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目的:研究中药青葙子中的齐墩果烷型三萜青葙苷A(celosin A,CA)对人肝癌HepG2细胞凋亡的诱导作用及其相关机制。方法:采用E-MEM培养基建立人肝癌HepG2细胞体外培养体系,采用细胞计数试剂盒(,CCK-8)检测细胞活力,噻唑蓝(MTT)法测定乳酸脱氢酶(LDH),免疫比色法检测细胞增殖情况,用4’,6-二脒基-2-苯基吲哚(4,6-2 amidine base-2-phenyl indole,DAPI)荧光染色观察细胞凋亡形态学变化,Western blot检测半胱氨酸天冬氨酸蛋白酶(Caspase)-3/7和核转录因子kappa B(NF-κB)蛋白表达水平。结果:用0.22-3.5μmol·L-1CA处理过的HepG2细胞,LDH释放增加,呈剂量和时间依赖性。在CCK-8和5-溴脱氧尿嘧啶核苷(BrdU)测定过程中,CA比标准药物抗HepG2活性更强,随浓度和作用时间的增加,细胞的活性不断下降,对HepG2增殖的抑制作用明显。DAPI染色观察用CA处理过的HepG2细胞浓缩明显,细胞核分散,染色质凝聚。Western blot结果显示用CA处理过的HepG2细胞Caspase-3/7的活性显著增加,NF-κB蛋白表达水平下降。结论:Celosin A具有诱导HepG2细胞凋亡的作用,其机制可能与激活Caspase-3/7和抑制NF-κB蛋白表达有关。
OBJECTIVE: To study the induction effect of oleanane-type triterpene glycoside A (CA) on HepG2 human hepatoma cell apoptosis and its related mechanisms. Methods: The human hepatocellular carcinoma HepG2 cells were cultured in vitro by using E-MEM medium. Cell viability was measured by cell counting kit (CCK-8), lactate dehydrogenase (LDH), immunochromaticity The cell proliferation was detected by fluorescence microscope. The morphological changes of apoptosis were observed by 4, 6-2 amidine base-2-phenylindole (DAPI) staining. Western blot The Caspase -3/7 and NF-κB protein expression levels were detected. Results: HepG2 cells treated with 0.22-3.5μmol·L-1CA showed an increase in LDH release in a dose and time-dependent manner. In CCK-8 and 5-bromodeoxyuridine (BrdU) determination, CA was more active than standard drugs in anti-HepG2 activity. With the increase of concentration and duration of action, the activity of cells was decreased and the inhibitory effect on HepG2 proliferation obvious. DAPI staining observed with CA-treated HepG2 cells concentrated significantly, the nucleus dispersed, chromatin condensation. Western blot results showed that the activity of Caspase-3/7 in HepG2 cells treated with CA increased significantly and the expression of NF-κB decreased. Conclusion: Celosin A can induce the apoptosis of HepG2 cells, which may be related to the activation of Caspase-3/7 and the inhibition of NF-κB protein expression.