摘要
目的观察蛇床子素增强肿瘤坏死因子对骨肉瘤细胞的凋亡能力及其机制。方法将骨肉瘤细胞系HOS分为对照组、TNF-α组及蛇床子素联合TNF-α组和蛇床子素+TNF-α+CYLD si RNA组;MTT法检测HOS细胞的细胞活力,Annexin V/PI染色检测HOS细胞的凋亡,免疫共沉淀联合Western blot法检测HOS细胞RIP1蛋白的泛素化,Western blot法检测HOS细胞caspase-8的活化和CYLD去泛素化酶的相对表达量。结果与对照组比较,TNF-α组、蛇床子素+TNF-α组对HOS细胞活力的抑制率增加[(12.5±0.9)%、(58.6±3.8)%比0,P<0.05],TNF-α组、蛇床子素+TNF-α组对HOS细胞凋亡诱导率提高(8.3±0.5)%、(39.9±2.5)%比(1.9±0.3)%,P<0.05],而蛇床子素+TNF-α+CYLD si RNA组的HOS细胞活力抑制率、HOS细胞凋亡诱导率均低于蛇床子素+TNF-α组[(22.1±1.2)%比(58.6±3.8)%,P<0.05;(13.5±0.9)%比(39.9±2.5)%,P<0.05]。与对照组、TNF-α组比较,蛇床子素+TNF-α组RIP1泛素蛋白相对表达量降低(0.28±0.02比1.00±0.07、0.81±0.06,P<0.05),蛇床子素+TNF-α组活化的caspase-8蛋白的相对表达量明显提高(0.46±0.04比0.01±0.01、0.08±0.02,P<0.05),蛇床子素+TNF-α组CYLD蛋白的相对表达量也明显增加(0.81±0.05比0.28±0.02、0.30±0.02,P<0.05)。与蛇床子素+TNF-α组比较,蛇床子素+TNF-α+CYLD si RNA组RIP1泛素蛋白相对表达量增加(0.77±0.05比0.28±0.02,P<0.05),显示CYLD si RNA能显著抑制蛇床子素对TNF-α的协同作用。结论蛇床子素通过上调去泛素化酶CYLD的表达水平,促进TNF-α依赖的RIP1蛋白的去泛素化,进而诱导骨肉瘤细胞caspase-8的活化和凋亡的发生。
Objective To investigate the role of osthole in TNF-α-induced apoptosis in osteosarcoma and the underlying mechanism. Methods HOS cells were divided into control group, TNF-α group, osthole group, osthole＋TNF-α group, and osthole＋TNF-α＋CYLD si RNA group. The cell viability, cell apoptosis, ubiquitination of RIP1 protein,and the relative expression of cleaved caspase-8 and CYLD were detected by MTT assay, Annexin V/PI staining, coimmunoprecipitation together with Western Blot, respectively. Results The inhibitory rate of cell viability and the apoptotic rate of cells in TNF-α group group and osthole＋TNF-α group were significantly higher than that in control group（12.5%±0.9%, 58.6%±3.8% vs 0; 8.3%±0.5%, 39.9%±2.5% vs 1.9%±0.3%; all P〈0.05）. Those index in osthole＋TNF-α＋CYLD si RNA group were significantly lower than those in osthole＋TNF-α group（22.1%±1.2% vs 58.6%±3.8%;13.5%±0.9% vs 39.9%±2.5%; all P〈0.05）. Compared with control group and TNF-α group, the relative expression of ubiquitin protein conjugated with RIP1 was significantly lower and the relative expression of cleaved caspase-8 was higher in osthole＋TNF-α group（RIP1： 0.28±0.02 vs 1.00±0.07, 0.81±0.06; caspase-8： 0.46±0.04 vs 0.01±0.01, 0.08±0.02;CYLD： 0.81 ±0.05 vs 0.28 ±0.02, 0.30 ±0.02; all P〈0.05）. The relative expression of ubiquitin protein conjugated with RIP1 in osthole＋TNF-α＋CYLD si RNA group was significantly higher than that in osthole＋TNF-α group（0.77±0.05 vs0.28±0.02, P〈0.05）, indicating that transfection with CYLD si RNA significantly impaired the synergistic effect of osthole on the TNF-α-induced cell death and apoptosis. Conclusion Osthole promotes TNF-α-dependent deubiquitination of RIP1 and the subsequent cleavage of caspase-8 and apoptosis by upregulating the expression of CYLD.
出处
《浙江中西医结合杂志》
2016年第11期984-987,共4页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
