香烟烟雾提取物对巨噬细胞线粒体功能的影响

Effects of cigarette smoke extract on mitochondrial function of macrophages

目的:研究香烟烟雾提取物(CSE)对巨噬细胞线粒体功能的影响。方法:本研究选取RAW264.7巨噬细胞进行实验。当细胞融合度达70%左右,弃旧培养液,将100%CSE原液用无血清的DMEM和FBS稀释成1%、5%、15%、25%和90%CSE加入到孔板中,采用CCK-8法检测不同浓度CSE处理RAW264.7细胞24 h的细胞活性;再选取最佳CSE浓度分别处理细胞0 h、24 h、48 h、72 h,CCK-8法检测CSE处理细胞不同时间组细胞活性。0%、5%和25%CSE处理24 h后,Annexin V-FITC/PI染色检测细胞坏死和凋亡情况;JC-1线粒体膜电位检测试剂盒检测细胞线粒体膜受损情况;ROS特异性染料DCFH-DA对小鼠巨噬细胞进行染色后,流式细胞计数法测定荧光度及ROS阳性巨噬细胞数目比例;增强型ATP检测试剂盒检测细胞内ATP浓度。结果:①和0%CSE组比较,1%CSE处理组细胞活性显著增加(P<0.01),当CSE浓度为5%以上时细胞活性显著下降(P<0.05);使用5%CSE处理巨噬细胞,随着处理时间的增加细胞活性显著下降(P<0.01)。②和0%CSE组比较,5%CSE和25%CSE处理主要导致巨噬细胞坏死,引起巨噬细胞线粒体膜电位显著下降、ROS生成显著增加(P<0.01)、ATP浓度显著减少(P<0.05或P<0.01),且25%CSE处理组的变化更为显著(P<0.05或P<0.01)。结论:CSE处理可能影响巨噬细胞线粒体功能,进而导致细胞活性下降并出现坏死。

Objective:To investigate the effects of cigarette smoke extract(CSE)on the mitochondrial function of macrophages.Methods:RAW264.7 macrophages were used for the experiment in this study.When the cell density was about 70%,the old culture medium was abandoned,and the 100%CSE stock solution was diluted with serum-free DMEM and FBS into 1%,5%,15%,25%and 90%CSE and added to the well plate.The cell activity of RAW264.7 cells treated with CSE at different concentrations for 24 h was detected by CCK-8 method.Then the optimal CSE concentration was selected to treat cells for 0 h,24 h,48 h or 72 h respectively,and CCK-8 assay was used to detect the cell activity of CSE treated cells at different time groups.After the cells were treated with 0%,5%and 25%CSE for 24 hours,cell necrosis and apoptosis was detected by Annexin V-FITC/PI staining;Mitochondrial membrane damage of RAW 264.7 was detected by mitochondrial membrane potential assay kit with JC-1;Macrophages were stained with ROS-specific dye DCFH-DA,and then Flow cytometer was used to determine the fluorescence and the proportion of ROS-positive macrophages;the enhanced ATP assay kit was used to detect the intracellular ATP concentration.Results:①Compared with 0%CSE,cell viability was increased significantly in 1%CSE group(P<0.01),cell viability was decreased significantly when CSE concentration was above 5%(P<0.05);Macrophages were treated with 5%CSE,and cell viability was decreased significantly with the increase of treatment time(P<0.01).②Compared with 0%CSE,5%CSE and 25%CSE mainly caused macrophage necrosis,decreased mitochondrial membrane potential,increased ROS production and decreased ATP significantly(P<0.05 or P<0.01),and the changes were more significant in 25%CSE treatment group(P<0.05 or P<0.01).Conclusion:CSE may affect mitochondrial function of macrophages,leading to decreased cell viability and necrosis.