Objective: To investigate the effects of ursolic acid on T cell lymphoma cell lines-Hut-78 cells and its mechanism.
Methods: Inhibition of Hut-78 cells proliferation by ursolic acid at different concentration (10, 20, 40 and 80 μmol/L) for different incubation time (4, 12, 24, 48 and 72 h)was examined by MTT method, and early apoptosis by flow cytometry. The protein expressions of p65, p50, p52 and p100, and caspase-8, caspase-3 and caspase-9 were detected by Western blot. VEGF and COX-2 mRNA expressions were measured by reverse transcription polymerase chain reaction (RT-PCR).
Results: It was showed that ursolic acid inhibited proliferation of Hut-78 cells (P<0.05). Apoptosis of Hut-78 cells was induced by 10, 20, 40 and 80 μmol/L ursolic acid treatment (P<0.01). Likewise, expression of p65 and p50 proteins were down-regulated by ursolic acid treatment (10, 20, 40 and 80 μmol/L) (P<0.01), but there was no significant change in the expression of p52 and p100. Moreover, ursolic acid could up-regulate expression of caspase-8, caspase-3 and caspase-9 protein (P<0.01). RT-PCR examination showed that VEGF and COX-2 mRNA expression decreased by ursolic acid treatment.
Conclusion: Inhibition of Hut-78 cells proliferation may be related to ursolic acid induced apoptosis through h death receptors and mitochondrial pathways. NF-κB classical signal pathway may be one of its mechanisms, and VEGF and cox-2 may also be involved.
目的: 探讨乌索酸对T细胞淋巴瘤细胞株Hut-78细胞体外增殖的影响及其作用机制。
方法: 采用MTT法观察不同浓度（10、20、40、80 µmol/L）乌索酸作用不同时间(4、12、24、48、72 h)对Hut-78细胞增殖的抑制作用，并采用流式细胞术分析其早期凋亡的情况。采用免疫印迹法检测乌索酸作用后Hut-78细胞细胞核因子-κB（NF-κB）p65、p50、p52和p100亚基蛋白，以及caspase-8、3、9蛋白表达变化，采用逆转录PCR法检测血管内皮生长因子(VEGF)和环氧合酶-2(COX-2）mRNA表达水平。
结果: 与不加药对照组比较，不同浓度的乌索酸作用组Hut-78细胞的增殖均受抑制（P值均<0.05）；Annexin Ⅴ+/PI−细胞比例均升高（P值均<0.05）；经10、20、40、80 µmol/L乌索酸处理Hut-78细胞72 h后，Hut-78细胞NF-κB p65、p50蛋白和VEGF、COX-2 mRNA表达水平均下降（P值均<0.05），caspase-8、3、9蛋白表达均增加（P值均<0.05）；NF-κB p100、p52蛋白表达差异均无统计学意义（P值均>0.05）。上述作用均呈剂量和（或）时间依赖性（P值均<0.05）。
结论: 乌索酸抑制Hut-78细胞增殖，作用可能与促其凋亡有关，经过死亡受体和线粒体途径完成；影响NF-κB经典信号路径可能是其机制之一，VEGF和COX-2可能也参与其中。