低频低强度超声对巨噬细胞内耻垢分枝杆菌的损伤效应

Low-frequency and low-intensity ultrasound suppresses activity of Mycobacterium smegmatis in macrophages

目的探讨低频低强度超声(low frequency low intensity ultrasound,LFLIU)对巨噬细胞内耻垢分枝杆菌的损伤效应及机制。方法将巨噬细胞RAW264.7分为超声辐照0、5、10 min和15 min组(n=3),应用MTT比色法分析不同超声强度对RAW264.7细胞存活率的影响,采用流式细胞术检测RAW264.7细胞经超声辐照以及吞噬细菌后的凋亡率和坏死率,使用平板计数法评估RAW264.7细胞内耻垢分枝杆菌活性,使用激光共聚焦显微镜观察RAW264.7细胞经超声辐照后活性氧(reactive oxygen species,ROS)的产生。结果超声辐照声强为0.14 W/cm^2,辐照时间为5、10 min时,RAW264.7细胞活性未见明显影响,存活率分别为(116.37±9.54)%、(97.14±9.26)%;经0.14 W/cm2超声辐照5 min后,RAW264.7细胞的凋亡率和坏死率显著大于超声辐照0 min组(P<0.05);平板菌落计数结果显示,RAW264.7细胞内耻垢分枝杆菌经声强0.14 W/cm^2超声辐照5、10 min后活性均低于超声辐照0 min组(P<0.05);激光共聚焦观察到0.14 W/cm^2超声刺激RAW264.7细胞产生了ROS。结论 LFLIU能诱导RAW264.7细胞凋亡和产生ROS,并导致RAW264.7细胞内耻垢分枝杆菌活性下降。

Objective To investigate the effect of low-frequency and low-intensity ultrasound（ LFLIU） in inducing damages of Mycobacterium smegmatis（ M. smegmatis） in macrophages and explore the mechanism. Methods Murine macrophage RAW264. 7 cells were exposed to ultrasound irradiation for 5,10 or 15 min,and the changes in the cell viability were analyzed with MTT assay. The apoptotic and necrosis rates of RAW264. 7 cells after ultrasonic irradiation and after phagocytosis of M. smegmatis were assessed using flow cytometry; the activity of M. smegmatis in RAW264. 7 cells following the exposure was evaluated with plate colony counting. Laser scanning confocal microscopy was used to observe the production of reactive oxygen species（ ROS） in RAW264. 7 cells after ultrasound exposure. Results Exposure of RAW264. 7 cells to a ultrasonic intensity of 0. 14 W/cm^2 for 5 min and 10 min resulted in cell survival rates of（ 116. 37 ±9. 54） % and（ 97. 14 ± 9. 26） %,respectively,suggesting no significant impact of LFLIU exposure on the cell viability. The apoptotic rate of RAW264. 7 cells increased significantly after irradiation with 0. 14 W/cm^2 ultrasound for 5 min compared with the control cells（ P〈0. 05）. The results of plate colony counting suggested that the viability of M. smegmatis in RAW264. 7 cells was significantly lowered after exposure to a ultrasound intensity of 0. 14 W/cm^2 for 5 and 10 min. Confocal laser scanning microscopy revealed obvious ROS production in RAW264. 7 cells in response to 0. 14 W/cm^2 ultrasound exposure. Conclusion Exposure to LFLIU induces apoptosis and ROS production in RAW264. 7 cells and causes decreased activity of M. smegmatis within the cells.