目的 研究新型一氧化氮(NO)供体,O2-{2,4-二硝基-5-〔4-(N-甲基氨基)苯甲酰氧基〕苯基}-1-(N,N-二甲基氨基)偶氮-1-鎓-1,2-二醇(PABA/NO)对人肝癌细胞凋亡的影响.方法 PABA/NO 7.5,15.0和30.0μmol·L-1处理细胞24 h后,CCK-8法检测细胞存活率,倒置显微镜下检测细胞形态,DAF-FM DA荧光染色法检测细胞内NO水平,膜联蛋白(Annexin)Ⅴ-FITC/PI双染法检测细胞凋亡率,罗丹明123(Rh123)染色法检测细胞线粒体膜电位,Western蛋白质印迹法检测Bcl-2、Bax、活化胱天蛋白酶3、细胞色素c(Cyt c)和凋亡诱导因子(AIF)蛋白表达.结果 与细胞对照组比较,PABA/NO可显著抑制HepG2细胞存活,24 h时IC50值为(10.8±0.6)μmol·L-1;形态上出现胞膜皱缩、细胞扁圆等变化;细胞内NO水平提高,荧光强度分别为121±9(P<0.05),174±31(P<0.05)和230±43(P<0.01);凋亡率由原来的(2.9±0.5)％增加至(17.0±4.5)％,(39.8±5.4)％和(74.3±45.2)％(P<0.01);细胞内Rh123的荧光强度由原来的668±69下降到605±73,420±65(P<0.05)和242±47(P<0.01);PABA/NO引起Bcl-2表达下调(P<0.01)、Bax及活化胱天蛋白酶3表达上调,胞浆中Cyt c的表达由原来的0.15±0.04升高到0.27±0.06(P<0.05),0.38±0.07(P<0.01)和0.82±0.16(P<0.01).胞核中AIF的表达由原来0.183±0.032升高至0.231±0.011,0.682±0.020(P<0.01)和0.966±0.090(P<0.01).加入NO清除剂羧基(carboxy)-PTIO后,可逆转PABA/NO引起的Bcl-2表达下调,对Bax、活化胱天蛋白酶3、胞浆中Cyt c和胞核中AIF蛋白表达的上调也有一定的逆转作用(P<0.01).结论 PABA/NO可能经线粒体途径诱导HepG2细胞凋亡.%OBJECTIVE To investigate the effects of nitric oxide (NO) donor, O2-{2,4-dinitro-5-[4-(N-methylamino) benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) on apop-tosis in human hepatocarcinoma cells. METHODS Proliferation of HepG2 cells treated with PABA/NO 7.5, 15.0 and 30.0μmol · L-1 was measured with Cell Counting Kit-8 (CCK-8) assay, the morphological features were observed under fluorescence microscopy, the level of NO was measured by DAF-FM DA staining, the apoptosis rate was determined by Annexin Ⅴ-FITC staining, mitochondrial membrane potential was determined by Rhodamine 123 staining, and protein expressions of Bcl-2, Bax, cleaved caspase 3, cytochrome c (Cyt c) and apoptosis inducing factor (AIF) were measured by Western blotting analysis. RESULTS Compared with cell control group, PABA/NO could obviously inhibit the proliferation of HepG2 cells (P<0.01). IC50 value of HepG2 cells treated with PABA/NO for 24 h was (10.8±0.6)μmol·L-1. The cells became round, deformed and appeared shrunken.The level of NO was increased and the fluores-cence intensity was 121 ± 9 (P<0.05), 174 ± 31 (P<0.05) and 230 ± 43 (P<0.01). The apoptosis rate was increased from (2.9 ± 0.5)％ to (17.0 ± 4.5)％ (P<0.01), (39.8 ± 5.4)％ (P<0.01) and (74.3 ± 45.2)％ (P<0.01). The fluorescence intensity of Rh123 was reduced from 668±69 to 605±73, 420±65 (P<0.05) and 242±47 (P<0.01). Compared with cell control group, PABA/NO down-regulated Bcl-2, up-regulated Bax and activated cleaved caspase 3. Meanwhile, the expression of Cyt c in the cytoplasm was increased from 0.15±0.04 to 0.27±0.06 (P<0.05), 0.38±0.07 (P<0.01) and 0.82±0.16 (P<0.01). The expression of AIF in the nucleus was increased from 0.183±0.032 to 0.231±0.011, 0.682±0.020 (P<0.01) and 0.966± 0.090 (P<0.01). Addition of carboxy-PTIO (NO scavenger) 50 μmol · L- 1 blocked PABA/NO-induced down-regulation of Bcl-2, up-regulation of Bax and cleaved caspase 3 activation. Additionally, up-regu-lation of Cyt c in the cytoplasm and up-regulation of AIF in the nucleus were also blocked by carboxy-PTIO in PABA/NO-treated HepG2 cells (P<0.01). CONCLUSION PABA/NO may induce HepG2 cell apoptosis through a mitochondrial pathway.
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