一氧化氮供体PABA/NO经线粒体途径诱导HepG2细胞凋亡

Nitric oxide donor PABA/NO induced mitochondrial-mediated apoptosis in HepG2 cells

目的研究新型一氧化氮(NO)供体,O^2-{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 apoptosis in human hepatocarcinoma cells. METHODS Proliferation of HepG2 cells treated with PABA/NO 7.5, 15.0 and 30.0 μmol·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, PABNNO 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 fluorescence 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-regulation 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.