2022 年第 8 期 第 17 卷

雷公藤红素对人恶性黑色素瘤细胞凋亡的影响

Effects of tripterine on inducing apoptosis of human malignant melanoma cells

作者：饶俊珍汪娟

英文作者：Rao Junzhen Wang Juan

单位：湖北省武汉市第一医院武汉市中西医结合医院药学部，武汉432000

英文单位：Department of Pharmacy Wuhan First Hospital Wuhan Hospital of Integrated Traditional Chinese and Western Medicine Hubei Province Wuhan 432000 China

关键词：性黑色素瘤细胞；雷公藤红素；细胞凋亡；凋亡蛋白；蛋白激酶R样内质网激酶抑制剂

英文关键词：Malignantmelanomacells;Tripterine;Apoptosis;Apoptosisprotein;ProteinkinaseR-likeERkinaseinhibitor

• 摘要：

• 目的 观察雷公藤红素对人恶性黑色素瘤MUM-2C细胞凋亡的影响，并探究其可能机制。方法 体外培养黑色素瘤MUM-2C细胞，采用不同浓度（0、0.125、0.25、0.5、1、2、4、8、16、32 μmol/L）的雷公藤红素处理黑色素瘤MUM-2C细胞，采用细胞计数试剂盒8（CCK-8）检测细胞活性确定实验浓度，根据选定浓度将黑色素瘤MUM-2C细胞分为对照组和低、中、高浓度组及抑制剂组；对照组无特殊处理，低、中、高浓度组分别采用1、2、4 μmol/L的雷公藤红素处理，抑制剂组采用蛋白激酶R样内质网激酶(PERK)抑制剂GSK2656157处理。采用流式细胞仪检测各组细胞周期及凋亡情况；采用蛋白质印迹法检测各组凋亡蛋白B淋巴细胞瘤2（Bcl-2）和Bcl相关X蛋白（Bax）表达情况；采用蛋白质印迹法检测各组细胞PERK信号通路蛋白及内质网应激标志蛋白免疫球蛋白重链结合蛋白（Bip）的表达情况。结果 根据CCK-8实验结果确定培养24、48、72 h的半抑制浓度分别为2.14、2.03、1.98 μmol/L，设置实验作用浓度为低剂量组1 μmol/L，中剂量组2 μmol/L，高剂量组4 μmol/L。使用低、中、高浓度雷公藤红素处理组和抑制剂组G0/G1期细胞比例、细胞的凋亡率［（15.2±3.0）%、（26.3±4.2）%、（37.3±5.0）%、（40.5±5.3）%比（8.4±2.1）%］、Bax蛋白相对表达水平明显高于对照组（均P<0.05），且对雷公藤红素呈剂量依赖性。使用雷公藤红素处理各组和抑制剂组G2/M期和S期细胞比例、Bcl-2、磷酸化PERK和Bip蛋白相对表达水平明显低于对照组（均P<0.05），且对雷公藤红素呈剂量依赖性。结论 雷公藤红素可以促进黑色素瘤MUM-2C细胞的凋亡，其作用机制可能与抑制PERK通路的激活介导细胞内质网应激，增加凋亡蛋白的表达诱导细胞凋亡相关。

• Objective To observe the effects of tripterine on the apoptosis of human malignant melanoma MUM-2C cells, and to explore its possible mechanism. Methods The melanoma MUM-2C cells were cultured in vitro and were treated with tripterine of different concentrations(0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 μmol/L). The cell activity was detected by cell counting kit-8(CCK-8) to determine the experimental concentration. According to the determined concentration, melanoma MUM-2C cells were divided into control group, low concentration group, medium concentration group, high concentration group and inhibitor group. The control group had no special treatment; the low, medium and high concentration groups were treated with 1, 2 and 4 μmol/L tripterine, respectively; the inhibitor group was treated with protein kinase R-like ER kinase(PERK) inhibitor GSK2656157. The cell cycle and apoptosis in each group were detected by flow cytometry. The expressions of apoptosis proteins B lymphocytoma 2(Bcl-2), Bcl-associated X protein(Bax), PERK signaling pathway proteins and endoplasmic reticulum stress marker immunoglobulin heary chain binding protein (Bip) in each group were detected by western blotting. Results According to CCK-8 results, the 50% inhibiting concentration concentrations for 24, 48 and 72 h were 2.14, 2.03 and 1.98 μmol/L, respectively. The experimental concentrations were set as 1 μmol/L in low dose group, 2 μmol/L in medium dose group, and 4 μmol/L in high dose group. Compared with control group, proportion of cells in G0/G1 phase, apoptosis rate［（15.2±3.0）%,（26.3±4.2）%,（37.3±5.0）%,（40.5±5.3）% vs （8.4±2.1）%］ and the relative expression level of Bax protein were significantly increased in low, medium and high concentration tripterine group and inhibitor group(all P<0.05), showing dose-dependence on tripterine. Compared with control group, proportion of cells in G2/M phase and S phase, and the relative expression levels of Bcl-2, phosphorylation PERK and Bip proteins were significantly decreased in each group treated with tripterine and inhibitor group(P<0.05), showing dose-dependence on tripterine. Conclusions  Tripterine can promote the apoptosis of melanoma MUM-2C cells, and its mechanism may be related to inhibiting the activation of PERK pathway, mediating endoplasmic reticulum stress, increasing the expression of apoptosis proteins and inducing apoptosis.