摘要
目的探讨海水对人肺泡上皮细胞中活性氧(ROS)和内质网应激(ER stress)水平的影响。方法以含不同浓度海水(20%、40%、60%、80%)的培养液刺激人肺泡上皮细胞系A549细胞1 h后更换培养液,3 h后CCK-8法检测海水对细胞的毒性;以25%浓度海水刺激A549细胞1 h后,不同时间点(2 h、4 h、6 h、8 h)应用CCK-8法检测海水对细胞的毒性,Western Blotting法检测内质网应激特异蛋白GRP78蛋白表达情况,DCFH-DA探针检测细胞内活性氧水平变化情况。结果以不同浓度海水刺激A549细胞时,细胞活性受抑制程度呈明显的浓度依赖性,海水处理组与对照组相比较,差异有统计学意义(P<0.05);细胞在受到短暂海水刺激后恢复时,细胞活性依然受到明显抑制,在4 h抑制最显著,之后逐渐恢复,2 h、8 h组与0 h组相比较,差异无统计学意义(P>0.05);而4 h、6 h组与0 h组相比较,差异有统计学意义(P<0.05);同时细胞内活性氧类(ROS)含量增多,内质网应激相关蛋白GRP78表达量增加,且都在4 h达到峰值。结论海水干预可抑制人肺泡上皮细胞增殖活性,而内质网应激和活性氧类参与了海水引起的A549细胞损伤过程。
Objective To investigate the effect of seawater on the levels of reactive oxygen species （ROS） and endoplasmic reticulum stress （ER stress） in human alveolar epithelial cells. Methods The A549 cells were stimulated with cell culture medium containing different concentrations of seawater （20%, 40%, 60%, 80%） for 1 hour, then the culture medium was replaced for 3 hours, and then the CCK-8 method was used to detect the cytotoxicity of seawater; CCK-8 assay was used to detect the cytotoxicity of seawater on the ceils at different time points （2 h, 4 h, 6 h, 8 h）after A549 cells were stimulated with 25% concentrations of seawater 1 hour. The expression of ER stress related protein （GRP78） was detected by Western blotting method. The level of intracellular ROS was detected by DCFH-DA probe. Results When the A549 cells were stimulated with different concentrations of seawater, the inhibition degree of the cell proliferation activity showed a concentration-dependent manner, compared with the control group, the difference was statistically significant （P〈O.05） ; When the cells were recovered from the sea water stimulation, the cell activity was still significantly inhibited, the most significant inhibition in 4 h, followed by a gradual recovery , Compared with the 0 h group, the 2 h and 8 h group had no significant difference （P〉0.05）, and the difference of 4 h and 6 h group wasstatistically significant （P〈 0.05 ） ; At the same time, the content of ROS increased, and the expression of GRP78 increased, and all of them reached the peak value at 4 h. Conclusion Seawater intervention can inhibit the proliferation of human alveolar epithelial cells, and the ER stress and ROS are involved in the process of A549 cell damage induced by seawater.
出处
《中华肺部疾病杂志（电子版）》
CAS
2016年第5期489-493,共5页
Chinese Journal of Lung Diseases(Electronic Edition)
基金
国家自然科学基金资助项目(81570067)
关键词
急性肺损伤
活性氧
内质网应激
海水
Acute lung injury
Reactive oxygen species
Endoplasmic reticulum stress
Seawater
