ATPase6,8在纳米氧化锌诱导HL-7702细胞线粒体能量代谢障碍中作用

Role of ATPase6,8 in the mitochondrial energy metabolism disorder of HL-7702 cells induced by nano-zinc oxide

目的分析ATPase6,8在纳米氧化锌(ZnO NP)诱导正常人肝细胞(HL-7702)线粒体氧化磷酸化能量代谢障碍中的作用,为ZnO NP疾病防控提供基础数据。方法透射电镜和马尔文粒径分析仪测定ZnO NP颗粒表征;ZnO NP染毒24 h后,3-(4,5-二甲基-2-噻唑)-2,5-二甲苯溴化四唑(MTT)法检测细胞活性及剂量选择;激光共聚焦显微镜检测线粒体膜电位改变;高效液相色谱法测定细胞内三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)的含量;紫外分光光度计检测线粒体呼吸链复合体Ⅴ(MRCCⅤ)活性;qRT-PCR检测ATPase6及ATPase8 mRNA表达量。结果 ZnO NP(2.5、5、10、20、40、80、160和320μg/mL)染毒24 h后,随着ZnO NP剂量增加,细胞存活率逐渐降低(r=-0.859,P<0.05),80、160、320μg/mL剂量组细胞存活率显著低于对照组(P<0.05),根据细胞存活率>70%,最终选择染毒剂量为0、2.5、10、40μg/mL;与对照组相比,10和40μg/mL剂量组红/绿色荧光比值明显减少(P<0.05),说明细胞线粒体膜电位(MMP)下降;同样40μg/mL剂量组细胞ATP、总腺普酸含量(TAN)、能荷值(EC)水平均明显下降,10~40μg/mL剂量组细胞AMP水平和2.5~40μg/mL剂量组细胞ATP/ADP水平也均明显下降(P<0.01);与对照组相比,2.5~40μg/mL剂量组细胞MRCCⅤ活性明显降低而ATPase6和ATPase8 mRNA表达水平均显著上升(P<0.01)。结论 ZnO NP能诱导HL-7702线粒体能量代谢障碍,可能是通过影响细胞线粒体ATPase6,8 mRNA的表达水平,进而引起MRCCⅤ活性降低及线粒体MMP下降,氧化磷酸化无法正常进行,ATP生成过程受到抑制,最终导致线粒体能量代谢障碍。

Objective To analyze the role of ATPase6,8 in the mitochondrial oxidative phosphorylation energy metabolism disorder of normal human hepatocytes(HL-7702) induced by nano zinc oxide(ZnO NP), and provide basic data for disease prevention and control of ZnO NP. Methods The particle characterization of ZnO NP was determined by transmission electron microscopy and Malvern particle size analyzer. After exposure to ZnO NP for 24 h, MTT method was used to detect cell activity and dosage selection. The changes of mitochondrial membrane potential were detected by laser confocal microscopy, and the contents of ATP,ADP and AMP in cells were measured by HPLC. The activity of mitochondrial respiratory chain complex V(MRCC V) was detected by ultraviolet spectrophotometer, and the expression of ATPase6 and ATPase8 mRNA was detected by qRT-PCR.Results After exposure to ZnO NP(2.5, 5, 10, 20, 40, 80, 160 and 320μg/mL) for 24 h, the cell survival rate decreased with the increase of Zn O NP dose(r=-0.859, P<0.05), the cell survival rate of 80, 160, 320 μg/mL groups were significantly lower than that of the control group(P<0.05). According to the cell survival rate > 70 %, the final exposure dose was 0, 2.5, 10, 40 μg/mL. Compared with the control group, the red/green fluorescence ratio decreased significantly in the 10 and 40 μg/mL dose group, indicating that the cell mitochondrial membrane potential(MMP) decreased. In the same, the levels of ATP, TAN and EC in the 40 μg/mL dose group,AMP in the 10-40 μg/mL dose group and ATP/ADP in the 2.5-40 μg/mL dose group were significantly decreased(P < 0. 01).Compared with the control group, the activity of MRCC V in the 2.5-40 μg/mL group were significantly decreased, while the expression of ATPase6 and ATPase8 mRNA were significantly increased(P<0.01). Conclusion ZnO NP can induce the mitochondrial energy metabolism disorder of HL-7702, which may be caused by affecting the expression level of mitochondrial ATPase6,8 m RNA in the cell, thereby causing the decrease of MRC Ⅴ activity and mitochondrial MMP, oxidative phosphorylation can not proceed normally, and the ATP production process is inhibited, and finally lead to mitochondrial energy metabolism disorder.