丹皮酚对人大肠癌HT-29细胞增殖、凋亡的影响及其分子机制

Effects of paeonol on apoptosis and proliferation of HT-29 cells and its molecular mechanism

目的:探讨丹皮酚(paeonol,Pae)对人大肠癌HT-29细胞的增殖抑制、凋亡诱导作用及可能的作用机制.方法:应用MTT法、荧光显微镜及透射电镜技术、TUNEL法和流式细胞仪技术观察不同浓度的Pae对HT-29细胞增殖的抑制及凋亡的诱导作用;应用免疫细胞化学技术检测用药前后凋亡相关基因Bcl-2,Bax及P53表达的变化.结果:HT-29细胞经Pae(浓度范围7.81-250 mg/L)作用后细胞生长明显受到抑制,呈明显的剂量依赖效应关系和时间依赖效应关系;荧光显微镜及透射电镜观察到Pae作用后HT-29细胞出现典型的细胞凋亡形态;Pae在15.63,62.5,250 mg/L 3种浓度下作用48 h均可诱导HT-29细胞凋亡.TUNEL法显示凋亡指数与Pae浓度呈正相依赖关系:流式细胞仪技术检测其凋亡率分别为7.6%,16.2%和34.5%,也有明显的剂量效应关系,同时Pae使细胞周期分布发生明显变化,表现为S期细胞比例上升,G_0/G_1期和G_2/M期细胞比例下降;免疫细胞化学结果显示Pae作用后HT-29细胞Bcl-2及P53蛋白表达显著降低,Bax蛋白表达无显著改变.结论:Pae能抑制HT-29细胞增殖并诱导其凋亡,呈现明显的剂量依赖效应关系和时间依赖效应关系.其作用可能与影响癌细胞的细胞周期、下调Bcl-2/Bax的比例及P53蛋白的表达有关.

AIM： To investigate the effects of paeonol in inhibiting proliferation of human colorectal cancer cell line HT-29 and inducing their apoptosis, and the possible molecular mechanisms involved. METHODS： The inhibitory effect of paeonol on HT-29 cell proliferation was detected by MTT assay. Induction of apoptosis of HT-29 cells was measured by fluorescence microscopy, transmission electron microscopy, TUNEL assay and flow cytometry （FCM）. Expression of apoptosis-associated genes bcl-2, bax and P53 was observed by immunocytochemical staining. RESULTS： Paeonol at a concentration of 7.81-250 mg/L inhibited the proliferation of HT-29 cells, with obvious concentration-and timeeffect relationships. Typical apoptosis morphology of HT-29 cells was observed by fluorescence and transmission electron microscopy after treatment with paeonol. Paeonol induced apoptosis of HT-29 cells when it was applied at a concentration of 15.63, 62.5 or 250 mg/L after 48 h. TUNEL assay showed a concentration-effect relationship between paeonol and apoptosis index. By FCM, the apoptosis rate of HT-29 cells was 7.6%, 16.2% and 34.5% respectively, which showed an obvious concentration-effect relationship. Cell cycle distribution was altered simultaneously. The S phase of the cells was increased, while the G0/G1 and G2/M phases were decreased after treatment with paeonol. Immunocytochemical staining showed that the expression of Bcl-2 and p53 was decreased significantly, while the expression of Bax protein was not significantly altered by paeonol CONCLUSION： Paeonol inhibits HT-29 cell proliferation and induces apoptosis. This may be mediated via changes in the cell cycle, downregulation of the Bcl-2/Bax ratio, and expression of p53 protein.