多房棘球蚴感染对巨噬细胞线粒体功能的影响

Effect of Echinococcus multilocularis infections on mitochondrial functions of macrophages

目的观察多房棘球蚴感染后巨噬细胞线粒体代谢功能变化,为探索多房棘球蚴病发病机制提供依据。方法根据处理方法不同设培养组和对照组,其中培养组按照500∶1比例将小鼠单核巨噬细胞白血病细胞(RAW264.7)与2 000个多房棘球蚴共培养,对照组RAW264.7细胞不做任何处理。根据培养时间不同,将对照组和培养组分为24 h对照组、72 h对照组、24 h培养组、72 h培养组。采用Mito Tracker;Deep Red FM线粒体深红色荧光探针标记线粒体并应用Cytation5细胞成像微孔板检测系统检测细胞线粒体平均荧光强度,用实时荧光定量PCR检测线粒体DNA拷贝数(mitochondrial DNA copy number,mt DNA-CN),采用Seahorse细胞能量代谢系统实时监测线粒体能量代谢功能,采用流式细胞术检测线粒体活性氧及线粒体膜电位。结果 24 h(15.341±2.532 vs. 17.823±3.429;t=6.379,P <0.01)、72 h(18.102±3.505 vs. 21.511±5.144;t=17.680,P <0.01)培养组线粒体平均荧光强度均较其相应对照组显著降低。72 h培养组mt DNA-CN(3.23×10^(9)±1.78×10^(7))较其对照组(4.39×10^(9)±3.70×10^(7))显著降低(t=8.85,P <0.001)。实时线粒体能量代谢功能分析结果示,24 h [(241.70±73.13) pmol/min vs.(69.05±52.30)pmol/min;t=7.89,P <0.01]、48 h [(249.50±42.06)pmol/min vs.(60.28±40.66)pmol/min;t=8.64,P <0.01]培养组较相应对照组细胞耗氧率升高,且48 h培养组细胞外酸化率较其对照组增高[(111.60±17.49) mp H/min vs.(35.05±7.57)mp H/min;t=16.90,P <0.01];72 h培养组较其对照组线粒体活性氧平均荧光强度显著升高(58 264±10 087 vs. 4 307±97;t=12.930,P <0.01)、线粒体膜电位显著降低(9.833%±2.285% vs. 2.667%±0.208%;t=6.645,P <0.01)。结论多房棘球蚴感染可损伤巨噬细胞线粒体功能、抑制巨噬细胞氧化磷酸化过程而使糖酵解增强,巨噬细胞代谢状态改变可能是多房棘球蚴病发生和发展的机制之一。

Objective To investigate the changes of mitochondrial metabolic functions of macrophages following Echinococcus multilocularis infections,so as to provide insights into the pathogenesis of alveolar echinococcosis.Methods Two groups were assigned according to different treatment methods. In the culture group,mouse leukemic monocyte macrophage RAW264.7 cells were cultured with 2 000 E. multilocularis at a ratio of 500∶1,while RAW264.7 cells in the control group were given no treatment. Then,both the culture and control groups were further divided into the 24 h and 72 h subgroups. Mitochondria were stained with MitoTracker?Deep Red FM and the mean fluorescence intensity of macrophage mitochondria was measured with the Cytation 5 Cell Imaging Multi-Mode Reader. The mitochondrial DNA copy number was quantified using the quantitative realtime PCR(qPCR)assay,and the mitochondrial energy metabolism was monitored using the Seahorse XF assay. In addition,the mitochondrial reactive oxygen species and mitochondrial membrane potential were detected using flow cytometry.Results The mean fluorescence intensities of macrophage mitochondria were significantly lower in the 24 h(15 341 ± 2 532 vs. 17 823 ± 3 429;t = 6.379,P < 0.01)and 72 h(18 102 ± 3 505 vs. 21 511 ± 5 144;t = 17.680,P < 0.01)culture subgroups than in the corresponding control subgroups,and lower mitochondrial DNA copy numbers were measured in the 72 h culture subgroup than in the 72 h control group [(3.23 × 10^(9));t = 8.85,P < 0.001]. The oxygen consumption rates were significantly greater in the 24 h [(241.70 ± 73.13)pmol/min vs.(69.05 ± 52.30)pmol/min;t = 7.89,P < 0.01] and48 h culture groups [(249.50 ± 42.06)pmol/min vs.(60.28 ± 40.66)pmol/min;t = 8.64,P < 0.01] than in the corresponding control groups,and a higher extracellular acidification rate was seen in the 48 h culture group than in the 48 h control group[(111.6 ± 17.49)mpH/min vs.(35.05 ± 7.57)mpH/min;t = 16.90,P < 0.01]. In addition,flow cytometry detected higher mean fluorescence intensity of mitochondrial reactive oxygen species(58 264 ± 10 087 vs. 4 307 ± 97;t = 12.930,P < 0.01)and lower mitochondrial membrane potential(9.833% ± 2.285% vs. 2.667% ± 0.208%;t = 6.645,P < 0.01)in the 72 h culture group than in the control group.Conclusions E. multilocularisinfection may impair mitochondrial functions and inhibit oxidative phosphorylation of macrophages,resulting in increased macrophage glycolysis. It is speculated that the alteration of macrophage metabolic states may contribute to the mechanisms underlying the development and progression of alveolar echinococcosis.