TGF-β1/Smad2信号通路参与海水淹溺肺损伤引起的细胞凋亡

doi:10.3877/cma.j.issn.1674-6902.2020.02.006

目的

探讨TGF-β1/Smad2信号通路是否参与海水淹溺肺损伤引发的细胞凋亡过程。

方法

体外培养A549人肺癌细胞，用浓度为20%、40%、60%的海水进行处理，根据MTT实验结果，筛选出最适浓度的海水用于后续研究；用抑制剂SB431542预处理细胞后，再用最适浓度的海水进行处理，然后采用Real-time PCR与Western blot检测凋亡相关指标Cleaved Caspase-3的表达水平、流式细胞术与Hoechst染色观察细胞凋亡情况，同时应用Real-time PCR与Western blot对TGF-β1、Smad2和p-Smad2的表达水平进行检测。

结果

随着海水浓度的增加，A549细胞的活力逐渐降低，最终选用浓度为40%的海水进行后续研究；海水处理可使细胞发生明显的凋亡现象，而抑制剂SB431542可明显恢复由海水引发的凋亡水平；同时，发现海水使TGF-β1的表达水平显著上调，抑制剂SB431542处理后这种异常上调可显著被缓解，而Smad2的表达水平无显著性变化，但海水可使p-Smad2表达水平显著上调，且抑制剂SB431542处理后p-Smad2的表达水平可明显被抑制。

结论

海水淹溺肺损伤可能通过激活TGF-β1/Smad2信号通路，调节凋亡执行蛋白Caspase-3的活性，从而促使肺组织细胞发生凋亡。

关键词: 海水淹溺肺损伤, 细胞凋亡, TGF-β1/Smad2信号通路

Objective

To investigate whether TGF-β1/Smad2 signal pathway is involved in the process of apoptosis induced by seawater-induced acute lung injury.

Methods

A549 human lung cancer cells were cultured in vitro and treated with seawater at the concentrations of 20%, 40%, and 60%, respectively. According to the MTT results, the optimal concentration of seawater was selected for subsequent studies. The cells were pretreated with the inhibitor SB431542, and then treated with the optimal concentration of seawater. Then the expression level of apoptosis-related molecule cleaved caspase-3 was detected by Real-time PCR and Western blotting, and the cell apoptosis was observed by flow cytometry and Hoechst staining. At the same time, the expression levels of TGF-β1, Smad2 and p-Smad2 were detected by Real-time PCR and Western blotting.

Results

As the concentration of seawater increased, the viability of A549 cells gradually decreased, and 40% of seawater was used for the subsequent research. Seawater treatment could cause obvious apoptosis of the cells, and the inhibitor SB431542 could significantly restore the level of apoptosis induced by seawater. At the same time, the expression level of TGF-β1 was significantly up-regulated by seawater, and the abnormal up-regulation was significantly decreased by the inhibitor. While the expression level of Smad2 was not significantly changed, but seawater can significantly up-regulate the expression level of p-Smad2, and the up-regulation of p-Smad2 can be significantly inhibited after the inhibitor treatment.

Conclusion

Seawater-induced acute lung injury may activate the TGF-β1/Smad2 signal pathway and regulate the activity of the apoptosis-executing protein Caspase-3, thereby promoting the apoptosis in the lung tissue cells.

Key words: Seawater drowning-induced acute lung injury, Cell apoptosis, TGF-β1/Smad2 signal pathway

图1 MTT法检测细胞活力结果

图2 Hoechst染色观察细胞凋亡结果(400×)

图3 TGF-β1/Smad2信号通路相关指标mRNA与蛋白表达水平；注：A：Real-time PCR检测相关指标mRNA表达水平；B：Western blot检测相关指标蛋白表达水平

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