李明+谭诗云

武汉大学人民医院消化内科，湖北武汉 430060

[摘要] 目的 观察丹皮酚对人结肠癌细胞系LoVo细胞增殖、凋亡的影响及探讨可能的作用机制。 方法 用不同浓度丹皮酚处理体外培养的LoVo细胞24、48、72、96 h，CCK-8比色法测定其对结肠癌LoVo细胞的活力的影响；倒置显微镜下观察细胞的形态学变化；根据CCK-8结果分为丹皮酚组（浓度31.25、62.50、125 mg/L）和对照组，处理48 h后，流式细胞仪检测细胞凋亡；激光共聚焦显微镜检测细胞内Ca2+浓度的变化；RT-PCR和Western blot法检测RUNX3基因表达情况。 结果 丹皮酚能显著降低LoVo细胞存活率，呈剂量、时间依赖性；倒置显微镜下可观察到典型的细胞凋亡形态；丹皮酚组（浓度31.25、62.50、125 mg/L）处理细胞48 h后，凋亡率分别为（12.3±1.3）%、（22.4±1.5）%、（36.2±2.1）%，与对照组[凋亡率（2.3±0.9）%]比较，差异均有统计学意义（P < 0.05）；丹皮酚组处理48 h后，细胞钙离子荧光强度分别为（41.36±4.62），（57.51±3.83）和（69.43±3.76），与对照组[荧光强度（24.45±3.74）]比较，差异均有统计学意义（P < 0.05）；丹皮酚能上调RUNX3基因的表达，呈剂量依赖性。 结论 丹皮酚能抑制LoVo细胞的增殖并诱导其凋亡，其作用机制可能与增加细胞内Ca2+含量和上调RUNX3基因的表达有关。

[关键词] 丹皮酚；LoVo细胞；Ca2+；RUNX-3

[中图分类号] R735.3 [文献标识码] A [文章编号] 1673-7210（2014）03（a）-0023-05

Effects of Paeonol on cell proliferation and apoptosis in human colorectal cancer LoVo cells and its mechanisms

LI Ming TAN Shiyun

Department of Gastroenterology， Renmin Hospital of Wuhan University， Hubei Province， Wuhan 430060， China

[Abstract] Objective To investigate the effect of Paeonol on the proliferation of colorectal cancer line LoVo and discuss its possible mechanisms. Methods LoVo cells were cultured in vitro. After treatment by Paeonol at different concentrations respectively at different time， the cell survival was determined by the CKK-8 method. The changes of cell morphology were observed by inverted microscope. Apoptosis was detected by flow cytometry. The concentration of intracellular calcium was investigated by laser confocal sanning microscope with calcium-flourecent probes-Fluo-3/AM. Reverse transcription-polymerase chain reaction （RT-PCR） was used for mRNA analysis. The change of protein expression of RUNX-3 was detected by western blot. Results From the data of CCK-8， the cell proliferation of human colorectal cancer LoVo cells was inhibited by Paeonol in a dose-dependent and time-dependent manner. Typical apoptosis morphology of LoVo cells was observed by inverted microscope. Flow cytometry assays showed that Paeonol significantly induced apoptosis in LoVo cells. After treated with Paeonol for 48 h， the apoptosis rate of Lovo cells was （12.3±1.3） %， （22.4±1.5） %， and （36.2±2.1） % respectively， which showed an obvious concentration-effect relationship. The Ca2+ relative fluorescence intensity of Paeonol group was （41.36±4.62）， （57.51±3.83） and （69.43±3.76） respectively， which indicated that the concentration of intracellular calcium was significantly elevated by Paeonol. The data of RT-PCR and western blot showed that Paeonol up-regulated RUNX3 in a dose-dependent manner in LoVo cells. Conclusion Paeonol can inhibit the proliferation of LoVo cells and induce apoptosis， and the mechanism of Paeonol on apoptosis may be related to the up regulation of RUNX3 expression and the increase of concentration of intracellular calcium.

[Key words] Paeonol； LoVo cell； Ca2+； RUNX-3

研究表明，非甾体类消炎药（non-steroid anti-inflammatory drugs，NSAIDs）能降低结肠癌的发生，减少家族性结肠息肉患者息肉数量和大小，并能预防结肠腺瘤的复发[1]。本室前期研究显示[2]乙酰水杨酸能抑制结肠癌细胞系环氧合酶-2（COX-2）表达而抑制生长，并诱导细胞凋亡。但临床研究表明长期应用NSAIDs可引起胃溃疡甚至胃出血及一系列心血管不良反应，限制了其在临床上的广泛应用。……