摘要目的 研究内皮素3(ET-3)对人恶性黑素瘤(MM)A375细胞核因子(NF)-κB/Bfl-1抗凋亡通路的调节.方法 用ET-3(100 nmol/L)刺激A375细胞24 h后,流式细胞仪检测细胞凋亡率.RT-PCR及Western印迹测定不同浓度ET-3(0、1、10、100 nmol/L)及其与内皮素受体B(ETRB)阻断剂BQ788联合干预A375细胞后,Bfl-1在mRNA和蛋白质水平的表达.Western印迹检测相同条件干预下磷酸化NF-κB(pNF-κB)的蛋白表达.结果 ET-3可以降低A375细胞的凋亡率(F=10.68,P＜0.05).ET-3对A375细胞Bfl-1 mRNA和蛋白表达水平的影响呈浓度依赖性上调,BQ788明显阻断ET-3上调Bfl-1的效应(P＜0.01).Western印迹检测结果显示,ET-3显著上调pNF-κB的表达(P＜0.05),BQ788干预下其表达水平降低,接近对照组,ET-3(100 nmol/L)+BQ788组与100 nmol/L ET-3组比较,差异有统计学意义(P＜0.01).结论 ET-3/ETRB通过激活NF-κB/Bfl-1抗凋亡通路抑制A375细胞凋亡.

abstractsObjective To investigate the modulation of ET-3 on the nuclear factor (NF)-κB/Bfl-1 antiapoptotic pathway in a malignant melanoma cell line A375. Methods Flow cytometry was performed to detect the apoptosis in cultured A375 cells after treatment with ET-3 of 100 nmol/L for 24 hours. ET-3 of various concentrations (0, 1, 10, 100 nmol/L) was used to treat some A375 cells with or without the pretreatment with the ETRB antagonist BQ788; after another 24-hour culture, RT-PCR and Western blot were conducted to examine the mRNA expression of Bfl-1 and protein expressions of Bfl-1 and ETRB, respectively. Results The 24-hour treatment with ET-3 of 100 nmol/L significantly reduced the apoptosis rate of A375 cells (F = 10.68, P ＜0.05). The mRNA and protein expressions of Bfl-1 were up-regulated by ET-3 in a concentration dependent manner (both P ＜ 0.01 ), while BQ788 significantly blocked the ET-3-induced up-regulation (F = 420.38,229.49, both P ＜ 0.01 ). The protein expression of pNF-κB in A375 cells was also enhanced by ET-3 of different concentrations (all P ＜ 0.05), but the enhancement was suppressed by BQ788, and there was a significant difference in the protein expression of pNF-κB between cells treated with ET-3 of 100 nmol/L and those treated with the combination of ET-3 of 100 nmol/L and BQ788 (F = 255.46, P ＜ 0.01 ). Conclusion ET-3/ETRB inhibits the apoptosis in A375 cells likely by activating the NF-κB/Bfl-1 anti-apoptotic pathway.