石榴皮鞣质对膀胱癌T24细胞增殖及凋亡的影响

目的：研究石榴皮鞣质对膀胱癌T24细胞增殖及凋亡的影响并探讨其作用机制。方法：采用CCK-8法测石榴皮鞣质对膀胱癌T24细胞的增殖抑制作用、计算半数抑制浓度（IC₅₀）并筛选浓度区间，Hoechst染色法观察石榴皮鞣质作用后细胞核形态变化，Annexin V/PI双染法流式细胞术（FCM）检测细胞凋亡率，JC-1染色标记后流式细胞仪测定线粒体膜电位水平的变化，蛋白免疫印迹法检测细胞色素C （Cyt-C）、Bcl-2、Bax、Caspase-3凋亡相关蛋白表达。结果：石榴皮鞣质抑制膀胱癌细胞增殖呈时间浓度依赖关系，24，48，72 h的IC₅₀分别是72.70，62.26，19.64 μg·mL^-1。Hoechst染色观察石榴皮鞣质作用24 h后，细胞核出现浓染和荧光碎片为典型凋亡细胞。流式细胞术结果表明石榴皮鞣质具有显著的诱导T24细胞早期凋亡，线粒体膜电位下降。蛋白免疫印迹实验证明石榴皮鞣质上调Cyt-C、Bax蛋白的表达、下调凋亡抑制蛋白Bcl-2的表达，并激活Caspase-3蛋白的表达。结论：石榴皮鞣质显著抑制膀胱癌T24细胞增殖并诱导其凋亡，其作用机制可能与线粒体膜电位下降及调控线粒体凋亡途径相关蛋白的表达相关。

Study on the proliferation and apoptosis activity of pomegranate peel tannins on bladder cancer T24 cells

OBJECTIVE To study the effect of pomegranate peel tannins on the proliferation and apoptosis of bladder cancer T24 cells and explore its mechanism.METHODS Using CCK-8 method to detect the inhibitory effect of pomegranate peel tannins on bladder cancer T24 cells, and calculate the half inhibitory concentration (IC₅₀) and select the concentration range. Hoechst staining method was used to observe the changes of nuclear morphology after pomegranate peel tannins treatment. Annexin V/PI double staining flow cytometry (FCM) was used to detect the apoptosis rate. The changes of mitochondrial membrane potential were measured by flow cytometry after JC-1 staining. Western Blot analysis was used to detect the expression levels of Cyt-C, Bcl-2m, Bax and Caspase-3.RESULTS Pomegranate peel tannins inhibited the proliferation of bladder cancer T24 cells with a time and concentration dependent manner, the IC₅₀ values of 24, 48 and 72 h were 72.70,62.26 and 19.64 μg·mL^-1. Hoechst staining showed that after the pomegranate peel tannins treatment, the nucleus appeared concentrated staining and fluorescent fragments, which were typical apoptotic cells. The results of flow cytometry showed that the pomegranate peel tannins could induce the early apoptosis of T24 cells and decrease the mitochondrial membrane potential. Western blot analysis showed that tannins in pomegranate peel increased the expressions of Cyt-C and Bax, downregulated the expression of Bcl-2, and activated the expression of Caspase-3 protein.CONCLUSION The pomegranate peel tannins significantly inhibited the proliferation and induced apoptosis of bladder cancer T24 cells, which may be related to the decrease of mitochondrial membrane potential and the regulation of the expression of mitochondrial apoptosis pathway related protein.