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目的研究NO供体——新型偶氮鎓二醇盐衍生物(JS-K)体外对人肝癌SMMC-7721细胞增殖抑制作用。方法用噻唑兰法检测JS-K对细胞增殖的影响;倒置显微镜观察24 h后细胞形态学变化;以Annexin V-FITC/PI双染法检测细胞凋亡率;以DAF-FM DA荧光探针检测细胞中NO水平;以蛋白免疫印迹法观察JS-K对B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(caspase-9)蛋白表达变化。结果 JS-K能显著抑制细胞增殖,呈一定的剂量、时间依赖性。倒置显微镜观察到JS-K组出现变圆、皱缩等形态学变化;3个浓度JS-K和对照组(5-FU 100μmol·L~(-1))作用细胞24 h,凋亡率分别为(19.12±0.07)%,(40.81±0.03)%,(75.53±0.12)%,(56.70±0.07)%,均高于空白组的(2.72±0.01)%,差异有统计学意义(P<0.05)。3个浓度JS-K作用细胞24 h后,细胞内NO水平分别是(151.2±0.1)%,(248.6±0.2%),(347.6±0.4)%,均高于空白组100%,差异有统计学意义(P<0.05);加入Carboxy-PTIO后,JS-K组NO水平由(318.6±0.2)%降至(307.6±0.3)%,差异有统计学意义(P<0.05)。Bax/Bcl-2比值也升高,3个浓度JS-K和对照组依次为(0.54±0.02)%,(1.19±0.03)%,(1.32±0.02)%,(1.20±0.01)%,均高于空白组的(0.20±0.02)%,差异有统计学意义(P<0.05)。加入caspase-9抑制剂(Z-LEHD-FMK)和caspase-3抑制剂(Z-VAD-FMK)后,可显著减少JS-K诱导的细胞凋亡。结论 JS-K可通过caspase依赖性途径诱导SMMC-7721细胞凋亡。
Objective To study the inhibitory effect of NO donor-new azobenzene diol derivative (JS-K) on the proliferation of human hepatoma SMMC-7721 cells in vitro. Methods The effect of JS-K on cell proliferation was detected by thiazolyl method. The morphological changes of cells were observed after 24 h by inverted microscope. The apoptosis rate was detected by Annexin V-FITC / PI double staining. The levels of Bcl-2, Bcl-2, Caspase-3 and Bcl-2 were determined by Western blotting. (caspase-3), caspase-9 protein expression changes. Results JS-K significantly inhibited cell proliferation in a dose-and time-dependent manner. The morphology of JS-K group was observed by inverted microscope. The apoptotic rates of JS-K group and control group (5-FU 100μmol·L -1) Were (19.12 ± 0.07)%, (40.81 ± 0.03)%, (75.53 ± 0.12)% and (56.70 ± 0.07)%, respectively, which were significantly higher than that of the blank group (2.72 ± 0.01)%, 0.05). The levels of NO in three concentrations of JS-K-treated cells for 24 h were (151.2 ± 0.1)%, (248.6 ± 0.2%) and (347.6 ± 0.4)%, respectively, which were all higher than those in the blank group (P <0.05). After adding Carboxy-PTIO, the level of NO in JS-K group decreased from (318.6 ± 0.2)% to (307.6 ± 0.3)% with statistical significance (P <0.05). Bax / Bcl-2 ratio also increased. The average concentrations of JS-K and control group were (0.54 ± 0.02)%, 1.19 ± 0.03%, 1.32 ± 0.02% and 1.20 ± 0.01% Which was significantly higher than that of the blank group (0.20 ± 0.02)% (P <0.05). The addition of caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-VAD-FMK) significantly reduced the apoptosis induced by JS-K. Conclusion JS-K can induce apoptosis of SMMC-7721 cells through a caspase-dependent pathway.