黄连对小鼠成纤维细胞L929的毒性实验研究

doi:10.5246/jcps.2015.02.013

摘要/Abstract

摘要：

黄连是一种广泛应用的传统中药, 历来被认为无毒, 很少人注意到黄连的毒性及其在细胞和基因水平上相关的毒性机制。本实验以体外培养的小鼠成纤维细胞L929为对象, 从细胞存活率、细胞周期、DNA损伤及活性氧(ROS)的生成方面研究黄连的毒性作用及其可能机制。L929细胞与不同浓度的黄连共培养24 h后, CCK-8法检测细胞存活率, 倒置显微镜观察细胞形态变化, 流式细胞仪检测细胞周期变化及细胞内ROS的生成情况, 彗星实验检测细胞DNA的损伤情况。结果显示, 黄连浓度高于1 mg/mL, 细胞存活率显著降低, 且呈浓度依赖性; 高于1 mg/mL的黄连可明显改变细胞的正常形态; 2 mg/mL的黄连可使G2/M期细胞比例及细胞内ROS生成显著增多; 0.05 mg/mL的黄连可引起DNA各损伤指标明显增加。本实验表明黄连对细胞有毒性作用, 其毒性机制与细胞周期阻滞、DNA损伤及细胞内ROS的积累有关。

关键词: 黄连, 细胞毒性, L929细胞, 细胞周期, DNA损伤, 活性氧ROS

Abstract:

Rhizoma Coptidis (RC), a widely used traditional Chinese medicine, is commonly believed to be non-toxic. However, little is known about its cytotoxicity and relevant mechanisms at cellular and genetic levels. The present study aimed to explore the cytotoxicity of RC and its possible mechanisms related to cell cycle arrest, DNA damage and reactive oxygen species (ROS) level in L929 murine fibroblast cells. The cells were cultured in vitro and treated with different RC concentrations for 24 h. Cell viability was determined by CCK-8 method, morphological changes were observed with an inverted microscope, cell cycle and ROS level were examined by flow cytometry, and DNA damages were detected by comet assay. Our results showed that cell viability was significantly decreased in a dose-dependent manner when the RC concentration was higher than 1 mg/mL. A RC concentration above 1 mg/mL altered the morphology of L929 cells. Both cells at G2/M phase and the ROS level increased in the 2 mg/mL group. Each DNA damage indicator score increased in the groups with the RC concentration of above 0.05 mg/mL. Taken together, our studysuggested that RC at a high dosage exhibited cytotoxicity on L929 cells, which was likely to be the consequences of cell cycle arrest, DNA damage and accumulation ofintracellular ROS.

Key words: Rhizoma Coptidis, Cytotoxicity, L929 cells, Cell cycle, DNA damage, ROS

中图分类号:

R962

Supporting:

顾曼曼, 何燕飞, 韩春杨, 周祺, 刘腾飞, 黄燕飞, 刘翠艳. 黄连对小鼠成纤维细胞L929的毒性实验研究[J]. 中国药学(英文版), 2015, 24(2): 111-120.

Manman Gu, Yanfei He, Chunyang Han, Qi Zhou, Tengfei Liu, Yanfei Huang, Cuiyan Liu. Cytotoxicity effects of Rhizoma Coptidis on L929 murine fibroblast cells[J]. Journal of Chinese Pharmaceutical Sciences, 2015, 24(2): 111-120.

参考文献

[1] Peng, F.; Yang, Y.; Zhang, B.X.; Qu, X.Y.; Zhong, G.Y.; Luo, W.Z. J. Chin. Pharm. Sci. 2012, 21, 81-87.

[2] Tomohisa, H.; Kazuya, F.; Toshiyuki, N.; Tadashi, N.; Mikio, I.; Yoshio, S. Jap. J. Pharmacol. 1992, 59, 159-169.

[3] Yu, Y.; Wang, B.; Peng, L.; Wang, J.; Zeng, C. J. Chongqing Univ. (Natural Science Edition). 2006, 2, 107-111.

[4] Choi, U.K.; Kim, M.H.; Lee, N.H. J. Microbiol. Biotechnol. 2007, 1880-1884.

[5] Lu, J.; Wang, J.S.; Kong, L.Y. J. Ethnopharmacol. 2011, 134, 911-918.

[6] Tang, J.; Feng, Y.; Tsao, S.; Wang, N.; Curtain, R.; Wang, Y. J. Ethnopharmacol. 2009, 126, 5-17.

[7] Jung, H.A.; Min, B.S.; Yokozawa, T.; Lee, J.H.; Kim, Y.S.; Choi, J.S. Biol. Pharm. Bull. 2009, 32, 1433-1438.

[8] Hu, Y.; Yang, Y.F.; Yang, W.Z.; Zhang, Y.T. J. Chin. Pharm. Sci. 2014, 23, 385-392.

[9] Ye, X.S.; Feng, Y.B.; Tong, Y.; Ng, K.M.; Tsao, S.; Lau, G.K.K. J. Ethnopharmacol. 2009, 124, 130-136.

[10] Tjong, Y.; Ip, S.; Lao, L.; Fong, H.H.; Sung, J.J.; Berman, B. J. Ethnopharmacol. 2011, 135, 754-761.

[11] Chen, H.Y.; Ye, X.L.; Cui, X.L.; He, K.; Jin, Y.N.; Chen, Z. Fitoterapia. 2012, 83, 67-73.

