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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(0~200 mg/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25.00 mg/L。用上述浓度的LY-EC染毒处理L-02肝细胞12 h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0.00 mg/L LY-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 effect of 10% liyangmycin emulsion (LY-EC) on the mitochondrial functional activity of L-02 hepatocytes. Methods The effect of different concentrations of LY-EC (0-200 mg / L) on the survival rate of L-02 hepatocytes was detected by MTT assay. The concentrations of the treated cells with the cell survival rate greater than 70% were selected as the follow-up experimental concentrations, which were 0, 0.39, 1.56, 6.25 and 25.00 mg / L, respectively. After L-02 hepatocytes were exposed to the above concentrations of LY-EC for 12 hours, the contents of malondialdehyde (MDA) and glutathione (GSH), the activities of superoxide dismutase (SOD) ), Succinate dehydrogenase (SDH) and ATPase activity. In addition, the effects of LY-EC on the openness of mitochondrial membrane permeability transition pore (PTP) and the content of adenosine monophosphate (ATP, ADP and AMP) in hepatocytes were also analyzed. The changes of ATP / ADP ratio and the energy load . The effect of LY-EC on mitochondrial ultrastructure was observed by transmission electron microscope. Results LY-EC induced a dose-dependent decrease in L-02 hepatocyte viability (r = 0.939, P <0.05). Compared with 0.006 mg / L LY-EC treatment group, MDA content of LY-EC-treated hepatocytes increased; GSH content, SOD, SDH and ATPase activity decreased and mitochondrial PTP openness increased (P <0.05); The contents of ATP, TAN and ATP / ADP decreased, and the energy load (EC) increased (P <0.05). Transmission electron microscopy showed that cytoplasmic edema of hepatocytes exposed to LY-EC, mitochondria swelled and deformed, and the ridges were incomplete or even disappeared. Conclusion LY-EC can induce oxidative stress in L-02 hepatocytes. After mitochondria oxidative damage, LY-EC has decreased functional activity and energy metabolism disorders.