葡萄冻干粉在线粒体应激介导肝细胞凋亡中的作用

The effect of freeze-dried grape powder on Huh7 cells apoptosis mediated by mitochondrial stress

目的了解葡萄冻干粉(FDG)对线粒体应激介导的肝细胞凋亡的阻抑作用,探讨其治疗肝细胞损伤的作用机制。方法用牛磺酸脱氧胆酸(TDCA)诱导Huh7细胞,建立线粒体应激凋亡模型,用不同剂量FDG进行干预。通过MTT、DNAladder、Western blot、细胞凋亡率检测等方法,了解FDG对Huh7细胞生长、对TDCA诱导的Huh7细胞的凋亡率、凋亡信号蛋白表达的影响。结果FDG(2μg/ml、20μg/ml、200μg/ml、400μg/ml和800μg/ml)与Huh7细胞共孵育,FDG可提高Huh7细胞的活力,以FDG400μg/ml时细胞活力最高。用400μM TDCA诱导Huh7细胞,随着诱导时间延长细胞活力下降、凋亡率增加,出现凋亡条带;Caspase-9和-7蛋白酶原被活化,PCNA表达下调。用FDG干预TDCA诱导Huh7细胞48h后发现,FDG明显提高Huh7细胞活力、使细胞凋亡减少、凋亡信号蛋白的表达减少。结论TDCA能触发Huh7细胞线粒体氧化应激凋亡,而FDG能促进细胞生长,减轻肝细胞损伤,阻断线粒体氧化应激介导的肝细胞凋亡。

Objective To analyze the mechanism of freeze dried grape powder （FDG） on mitochondrial pathway stress mediated Huh7 cells apoptosis and to evaluate the potential role of FDG in the treatment of liver injury. Methods We treated Huh7 cells with taurodeoxycholic acid （TDCA） to make the model of oxidative mitochondrial stress mediated apoptosis and to assert the optimal dosage and incubation time of FDG to intervene in apoptosis. We performed various methods such as MTT, DNA ladder, Cell death detection, Western blot and assess molecular events in the development of liver injury. Results The activity of cells decreased in time-dependent manner induced by TDCA. The ratio of apoptotic cells increased along with the prolonged treatment of TDCA. DNA ladder was observed at same concentration and time corresponds. The Huh7 cells show typical morphologic change of mitochondrial stress induced by 400uM TDCA at 48 hr. By Western Blot, the activity of caspase 9, -7 was higher in the group of TDCA treatments than in control. The expression of PCNA was downregulated. FDG could protect cells from apoptosis by concentration related and the up-limit of FDG was 400μg/L Conclusion TDCA induced significant oxidative stress in association with mitochondrial-mediated apoptosis. FDG can inhibit the apoptosis of hepatocytes and can ameliorate liver injury.