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目的观察铜绿假单胞菌外毒素A(PEA)对MLE-12细胞的氧化应激损伤及其自噬活动的影响。方法体外培养MLE-12细胞至对数生长期,分别采用PEA 0、200、400、800和1 000ng/mL处理细胞1h和2h,之后进一步采用PEA 1 000ng/mL处理细胞15min、30min、1h、2h和3h,应用过氧化氢试剂盒检测H2O2的生成量,Western-blot法检测微管相关蛋白1轻链3(LC3)的表达,分析LC3II与β-actin之间的比值变化。结果与对照组细胞相比较,随着PEA浓度的增高,细胞的H2O2检出量逐渐增加,PEA处理2h后,各组细胞H2O2检出量分别是对照组的1.73、2.09、3.82和5.06倍(P<0.01)。与此同时,细胞中自噬标记物LC3II的表达量显著增加。此外,高浓度PEA(1 000ng/mL)处理细胞后,随着时间的延长,H2O2检出量明显升高,同时伴随着细胞自噬活动的增强。结论 PEA可以诱导MLE-12细胞产生氧化应激损伤,并激活细胞的自噬活动。
Objective To investigate the effects of Pseudomonas aeruginosa exotoxin A (PEA) on oxidative stress injury and autophagy in MLE-12 cells. Methods MLE-12 cells were cultured in vitro until logarithmic growth phase. Cells were treated with PEA at 0, 200, 400, 800 and 1 000 ng / mL for 1 h and 2 h, respectively, and then treated with PEA at 1 000 ng / mL for 15 min, 30 min, H2O2 was used to detect the production of H2O2 at 2h and 3h. The expression of LC3 and LC3II was detected by Western-blot, and the ratio of LC3II and β-actin was analyzed. Results Compared with the control group, the detection of H2O2 increased gradually with the increase of PEA concentration. After 2 hours of PEA treatment, the detected amount of H2O2 in each group was 1.73, 2.09, 3.82 and 5.06 times respectively P <0.01). In the meantime, the expression level of autophagy marker LC3II in cells increased significantly. In addition, after treated with high concentration of PEA (1 000 ng / mL), the detected amount of H2O2 increased significantly with the prolongation of time, accompanied by the increase of autophagy. Conclusion PEA can induce oxidative stress injury in MLE-12 cells and activate cell autophagy.