小檗碱及其类似物体外缺糖缺氧条件下对PC12细胞生存及其对COX-2抑制作用的比较

doi:10.5246/jcps.2014.09.079

中国药学(英文版) ›› 2014, Vol. 23 ›› Issue (9): 617-625.DOI: 10.5246/jcps.2014.09.079

小檗碱及其类似物体外缺糖缺氧条件下对PC12细胞生存及其对COX-2抑制作用的比较

庞雨浓¹, 胡珺¹, 柴玉爽¹, 吴昊², 王玉刚¹, 雷帆¹, 邢东明¹, 杜力军^1*

1. 清华大学教育部蛋白质科学重点实验室, 生命科学学院医学院药物药理研究室, 北京 100084
2. NGM生物制药公司, 美国, 加利福尼亚州 94080, 南旧金山

收稿日期:2014-04-14 修回日期:2014-05-15 出版日期:2014-09-23 发布日期:2014-05-20
通讯作者: Tel./Fax: 86-10-62773630
基金资助:
National Natural Science Foundation of China (Grant No. 81374006, 81073092 and 90713043) and the National S&T Major Special Project for New Drug R&D Program of China (Grant No. 2012ZX09103-201-041, 2012ZX09102-201-008 and 2011ZX09101-002-11).

Comparison of berberine and its five analogues on cell viability and COX-2 expression during glucose-oxygen deprivation and reperfusion in PC12 cells

Yunong Pang¹, Jun Hu¹, Yushuang Chai¹, Hao Wu², Yugang Wang¹, Fan Lei¹, Dongming Xing¹, Lijun Du^1*

1. MOE Key Laboratory of Protein Sciences, Laboratory of Molecular Pharmacology and Pharmaceutical Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China
2. NGM Biopharmaceuticals, Inc., South San Francisco, California 94080, United States

Received:2014-04-14 Revised:2014-05-15 Online:2014-09-23 Published:2014-05-20
Contact: Tel./Fax: 86-10-62773630
Supported by:
National Natural Science Foundation of China (Grant No. 81374006, 81073092 and 90713043) and the National S&T Major Special Project for New Drug R&D Program of China (Grant No. 2012ZX09103-201-041, 2012ZX09102-201-008 and 2011ZX09101-002-11).

摘要/Abstract

摘要：

本文对小檗碱及其5个类似物对体外缺糖缺氧再灌注PC12细胞的保护作用及其对COX-2的抑制作用进行了实验观察, 并就它们的构效关系进行了分析。造模方法为缺糖缺氧4小时复灌24小时。以MTT法检测小檗碱及其类似物对细胞的保护作用。以实时定量PCR和Western blot方法检测COX-2的mRNA和蛋白的表达。结果表明, 小檗碱及其类似物均能够对抗缺糖缺氧再灌所引起的细胞损伤、抑制COX-2的表达。小檗碱和小檗红碱作用最强, 其最大有效剂量为0.31 μg/mL, 其最小有效剂量分别为0.02和0.04 μg/mL。巴马汀作用较弱。R₂和R₃位的亚甲二氧基是小檗碱及其类似物的活性基团。而且R₂, R₃, R₉和R₁₀位的亚甲二氧基影响小檗碱及其类似物对COX-2的亲和力。R₉位的羟基取代不影响其活性。

关键词: 小檗碱, 类似物, 环氧合酶-2, 构效关系

Abstract:

Berberine, an isoquinoline alkaloid component of Rhizoma Coptidishas been demonstrated to be the key active ingredient involved in its protective effect against cerebral ischemia-reperfusion. However, the comparison among the analogues to the protective effect against oxygen and glucose deprivation/reoxygenation (OGD-R) was mediated by inhibition of cyclooxygenase-2 (COX-2) has never been reported. The aim of this study is to investigate the protective effect of berberine and its five analogues against OGD-R in PC12 cells, as well as to determine whether the protective effect was regulated through COX-2. An established in vitro OGD-R model of PC12 cells by oxygen glucose deprivation of 4 h and reperfusion of 24 h was used in our study. After cells were treated with berberine or its five analogues, we examined the cell viability assay by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were also collected to determine the levels of mRNA and protein of COX-2 by real time PCR and Western blot. We found that berberine and its analogues improved the viability of PC12 cells against OGD-R. Whereas berberine and berberrubine presented stronger activity with the most effective dose of 0.31 μg/mL and the minimum effective doses of 0.02 and 0.04 μg/mL. Palmatine possessed potentially weaker protective effect. The mRNA level of COX-2 in cells treated with berberine, coptisine and epiberberine was decreased significantly. The protein level of COX-2 was significantly down-regulated in cells treated with berberine. Studies suggested the important role of methylenedioxy groups (R₂ and R₃) of berberine analogues in COX-2 inhibitory effect, and methylenedioxy groups (R₂, R₃, R₉ and R₁₀) in berberine analogues in binding affinity with COX-2. Substituted hydroxyl group at R₉ did not affect the activity of berberine. In summary, our study illustrated the protective effects of berberine and its analogues in PC12 cells against OGD-R and to elucidate the structure-activity relationships. Docking analysis indicates that methylenedioxys at R₂ and R₃is involved in the effect. More studies in other cells are needed to confirm our results.

Key words: Berberine, Analogues, COX-2, Structure-activity relationships

中图分类号:

R963

Supporting:

庞雨浓, 胡珺, 柴玉爽, 吴昊, 王玉刚, 雷帆, 邢东明, 杜力军. 小檗碱及其类似物体外缺糖缺氧条件下对PC12细胞生存及其对COX-2抑制作用的比较[J]. 中国药学(英文版), 2014, 23(9): 617-625.

Yunong Pang, Jun Hu, Yushuang Chai, Hao Wu, Yugang Wang, Fan Lei, Dongming Xing, Lijun Du. Comparison of berberine and its five analogues on cell viability and COX-2 expression during glucose-oxygen deprivation and reperfusion in PC12 cells[J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(9): 617-625.

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小檗碱及其类似物体外缺糖缺氧条件下对PC12细胞生存及其对COX-2抑制作用的比较

Comparison of berberine and its five analogues on cell viability and COX-2 expression during glucose-oxygen deprivation and reperfusion in PC12 cells

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