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目的探讨龙葵碱诱导HepG2细胞凋亡的作用机制。方法透射电镜观察凋亡细胞形态变化,原位缺口末端检测法(TUNEL法)检测DNA断裂情况,流式细胞术检测细胞凋亡率,间接免疫荧光法激光共聚焦扫描显微术检测Bcl-2与Bax蛋白表达,比色法检测caspase-3活性的变化。结果在透射电镜下观察,龙葵碱组细胞出现细胞固缩,染色质致密,核凝聚固缩,染色体断裂形成核碎块,凋亡小体形成等细胞凋亡特征形态。TUNEL法发现龙葵碱高、中、低剂量组HepG2细胞均有绿色荧光,阴性对照组无荧光。流式细胞术分析表明0.4、2、10μmol/L龙葵碱作用HepG2细胞24h凋亡率分别为4.0%、8.5%、20.1%。同时,龙葵碱升高caspase-3活性,下调Bcl-2蛋白表达,上调Bax蛋白表达。结论龙葵碱通过降低Bcl-2/Bax的值,激活caspase-3酶活性诱导HepG2细胞凋亡。
Objective To investigate the mechanism of solanine induced HepG2 cell apoptosis. Methods The morphological changes of apoptotic cells were observed by transmission electron microscopy. The DNA fragmentation was detected by TUNEL, the apoptosis rate was detected by flow cytometry, and the expression of Bcl-2 was detected by indirect immunofluorescence confocal laser scanning microscopy. And Bax protein expression, colorimetric assay caspase-3 activity changes. Results Under the transmission electron microscope, the cells of Solanum nigrum appeared cell apoptosis, chromatin condensation, nuclear condensation and solidification, chromosome rupture and nuclear fragmentation, apoptotic body formation and other apoptosis morphological features. TUNEL method was used to find that HepG2 cells in high, medium and low dose groups of Solanum nigrum had green fluorescence, while negative control group had no fluorescence. Flow cytometry analysis showed that the apoptotic rates of HepG2 cells treated with 0.4, 2, 10 μmol / L solanine for 4 h were 4.0%, 8.5% and 20.1%, respectively. Meanwhile, solanine increased caspase-3 activity, down-regulated Bcl-2 protein expression and up-regulated Bax protein expression. Conclusion Solanine can induce the apoptosis of HepG2 cells by decreasing the value of Bcl-2 / Bax and activating the activity of caspase-3.