，學名為Indigofera suffruticosa Mill （ISM），因可從染布青莖葉當中提煉出靛藍染料而稱之。先前本實驗室發現染布青可抑制脂多醣（lipopolysaccharides, LPS）所誘發小鼠巨噬細胞（RAW264.7）的發炎作用，但染布青對肝臟解毒效素pi屬麩胱甘肽轉移酶（glutathione S-transferase, GSTP）的影響與效應仍不清楚。首先分析兩種染布青萃出物總酚類與總類黃酮含量，總酚類分析顯示，染布青水萃出物（ISMWE）與染布青酒精萃出物（ISMEE）分別為53.22±2.71和53.35±3.70 mg 沒食子酸當量/g；總類黃酮分析顯示，ISMWE與ISMEE類黃酮含量分別為0.25±0.05和2.06±0.37mg類黃酮當量/g。進一步利用高壓液相層析儀（high pressure liquid chromatography, HPLC）以標準品分析ISMWE與ISMEE之成分，發現染布青內的syringaldehyde可能是誘發GSTP表現的可能有效成分之一。本研究以給予大鼠Clone 9肝細胞株不同濃度的ISMWE （250、500或1000 μg/ml）與ISMEE （250或500μg/ml），結果證實ISMWE和ISMEE皆具有增加胞內還原態glutathione含量之現象（p＜0.05）。利用西方墨點法分析發現染布青萃出物皆有誘發Clone 9肝細胞GSTP蛋白質，證明染布青具有提升解毒酵素GSTP之作用（p＜0.05），此外，ISMWE與ISMEE對phase II酵素NAD(P)H quinone oxidoreductase 1 （NQO1）也有誘發之現象（p＜0.05）。再利用Quantitative Real-Time PCR ( real-time PCR)證實ISMWE與ISMEE皆會誘發GSTP mRNA之表現（p＜0.05）。進一步探討ISMWE和ISMEE調控GSTP表現的可能訊息傳遞途徑，發現Clone 9細胞處理1000 μg/ml ISMWE或500 μg/ml ISMEE皆可促進extracellular signal-regulated kinase （ERK）磷酸化作用（p＜0.05），但不影響p38與c-Jun-NH2-terminal kinase （JNK）蛋白質。Clone 9細胞以20 μM PD98059 （ERK抑制劑）預處理，能抑制ISMEE誘發的GSTP蛋白質表現。另外利用Electrophoretic Mobility Shift Assay （EMSA）分析，結果證實細胞處理1000 μg/ml ISMWE或500 μg/ml ISMEE會增加胞內核蛋白與GSTP enhancer I （GPE I）之結合。利用暫時轉染（Transient Transfection），以四個不同長度GSTP promoter質體DNA （pTA-GSTP-Luc （-2713）、pTA-GSTP -/--Luc （-2604）、pTA-GSTP-/--Luc（-2375）及pTA-GPE I-Luc（-2713 to -2605））證實GPE I是染布青誘發GSTP蛋白質與mRNA表現的重要調控位置（p＜0.05）。另外，EMSA結果顯示ISMEE亦能促進AP-1與TRE序列之結合。總之，我們的數據顯示，ISM可能誘發 GSTP表現。

關鍵字

染布青； pi屬麩胱甘肽轉移酶大鼠Clone 9肝細胞

並列摘要

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.

並列關鍵字

Indigofera suffruticosa Mill ； pi-glutathione S-transferase ； Clone 9 liver cell

參考文獻

1. 何玉鈴, 郭昭麟, 高國清, 陳忠川, 張永勳: 板藍根藥材之鑑別研究. J Chin Med 2001;12.

2. 雛依芸: 大黃與板藍根抗日本腦炎病毒功效之研究. 中國醫藥大學藥學院藥學系碩士班碩士論文 2008.

3. Leite SP, de Medeiros PL, da Silva EC, de Souza Maia MB, de Menezes Lima VL, Saul DE: Embryotoxicity in vitro with extract of Indigofera suffruticosa leaves. Reprod Toxicol 2004;18:701-705.

4. Vieira JR, de Souza IA, do Nascimento SC, Leite SP: Indigofera suffruticosa: An Alternative Anticancer Therapy. Evid Based Complement Alternat Med 2007;4:355-359.

6. Lopes FC, Calvo TR, Colombo LL, Vilegas W, Carlos IZ: Immunostimulatory and cytotoxic activities of Indigofera suffruticosa (Fabaceae). Nat Prod Res 2011:1-11.

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染布青萃出物對大鼠Clone 9 肝細胞pi屬麩胱甘肽轉移酶表現之影響

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