姜黄素衍生物对原代大鼠肝星状细胞作用的体外研究

中国临床药理学与治疗学 ›› 2014, Vol. 19 ›› Issue (10): 1086-1092.

姜黄素衍生物对原代大鼠肝星状细胞作用的体外研究

孙永¹, 黄小华¹, 沈能², 唐华东³, 任红¹, 彭明利¹

¹ 重庆医科大学感染性疾病分子生物教育部重点实验室,重庆 400010;
² 重庆市肿瘤医院,重庆 400030;
³ 浙江工业大学,杭州 310014,浙江

收稿日期:2014-04-21 修回日期:2014-05-21 出版日期:2014-10-26 发布日期:2014-10-29
通讯作者: 彭明利,女,博士,硕士生导师,副研究员,研究方向:肝病药物的开发与应用、纳米粒药物递送系统的研究、乙型肝炎免疫调控机理研究。 E-mail: sallypeng2002@gmail.com
作者简介:孙永,男,硕士研究生,研究方向:肝纤维化药物研究。 Tel: 18716246098 E-mail: cysunyong@163.com
基金资助:
国家自然科学基金项目(30771921)

Effects of Curc-OEG on primary rat hepatic stellate cells in vitro

SUN Yong¹, HUANG Xiao-hua¹, SHEN Neng², TANG Hua-dong³, REN Hong¹, PENG Ming-li¹

¹ Key Laboratory of Molecular Biology for Infectious Diseases, Chongqing Medical University, Chongqing 400010, China;
² Chongqing Cancer Institute, Chongqing 400030, China;
³ Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China

Received:2014-04-21 Revised:2014-05-21 Online:2014-10-26 Published:2014-10-29

摘要/Abstract

摘要： 目的观察姜黄素衍生物(Curc-OEG)抑制原代肝星状细胞(HSC)增殖、活化以及诱导其凋亡的作用,探讨其可能的作用机制。方法采用IV型胶原酶、DNA酶消化SD雄性大鼠肝脏,percoll梯度离心得到纯化的肝星状细胞。细胞分离后 1 d 分别加入0、6.25、12.5 μg/mL 的姜黄素衍生物,作用 7 d 后用RT-PCR及Western blot检测细胞α-SMA、TGF-β₁、Smad2的mRNA水平和蛋白表达量。活化的肝星状细胞在第14天分别加入0、6.25、12.5、25、50、75 μg/mL 的姜黄素衍生物,作用 24 h 后RT-PCR检测凋亡基因Bax、Bcl-2的mRNA水平和纤维化相关基因TGF-β₁、collagen I、NF-κB及TIMP-1的mRNA水平。结果药物作用 7 d 后,6.25 μg/mL 和 12.5 μg/mL 浓度药物处理组与对照组相比,细胞数目分别减少了56%和86%。在 12.5 μg/mL 浓度药物作用下,原代肝星状细胞α-SMA、TGF-β₁及Smad2的mRNA表达水平分别下调83%、85%及75%,蛋白表达水平分别下调94%、92%及73%(P<0.05)。25 μg/mL 浓度药物作用 24 h 后,细胞凋亡明显。在 50 μg/mL 浓度药物作用下,活化的肝星状细胞Bax的mRNA表达水平上调约 2.3 倍,Bcl-2的mRNA表达水平下调约 5.6 倍;TGF-β₁、collagen I、NF-κB及TIMP-1的mRNA表达水平分别下调90%、83%、74%、65%(P<0.05)。结论姜黄素衍生物可以明显抑制原代肝星状细胞的增殖和活化,促进活化的肝星状细胞凋亡及减少细胞外基质的沉积。

关键词: 肝纤维化, 姜黄素, 肝星状细胞, 活化, 凋亡

Abstract: AIM: To investigate the effects and mechanisms of Curc-OEG on the proliferation, activation and apoptosis of hepatic stellate cells (HSCs). METHODS: The HSCs were isolated from male SD rats by collagenase IV and DNase digestion of liver, and further purified by percoll gradient centrifugation. Freshly isolated cells at day 2 were treated with Curc-OEG at concentrations of 0, 6.25 and 12.5 μg/mL for 7 days. The expression of α-SMA, TGF-β₁ and Smad2 was confirmed by RT-PCR and Western blot. Activated HSCs at day 14 were treated with Curc-OEG at concentrations of 0, 6.25, 12.5, 25, 50 and 75 μg/mL for 24 h. The expression of Bax and Bcl-2 and other genes associated with fibrogenesis including TGF-β₁, collagen I, NF-κB and TIMP-1 was observed with RT-PCR. RESULTS: After 7 days cultivation, Curc-OEG caused a significant concentration-dependent reduction in cell numbers of 56% and 86% at 6.25 and 12.5 μg/mL respectively compared with the control. At 12.5 μg/mL, Curc-OEG reduced α-SMA, TGF-β₁ and Smad2 mRNA levels by 83%, 85% and 75%, and protein levels by 94%, 92% and 73%, respectively (P<0.05). Cell apoptosis was significantly increased at a concentration of 25 μg/mL Curc-OEG. At 50 μg/mL, Curc-OEG significantly upregulated the mRNA levels of the proapoptotic Bax by 2.3-fold,downregulated the antiapoptotic Bcl-2, TGF-β₁, collagen I, NF-κB and TIMP-1 by 5.6-fold, 90%, 83%, 74% and 65%, respectively (P<0.05). CONCLUSION: Curc-OEG not only significantly inhibited the proliferation and activation of primary HSCs, but also induced apoptosis of activated HSCs and reduced ECM accumulation.

Key words: hepatic fibrosis, curcumin, hepatic stellate cell, activation, apoptosis

中图分类号:

R965.2

孙永, 黄小华, 沈能, 唐华东, 任红, 彭明利. 姜黄素衍生物对原代大鼠肝星状细胞作用的体外研究[J]. 中国临床药理学与治疗学, 2014, 19(10): 1086-1092.

SUN Yong, HUANG Xiao-hua, SHEN Neng, TANG Hua-dong, REN Hong, PENG Ming-li. Effects of Curc-OEG on primary rat hepatic stellate cells in vitro[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(10): 1086-1092.

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