哇巴因对血管内皮细胞ECV304细胞凋亡及胞内游离钙离子和活性氧浓度的影响

目的研究哇巴因(毒毛花苷G)对人脐静脉血管内皮细胞ECV304凋亡的诱导作用,并探讨其可能的作用机制。方法哇巴因0.01,0.05,0.1,0.5,1和10μmol·L-1与ECV304细胞作用24,48和72h,MTT法检测细胞存活率,Hoechst33342/碘化丙锭双荧光染色法和流式细胞仪检测细胞凋亡百分率,激光共聚焦显微镜观察细胞内游离Ca2+浓度(Ca2+]i)和活性氧(ROS)浓度,逆转录PCR和Western印迹法检测胱天蛋白酶3mRNA和蛋白表达。结果哇巴因在0.01～10μmol·L-1浓度范围内与ECV304细胞分别作用24,48和72h,对细胞存活的抑制率明显增加,且呈浓度和时间依赖性,24,48和72h浓度-效应相关系数分别为0.984,0.994和0.997(P<0.05);哇巴因作用24,48和72h的IC50值分别为0.624,0.184和0.041μmol·L-1,时间-效应相关系数为0.974(P<0.05)。哇巴因0.1μmol·L-1与ECV304细胞作用24h,细胞凋亡百分率由正常对照组的(4.2±0.5)%升高到(26.0±3.2)%,作用48h,细胞凋亡率由(4.7±0.5)%升高到(36.5±5.3)%,差异有统计学意义(n=3,P<0.01);同时细胞出现染色质凝集。哇巴因0.01,0.1和0.5μmol·L-1分别与ECV304细胞作用12,24和36h,Ca2+]i和ROS浓度呈浓度和时间依赖性增加,在哇巴因0.5μmol·L-1时Ca2+]i和ROS浓度的时间-效应相关系数分别为0.912和0.924,作用36h时Ca2+]i和ROS浓度的浓度-效应相关系数分别为0.889和0.907(P<0.05)。逆转录PCR和Western印迹法分析显示,哇巴因0.1和0.5μmol·L-1作用ECV304细胞24h后,胱天蛋白酶3mRNA表达增加,差异有统计学意义(P<0.05);哇巴因0.01,0.1和0.5μmol·L-1作用ECV304细胞24h,胱天蛋白酶3蛋白表达明显增加,差异有统计学意义(P<0.05)。结论哇巴因可诱导人脐静脉血管内皮细胞ECV304凋亡,其机制可能与增加Ca2+]i和ROS浓度及胱天蛋白酶3表达有关。

Effect of ouabain on human vascular endothelial ECV304 cell apoptosis and on concentrations of intracellular free calcium ion and reactive oxygen species

OBJECTIVE To study the effect of ouabain (strophanthin G) on human vascular endothelial ECV304 apoptosis and the possible mechanism. METHODS ECV304 cells were treated with ouabain 0.01, 0.05, 0.1, 0.5, 1 and 10 μmol·L^-1 for 24，48 and 72 h. The survival rates of cells were detected by MTT assay; cells morphological changes were studied by Hoechst 33342/propidium iodide staining; cell cycle distribution was detected by flow cytometry; and the concentrations of the intracellular free calcium ion (［Ca²⁺］_i) and reactive oxygen species (ROS) in ECV304 cells were measured by the laser confocal microscope. The caspase 3 mRNA and protein expression level on ECV304 cells were detected by RT-PCR and Western blotting, respectively. RESULTS Ouabain 0.01-10 μmol·L^-1 was incubated with ECV304 cells for 24，48 and 72 h. The inhibition rate of cell survival was increased in a concentration- and time-dependent manner. The coefficient of concentration-correlation was 0.984, 0.994 and 0.997(P<0.05), respectively. IC50 of ouabain at 24，48 and 72 h was 0.624, 0.184 and 0.041 μmol·L^-1 and the coefficient of time-correlation was 0.974(P<0.05). Compared with normal control group, the apoptosis rate of ECV304 cells treated with ouabain 0.1 μmol·L^-1, from increased from （4.2±0.5）% to （26.0±3.2）% at 24 h, and from (4.7±0.5)% to (36.5±5.3)% at 48 h, being statistically significant (n=3, P<0.01). The cells showed obviously defluxion and nuclear chromatin condensation when treated with ouabain 0.1 μmol·L^-1 for 24 and 48 h. The ［Ca²⁺］_i and ROS concentration in ECV304 cells significantly increased in a concentration- and time-dependant manner after ECV304 cells were incubated with ouabain 0.01, 0.1 and 0.5 μmol·L^-1 for 12, 24 and 36 h, respectively. When ouabain was 0.5 μmol·L^-1, the coefficient of time correlation of ［Ca²⁺］_i and ROS concentration were 0.912 and 0.924, respectively. When the ECV304 cells was incubated with ouabain for 36 h, the coefficient of concentration-correlation of ［Ca²⁺］_i and ROS concentration were 0.889 and 0.907(P<0.05), respectively. The RT-PCR and Western blotting results showed that caspase 3 mRNA expression was up-regulated after ECV304 cells were treated with ouabain 0.1 and 0.5 μmol·L^-1 for 24 h, being of statistical significance (P<0.05); caspase 3 protein expression was up-regulated after ECV304 cells were treated with ouabain 0.01, 0.1 and 0.5 μmol·L^-1 for 24 h, statistically significant (P<0.05). CONCLUSION Ouabain induces ECV304 apoptosis by increasing ［Ca²⁺］_i, ROS concentration and up-regulating caspase 3 expression.