香烟烟雾水溶性提取物对肝细胞线粒体的损伤

Effect of water-soluble fraction of cigarette smoke(CSW) on hepatocyte mitochondria

目的探讨香烟烟雾水溶性提取物(CSW)对L-02肝细胞线粒体结构与功能的影响。方法采用噻唑蓝(MTT)检测CSW处理L-02肝细胞24h后的生存率,以确定后续试验CSW染毒浓度为0、8×10-3、16×10-3、32×10-3、64×10-3支/ml培养基。CSW染毒处理L-02肝细胞24h后用MTT法检测线粒体总酶活力抑制率,荧光分光光度法检测线粒体膜通透性转运孔(PTP)开放度与细胞线粒体膜电位(ΔΨm)的变化,透射电子显微镜观察细胞线粒体形态学变化。结果8×10-3、16×10-3、32×10-3、64×10-3支/ml4个不同浓度处理组CSW对L-02肝细胞具有一定的细胞毒性,其细胞生存率随CSW处理浓度的增高而降低,且二者呈明显负相关(相关系数r=-0.957);线粒体PTP开放程度则随CSW浓度的增高而增加,32×10-3支/ml及其以上CSW浓度组开放度与对照组比较,差异有统计学意义(P<0.05);线粒体总酶活力与细胞线粒体膜电位(ΔΨm)随CSW浓度的增加而下降,二者分别在8×10-3、16×10-3支/ml及其以上CSW浓度组与对照组比较,差异均有统计学意义(P<0.05)。透射电子显微镜观察线粒体形态发生显著改变即表现为肿胀、空泡变性、膜和嵴破损。结论香烟烟雾水溶性提取物可对L-02肝细胞线粒体的结构与功能造成一定程度损伤。

Objective To explore the effects of water-soluble fraction of cigarette smoke （CSW） on mitochondria structure and function in L-02 hepatocyte. Methods The effects of CSW on cell survival rates of hepatocytes were assessed by the reductions of tetrazoliumdye（MTT）. According to cell survival rates, L-02 hepatocytes were incubated with CSW at the concentrations of 0.8×10^-3,16×10^-3, 32×10^-3, 64×10^-3 cig/ml respectively for 24 hours in the following tests. The permeability transition pore（PTP） of mitochondria and mitochondrial membrane potentials （△ψm）, and the inhibition rates of mitochondria enzymes were measured by fluorescent spectrometer and the reductions of MTT respectively. The changes of mitochondria morphology were examined by electron microscope. Results CSW resulted in severe cytotoxicity on L-02 hepatocytes. The survival rates of CSW-treated cells were decreased with the increases of the concentration of CSW. The relationship between the concentration and survival rates was significant negative correlation（r=-0.957）. In Comparition with the control groups, the permeability transition pore（PTP） of mitochondria significantly enlarged（P 〈 0.05）at the concentration of 32×10^-3, 64×10^-3 cig/ml of CSW, and the activities of mitochondria enzymes and mitochondrial membrane potentials（△ψm） were significantly decreased at the concentrations of 8×10^-3 and 16×10^-3 cig/ml of CSW respectively（ P 〈 0.05）. The changes of mitochondrial morphology in L-02 hepatocytes treated with CSW presented swelling, vacuolar degeneration and damages of membrane and cristae. Conclusion These results demonstrated that CSW could induce the damages to mitochondria in cultured L-02 hepatocytes.