白鲜碱对HepG2细胞的毒性作用及其机制

目的研究白鲜碱的肝细胞毒性作用及其毒性机制。方法白鲜碱2.5～800μmol·L-1与HepG2细胞作用24 h,用MTT法检测细胞存活率并计算IC50值,用乳酸脱氢酶(LDH)释放实验检测细胞膜损伤。白鲜碱25～100μmol·L-1与HepG2细胞作用4,24或48 h,用试剂盒方法分别检测细胞培养液中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、谷胱甘肽S-转移酶(GST)和谷氨酰转肽酶(GGT)活性。用倒置显微镜观察细胞形态变化;激光共聚焦扫描显微镜检测细胞线粒体膜电位的变化。结果白鲜碱25～800μmol· L-1与HepG2细胞作用24 h对HepG2细胞存活的抑制作用随着浓度的增加而降低(r=0.965,P<0.05),IC50值为(283±27)μmol·L-1。与溶剂对照组相比,白鲜碱12.5～50μmol·L-1作用24 h,HepG2细胞LDH释放率显著升高(P<0.01)。白鲜碱100和200μmol·L-1可引起HepG2细胞形态发生明显变化,细胞皱缩脱落,细胞数目减少,并使HepG2细胞线粒体膜电位下降(P<0.05,P<0.01)。白鲜碱100和200μmol·L-1与HepG2细胞作用24 h可使细胞培养液中ALT和AST活性显著升高,并呈浓度依赖性(r=0.995,P<0.05和r=0.996,P<0.05),线粒体膜电位亦明显下降(r=0.978,P<0.05)。与溶剂对照组相比,白鲜碱100和200μmol·L-1与HepG2细胞作用4 h,细胞培养液中GST活性明显升高(P<0.05);作用24 h,GST活性升高呈浓度依赖性(r=0.987,P<0.05)。白鲜碱200μmol· L-1作用48 h导致HepG2细胞培养液中GGT活性升高(P<0.05)。结论较高浓度的白鲜碱(≥100μmol· L-1)具有潜在的肝毒性,细胞膜损伤和线粒体损伤可能是其肝毒性作用机制之一。

Cytotoxic effect of dictamnine on HepG2 cells and its possible mechanism

OBJECTIVE To study the dictamnine-induced hepatotoxicity in vitro and to explore its mechanism. METHODS HepG2 cells were exposed to dictamnine 2.5-800 μmol·L^-1 for 24 h, and cell viability was examined by MTT assay. Cell membrane injury was examined by detecting the release rate of lactate dehydrogenase (LDH), and the morphological changes were observed under a contrast microscope. The activities of alanine transaminase (ALT), aspartate aminotransferase (AST), glutathione S-transferase (GST) and glutamyltranspeptidase (GGT) in HepG2 cell cultures were measured using enzyme-labeled instrument, respectively. The mitochondrial membrane potential was measured by laser scanning confocal fluorescence microscope. RESULTS HepG2 cell viability was significantly reduced following exposure to dictamnine 25-800 μmol·L^-1 for 24 h in a concentration-dependent manner (r=0.965, P<0.05), and IC₅₀ value was (283±27)μmol·L^-1. The LDH release rate of HepG2 cells was significantly increased after exposure to dictamnine 12.5-50 μmol·L^-1 for 24 h (P<0.01). The morphology of HepG2 cells after 24 h exposure to dictamnine 100 and 200 μmol·L^-1 was changed greatly. The cells wrinkled up and dropped. Compared with control group, ALT and AST activities in HepG2 cell culture were significantly increased (P<0.05) in a concentration-dependent manner (r=0.995,P<0.05 and r=0.996,P<0.05) following exposure to dictamnine 100 and 200 μmol·L^-1 for 24 h, and the mitochondrial membrane potential markedly declined(r=0.978,P<0.05). After exposure to dictamnine 100 and 200 μmol·L^-1 for 4 h, GST activity in HepG2 cell culture was significantly increased(P<0.05); and for 24 h, GST activity in dictamnine 50, 100 and 200 μmol·L^-1 groups was also increased in a concentration-dependent manner (r=0.987, P<0.05). After exposure to dictamnine 200 μmol·L^-1 for 48 h, GGT activity in HepG2 cell culture was increased(P<0.05). CONCLUSION Dictamnine at higher concentrations(≥100 μmol·L^-1)has potential hepatotoxicity. The cell membrane damage and mitochondrial membrane damage may be involved in the dictamnine-induced hepatotoxity mechanism.