[12] Yeung, C.Y.; Lee, F.T.; Wong, H.N. Biol. Neonate. 1990, 58, 98-103.

[13] Yang, Z.Y. Chin. J. Pediatrics. 2000, 38, 188-189.

[14] Yu, P.; Zheng, F.M.; Wu, J.L.; Zhou, Z.L.; Fu, Z.Q.; Zhao, L. Chin. J. Trad. Med. Sci. Technol. 2001, 8, 186-187.

[15] Li, F.Y.; Qiu, S.H.; Yin, J.K.; Sun, B.Q.; Cai, Y. J. Hunan Univ. Chin. Med. 2007, 27, 9-11.

[16] Qiu, S.H.; Li, F.Y.; Yin, J.K.; Wu, H.J.; Xiao, J.R. J. TCM Univ. Hunan. 2004, 24, 1-6.

[17] Zhang, X.Y.; An, L.H.; Shao, Z. Lishizhen Med. Mat. Med. Res. 2003, 14, 714.

[18] Feng, R.J. Med. J. Gentral Equipment. 2004, 6, 57.

[19] Liu, Y.Q.; Cheng, M.C.; Wang, L.X.; Xiao, H.B. J. Ethnopharmacol. 2010, 129, 121-126.

[20] Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L. Experimental Cell Res. 1988, 175, 184-191.

[21] Gao, J.J.; Feng, S.M.; Jin, W.F. Fudan Univ. J. Med. Sci. 2004, 31, 311-312.

[22] Nandhakumar, S.; Parasuraman, S.; Bhat, B.V. J. Pharmacol. Pharmacother. 2011, 2, 107-111.

[23] Xin, C.H.; Li, G.L.; Ji, Z.Y.; Gao, Y.; Yin, S.N. J. Toxicol. 2005, 19, 141-143.

[24] Liao, C.L.; Le, J.R.; Liu, S. China Med. College Annu. Bull. (Taiwan). 1982, 13, 1.

[25] Shen, T.; Jiang, T.R.; Wu, S.G. J. Sichuan Trad. Chin. Med. 2006, 24, 16-17.

[26] Yi, J.; Ye, X.L.; Wang, D.Z.; He, K.; Yang, Y.; Liu, X.J. J. Ethnopharmacol. 2013, 145, 303-310.

[27] Lee, Y.H.; Kim, D.; Lee, M.J.; Kim, M.J.; Jang, H.S.; Park, S.H. J. Ethnopharmacol. 2014, 152, 457-463.

[28] Cheong, H.I.; Johnson, J.; Cormier, M.; Hosseini, K. Toxicol. In Vitro. 2008, 22, 1070-1076.

[29] Han, J.; Back, S.H.; Hur, J.; Lin, Y.H.; Gildersleeve, R.; Shan, J. Nature Cell Biol. 2013, 15, 481-490.

[30] Stoddart, M.J. Methods Mol. Biol. 2011, 740, 21-25.

[31] Li, J.C.; Meng, X.L.; Fan, X.J.; Lai, X.R.; Zhang, Y.; Zeng, Y. China J. Chin. Mater. Med. 2010, 35, 1855-1858.

[32] Ma, B.L.; Ma, Y.M.; Shi, R.; Wang, T.M.; Zhang, N.; Wang, C.H. J. Ethnopharmacol. 2010, 128, 357-364.

[33] Ma, B.L.; Ma, Y.M. Exp. Opin. Drug Metab. Toxicol. 2013, 9, 51-61.

[34] Weinert, T. Cell. 1998, 94, 555-558.

[35] Ghosh, P.; Banerjee, M.; Giri, A.K.; Ray, K. Mutation Res. 2008, 659, 293-301.

[36] Leland, H.; Kastan, H.; Kastan, M.B. Am. Assoc. Adv. Sci. 1994, 266, 1821-1828.

[37] Yang, J.; Yu, Y.; Duerksen-Hughes, P.J. Mutation Res./Rev Mutation Res. 2003, 543, 31-58.

[38] Kruszewski, M.; Iwanenko, T.; Machaj, E.K.; Ołdak, T.; Wojewódzka, M.; Kapka-Skrzypczak, L. Mutagenesis. 2012, 27, 551-558.

[39] Liu, F.; Ng, T.B.; Zhao, L. J. Neural. Transm. 2000, 107, 1243-1251.

[40] Song, Y.; Lu, T.; Xie, L.; Liu, X.D. Chin. J. Exp. Trad. Med. Form. 2010, 16, 118-122.

[41] Bindhumol, V.; Chitra, K.C.; Mathur, P.P. Toxicology. 2003, 188, 117-124.

[42] Barzilai, A.; Yamamoto, K.I. DNA Repair. 2004, 3, 1109-1115.

[43] Hsu, W.H.; Hsieh, Y.S.; Kuo, H.C.; Teng, C.Y.; Huang, H.I.; Wang, C.J. Arch. Toxicol. 2007, 81, 719-728.

[44] Wood, C.D.; Thornton, T.M.; Sabio, G.; Davis, R.A. Int. J. Biol. Sci. 2009, 5, 428-437.

[45] Zhang, X.; Silva, D.D.; Sun, B.; Fisher, J.; Bull, R.J.; Cotruvo, J.A. Toxicology. 2010, 269, 13-23.