中藥水提出物在中國倉鼠細胞及人類血管內皮細胞之抗氧化活性研究

體內氧化壓力上升和DNA修補能力下降，與老化與癌症的形成發展有密切的相關性。本研究選取先前篩選具有體外抗氧化活性的五種中藥，包括(川芎、茵陳蒿、生地黃、牡丹皮及山藥)，探討其水提出物對中國倉鼠卵巢細胞(CHO-K1)及人類血管內皮細胞(ECV304)氧化傷害修補可能的調節活性及作用機轉。分別以彗星分析法測量中藥水提出物對過氧化氫及硫酸銅造成DNA傷害之保護潛力；以西方墨點法分析中藥水萃物對(1)細胞存活蛋白質(p53, Bcl-2, Bax, Hsp70)、(2)DNA傷害修補蛋白質(8-oxoguanine DNA glycosylase, mutT homolog 1, mutY Homolog )、(3)抗氧化蛋白質(heme oxygenase-1, superoxide dismutase -1, catalase)在ECV304細胞表現程度之影響；以TMM (tetramethylmurexide) 之方法測試水解物對銅離子的螯合能力。結果發現，在CHO-K1及ECV304細胞，山藥、川芎水提出物與硫酸銅的共同處理，可有效抑制硫酸銅所造成之DNA傷害，抑制百分比範圍從22%~38%；山藥、川芎、生地黃、牡丹皮、茵陳蒿水提出物，分別前處理CHO-K1及ECV304細胞48小時，可有效抑制過氧化氫所造成之DNA傷害，抑制百分比範圍從18%~63%。ECV304細胞前處理山藥、川芎、生地黃、牡丹皮或茵陳蒿水提出物48小時，明顯增加Bax、Bcl-2、MUTYH、MTH1或HO-1蛋白質的表現。此外，在五種中藥中，山藥及川芎水提出物更具有良好的螫合銅離子能力。由這些結果推測，川芎及山藥抑制過氧化氫或硫酸銅造成DNA氧化傷害的抗氧化活性，至少與螫合銅離子的能力，以及誘導細胞存活、DNA傷害修補、抗氧化相關蛋白質表現的增加有關。

關鍵字

山藥；牡丹皮；川芎；茵陳蒿；生地黃； DNA傷害修補； DNA修補酵素； CHO-K1細胞； ECV304細胞

並列摘要

BACKGROUND: A variety of evidence suggests that chronic oxidative stress may accelerate aging and increase risk of carcinogenesis. Therefore, exploring natural compounds that possess high antioxidant activity should be useful in both treating cancer and delaying aging. OBJECT: The object of this study was to evaluate the anticlastogenic effects of aqueous extracts from five traditional Chinese medicines with high free radical scavenging activity on two different cell models (CHO-K1 and ECV304 cells). METHODS: The comet assay was used for DNA damage detection, crystal violet stain was used for cell proliferation detection, Western blotting was used to detect the protein level of antioxidant enzymes (HO-1, catalase, SOD-1) and DNA repair proteins (Ogg1, MTH-1, and MYH). RESULTS: Pre-treatment of aqueous extracts derived from five different Chinese medicinal herbs (Dioscorea alata山藥, Ligusticum chuanxiong 川芎, Rehmania glutinosa 生地黃, Paeonia suffruticosa 牡丹皮, and Artemisia scoparia 茵陳蒿) for 48-hr decreased the DNA strand breaks level induced by H2O2 in CHO-k1 and ECV304 cells as compared with untreated control cells. The percent inhibition of H2O2-induced DNA strand breaks was found to be in the range 18-63%. Co-treatment of aqueous extracts derived from five different Chinese medicinal herbs, only D. alata and L. chuanxiongcould significantly inhibit the CuSO4-induced DNA strand breaks in CHO-K1 and ECV304 cells. However, the inhibition of CuSO4-induced DNA strand breaks by R. glutinosa and Artemisia scoparia 茵陳蒿was found only in ECV304 cells but not CHO-K1 cells. The percent inhibition of CuSO4-induced DNA strand breaks by extracts of TCM was found to be in the range 15-38%. In ECV304 cells, forty-eight hours pre-treatment of five different traditional Chinese medicines could significantly up-regulate Bax, Bcl2, MUTYH, MTH1 or HO-1 protein expression The level of L. chuanxiong -induced HO-1 expression was attenuated by Wortmannin (a phosphatidylinositol 3-kinase, PI3K inhibitor), SR11302 (a AP-1 inhibitor), and Bay 11-7082 (a NFκB inhibitor). CONCLUSION: These results suggest that the anticlastogenic activities of aqueous extracts of L. chuanxiong, A. scoparia, R. glutinosa, and P. suffruticosa may be partially through the up-regulation of antioxidant and DNA repair enzymes expression and copper-chelating activity. The data also suggest that L. chuanxiong- induced HO-1 expression, at least in part, through PI3K, AP-1 and NFκB signaling pathways.

並列關鍵字

DNA damage & repair ； Comet assay ； Anticlastogenic effect ； Dioscorea alata ； Paeonia suffruticosa ； Ligusticum chuanxiong ； Artemisia capillaries ； Rehmannia glutinosa, Bax ； Bcl-2 ； MUTYH ； MTH-1 ； HO-1 ； PI3K ； NFkB ； AP-1 ； CHO-K1 ； ECV304

參考文獻

Afonso, V., Santos, G., Collin, P., Khatib, A. M., Mitrovic, D. R., Lomri, N., Leitman, D. C., and Lomri, A. (2006). Tumor necrosis factor-alpha down-regulates human Cu/Zn superoxide dismutase 1 promoter via JNK/AP-1 signaling pathway. Free Radic Biol Med 41, 709-721.

Alfonso-Prieto, M., Biarnes, X., Vidossich, P., and Rovira, C. (2009). The Molecular Mechanism of the Catalase Reaction. J Am Chem Soc.

Ando, T., Mimura, K., Johansson, C. C., Hanson, M. G., Mougiakakos, D., Larsson, C., Martins da Palma, T., Sakurai, D., Norell, H., Li, M., et al. (2008). Transduction with the antioxidant enzyme catalase protects human T cells against oxidative stress. J Immunol 181, 8382-8390.

Aronovitch, Y., Godinger, D., Israeli, A., Krishna, M. C., Samuni, A., and Goldstein, S. (2007). Dual activity of nitroxides as pro- and antioxidants: catalysis of copper-mediated DNA breakage and H2O2 dismutation. Free Radic Biol Med 42, 1317-1325.

Arsova-Sarafinovska, Z., Matevska, N., Eken, A., Petrovski, D., Banev, S., Dzikova, S., Georgiev, V., Sikole, A., Erdem, O., Sayal, A., et al. (2009). Glutathione peroxidase 1 (GPX1) genetic polymorphism, erythrocyte GPX activity, and prostate cancer risk. Int Urol Nephrol 41, 63-70.

國際替代計量

中藥水提出物在中國倉鼠細胞及人類血管內皮細胞之抗氧化活性研究

全文下載

主題瀏覽