雷公藤红素抑制人胆囊癌NOZ细胞增殖及诱导凋亡的研究

Celastrol inhibits growth and induces apoptosis of human gallbladder cancer NOZ cells

目的研究雷公藤红素对人胆囊癌NOZ细胞增殖及凋亡的影响，探讨其可能的分子作用机制。方法体外培养NOZ细胞。细胞接受药物处理后，采用CCK-8实验、AnnexinV—FITC／PI双染细胞凋亡检测及细胞周期检测实验研究雷公藤红素对NOZ细胞增殖及凋亡的影响。采用罗丹明123测定细胞线粒体膜电位变化，最后采用蛋白印迹法检测Bax和Bcl蛋白表达水平。结果雷公藤红素可以抑制人胆囊癌NOZ细胞增殖，其IC50值为5．3μmol／L。Annexin V—FITC／PI双染实验显示雷公藤红素可诱导细胞凋亡，对照组细胞凋亡比例为4．4％，而给药组（2、5、10μmol／L）细胞的凋亡比例分别为7．4％、27．1％和43．4％。细胞周期实验显示雷公藤红素可诱导NOZ细胞出现G1期阻滞，其中对照组处于G1期细胞比例为25．6％，而药物处理组（2、5、10μmol／L）细胞处于G1期的比例分别为36．5％、45．7和92．5％。细胞经过药物处理后，细胞线粒体跨膜电位明显下降。蛋白印迹实验显示NOZ细胞接受药物处理之后，细胞中Bax蛋白表达水平上升，Bcl-2蛋白表达水平下降，并呈现一定的时间依赖性。结论雷公藤红素能显著抑制人胆囊癌NOZ细胞增殖并诱导其凋亡。雷公藤红素可能通过增强线粒体通透性来诱导细胞凋亡。

Objective To investigate the effects of celastrol on the cell growth and apoptosis of human gallbladder cancer NOZ cells, and explore its potential molecular mechanism. Methods NOZ cell were cultured in vitro. And CCK-8 assay, Annexin V-FITC/PI staining method, cell cycle analysis were conducted to investigate the effects of celastrol on the growth and apoptosis of NOZ ceils after being treated with drugs. The mitochondrial membrane potential and Bax and Bcl-2 protein expression level were determined by Rhodamine 123 and Western blot, respectively. Results Celastrol could inhibit NOZ cell growth, and the IC50 value was 5.3 μmol/L. Annexin-V/PI staining showed that cell apoptosis of NOZ cells were induced as the celastrol concentration increased, and the apoptosis ratio of control group was 4.4% , while the apoptosis rates of the test groups （2, 5, 10μmol/L） were 7.4% , 27.1% and 43.4%, respectively. In addition, cell cycle analysis revealed that celastrol could induce Gl-phase arrest, The G1-phase rate of control group was 25.6%, while the G1-phase rates of the test groups （2, 5, 10 μmol/L） were 36. 5%, 45.7% and 92. 5%, respectively. The mitochondrial membrane potential was measured after treatment with celastrol and the results indicated that the mitochondrial membrane potential was significantly decreased. Western Blot showed that the protein expression of Bax increased and Bcl-2 decreased in a time-dependent manner after treatment with eelastrol. Conclusions Celastrol may inhibit cell proliferation of human gallbladder cancer NOZ cells and induce cell apoptosis partly by inducing the loss of mitochondrial membrane potential.