胶霉毒素对肺泡Ⅱ型上皮细胞的作用研究

A study on the damaging effects of gliotoxin on alveolar type Ⅱ epithelial cell

摘要:

目的 探讨胶霉毒素（GT）对肺泡Ⅱ型上皮细胞损伤的影响及其潜在作用机制。方法 以GT作用于A549细胞制备体外模型。A549细胞与不同浓度GT共同孵育24 h后，采用CCK-8法检测细胞活性；采用流式细胞术检测细胞内活性氧（ROS）含量和细胞凋亡水平；采用Western blotting检测凋亡相关蛋白Caspase-3、Bax、Bcl-2的表达。结果 与对照组相比，不同浓度的GT均能抑制A549细胞的增殖活性，0.500 000 μmol/L组、1.000 000 μmol/L组、2.000 000 μmol/L组、4.000 000 μmol/L组的A549细胞活性均低于对照组（P <0.05），可以认为在浓度达到0.5 μmol/L时A549细胞活性开始下降，并随着药物浓度的增加呈浓度依赖性；与对照组相比，2.0 μmol/L组和4.0 μmol/L组的细胞内ROS含量、细胞凋亡率、Caspase-3和Bax表达均升高，Bcl-2表达降低（P <0.05）。结论 GT能降低A549细胞活性，使其ROS活性增强，并通过Caspase-3和Bax表达升高，Bcl-2表达降低，经线粒体介导的内源性凋亡途径导致细胞凋亡。

Abstract:

Objective This study aimed to investigate the effects of gliotoxin on type Ⅱ alveolar epithelial cell injury and explore its underlying mechanisms.Methods An in vitro model was established by treating A549 cells with gliotoxin (GT). The CCK-8 assay was used to detect the viability of A549 cells after 24-hour co-incubation with different concentrations of GT. Intracellular reactive oxygen species (ROS) content and apoptosis levels were measured by flow cytometry. Western blotting was performed to detect the expression of apoptosis-related proteins Caspase-3, Bax, and Bcl-2.Results As compared with the control group, different concentrations of GT exhibited an inhibitory effect on the proliferative activity of A549 cells. The viability of A549 cells in the 0.500 000 μmol/L, 1.000 000 μmol/L, 2.000 000 μmol/L, and 4.000 000 μmol/L GT groups was all lower than that in the control group, with statistically significant differences in all pairwise comparisons (P < 0.05). These results indicate that the viability of A549 cells starts to decrease at a GT concentration of 0.5 μmol/L, and the inhibitory effect shows a concentration-dependent manner with the increase in GT concentration. Compared with the control group, the 2.0 μmol/L and 4.0 μmol/L groups exhibited increased intracellular ROS content, apoptosis rate, and expression levels of Caspase-3 and Bax, while the expression of Bcl-2 was decreased (P < 0.05).Conclusion GT can reduce the activity of A549 cells, increase their ROS activity, and induce cell apoptosis through the mitochondria-mediated intrinsic apoptotic pathway by upregulating the expressions of Caspase-3 and Bax and downregulating the expression of Bcl-2.