丹皮酚对人黑素瘤A375细胞增殖和凋亡的影响

目的 探讨丹皮酚对体外培养人黑素瘤A375细胞增殖和凋亡的影响及其作用机制。 方法 CCK8法检测0.5，1，2，4，8 mmol/L丹皮酚作用24，48 和72 h后A375细胞增殖水平。用0、1.25、2.5和5 mmol/L浓度丹皮酚作用24 h后，以Annexin-Ⅴ/PI法观察A375细胞凋亡的变化、分别测定caspase 3，caspase 8和caspase 9的活性，并以Western印迹检测p53，NF-κB及相关蛋白水平的变化。 结果 与空白对照组比较，0.5，1，2，4，8 mmol/L丹皮酚作用24，48 和72 h后均对A375细胞的增殖有抑制作用，且与时间、浓度呈依赖性。1.25、2.5、5 mmol/L丹皮酚在作用24 h时，A375细胞早期凋亡率由对照组的（3.11 ± 0.53）%分别上升至（13.74 ± 1.73）%、（25.95 ± 0.57）%、（46.44 ± 0.81）%，各组与对照组间差异均有统计学意义（P < 0.05或 < 0.01）。2.5 mmol/L和5 mmol/L作用组的caspase 3，caspase 8，caspase 9活性升高，且与对照组间比较，差异均有统计学意义（P < 0.05或< 0.01）。p53及Bax的蛋白水平随药物浓度的增加而升高，NF-κB、bcl-2、bcl-XL的蛋白水平随药物浓度增加而降低。 结论 丹皮酚对黑素瘤A375细胞具有抑制增殖和诱导凋亡的作用。可通过细胞内和细胞外两条途径发挥促凋亡作用，其机制可能与调节p53及NF-κB基因有关。

Effects of paeonol on the proliferation and apoptosis of A375 human melanoma cells

Tao Yue*, Zhang Mengli, Ma Pengcheng, Sun Jianfang, Zhou Wuqing, Bao Jun. *Department of Dermatology, Nanjing Drum Tower Hospital, Nanjing 210008, China Corresponding authors: Ma Pengcheng, Email: mpc815@163.com；Sun Jianfang, Email: fangmin5758@aliyun.com 【Abstract】 Objective To study the effect of paeonol on the proliferation and apoptosis of A375 human melanoma cells and its mechanism. Methods Cell counting kit-8 （CCK8） was used to evaluate the proliferative activity of A375 cells treated with paeonol of 0.5，1，2，4，8 mmol/L for 24, 48 and 72 hours respectively. Subsequently, A375 cells were treated with paeonol of 1.25, 2.5 and 5 mmol/L for 24 hours followed by double staining with annexin Ⅴ and propidium iodide for the detection of cell apoptosis, fluorometric assay for the estimation of caspase 3, caspase 8 and caspase 9 activity, and Western blot for the determination of the levels of p53, nuclear factor-κB proteins and some of their target proteins. The A375 cells receiving no treatment served as the blank control group. Statistical analysis was carried out by t test. Results Within the investigated concentration and time ranges, paeonol significantly inhibited the proliferative activity of A375 cells in a concentration- and time-dependent manner. Compared with the blank control group, a significant increase was observed in the early apoptosis rate in A375 cells treated with paeonol of 1.25, 2.5 and 5 mmol/L for 24 hours （13.74% ± 1.73%, 25.95% ± 0.57% and 46.44% ± 0.81% vs. 3.11% ± 0.53%, P < 0.05 or 0.01）, as well as in the activity of caspase 3, 8 and 9 in A375 cells treated with paeonol of 2.5 and 5 mmol/L for 24 hours （P < 0.05 or 0.01）. After 24-hour treatment, the protein levels of p53 and Bax were elevated, but those of nuclear factor-κB, Bcl-2 and Bcl-XL were decreased in A375 cells with the increase of paeonol concentration. Conclusions Paeonol can inhibit the proliferation but induce the apoptosis of A375 cells, and the apoptosis-inducing effect may be realized through intrinsic and extrinsic pathways by modulating nuclear factor-κB and p53 genes.