10%浏阳霉素乳剂对肝细胞线粒体损伤效应的研究

Effects of 10% Liuyangmycin emulsifiable on mitochondrial function in L-02 hepatocytes

目的研究10％浏阳霉素乳剂（LY-EC）对L-02肝细胞线粒体功能活性的影响。方法用MTT法检测不同浓度的LY-EC（0～200mg／L）对L-02肝细胞生存率的影响。选取细胞生存率大于70％的处理组浓度作为后续实验浓度，分别为0、0．39、1．56、6．25和25．00mg／L。用上述浓度的LY—EC染毒处理L-02肝细胞12h后，用化学比色法测定肝细胞丙二醛（MDA）及谷胱甘肽（GSH）含量，超氧化物歧化酶（SOD）、琥珀酸脱氢酶（SDH）和ATP酶的活性。另外还测定LY．EC对线粒体膜通透性转运孔（PTP）开放度和肝细胞腺苷酸（ATP、ADP和AMP）含量的影响，并分析ATP／ADP比值以及负荷能量（EC）的变化。用透射电镜观察LY-EC对线粒体超微结构的影响。结果LY—EC可剂量依赖性地诱导L-02肝细胞生存率下降（r=0．939，P〈0．05）。与0．00mg／LLY—EC处理组相比，不同浓度LY—Ec处理的肝细胞MDA含量增加；GSH含量及SOD、SDH和ATP酶的活性降低，线粒体PTP开放度增加（P〈0．05）；肝细胞ATP、TAN含量及ATP／ADP比值降低，能量负荷（EC）增加（P〈0．05）。透射电镜观察发现LY—EC染毒的肝细胞胞浆水肿，线粒体肿胀变形，嵴不完整甚至消失。结论LY-EC可诱导L-02肝细胞氧化应激，线粒体受氧化损伤后，功能活性下降，能量代谢障碍。

Objective To investigate the effects of 10% Liuyangmycin emulsifiable （LY-EC） on mitochondrial function in L-02 hepatocytes. Methods To detect the effect of different concentration of LY-EC （0 -200 mg/L） on L-02 hepatocytes survival rate with MTT method, and choose proper LY-EC treated concentrations （survival rate 〉 70% ） for the following experiments. After hepatoeytes were treated with LY-EC at 0. 39,1.56,6.25 and 25.00 mg/L respectively for 12 h, MDA and GSH contents, SOD, SDH, ATPase activities were respectively analyzed by their assay kits using colorimetry. Then mitochondrial PTP open percentage and the contents of ATP, ADP and AMP were measured with HPLC assays, and ATP/ADP ratio and energy charge （EC） values were calculated. The effects of LY-EC on mitochondrial ultra-structures were observed by transmission electron microscopy. Results LY-EC induced the decrease of cell survival rates in a concentrate- dependent manner （r = 0. 939,P 〈 0.05）. Compared with control group, GSH contents and SOD, SDH and ATPase activities in groups treated with LY-EC decreased significantly, MDA contents and mitochondrial PTP open percentages increased （P 〈 0.05 ）, the levels of cellular ATP, TAN and ATP/ADP ratio in L-02 hepatocytes decreased, while energy charge （EC） increased significantly （P 〈 0.05）. Hepatocytes treated with LY-EC were observed cytoplasmic edema, mitochondria swelling and the deformation of mitochondrial cristae with transmission electron microscope. Conclusion LY-EC induced oxidative stress, mitochondrial function damage and energy metabolism disorder in L-02 hepatocytes.