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中华危重症医学杂志(电子版) ›› 2021, Vol. 14 ›› Issue (05) : 355 -361. doi: 10.3877/cma.j.issn.1674-6880.2021.05.002

论著

连接蛋白43通过蛋白激酶A介导丝氨酸373调控脓毒症急性肺损伤肺泡Ⅱ型上皮细胞屏障功能的研究
赵希伟1, 周佳伟2, 刘凯3, 侯林义3, 张文凯3,†()   
  1. 1. 030001　太原，山西医科大学第二医院心胸外科
    2. 430060　武汉，湖北省人民医院肝胆外科
    3. 030001　太原，山西医科大学第二医院重症医学科
  • 收稿日期:2020-12-03 出版日期:2021-10-31
  • 通信作者: 张文凯
  • 基金资助:
    山西省太原市科技项目(12016905)

Connexin 43 regulates the barrier function of alveolar type Ⅱ epithelial cells in sepsis-induced acute lung injury through serine 373 mediated by protein kinase A

Xiwei Zhao1, Jiawei Zhou2, Kai Liu3, Linyi Hou3, Wenkai Zhang3,†()   

  1. 1. Department of Cardiothoracic Surgery, the Second Hospital of Shanxi Medical University, Taiyuan 030001, China
    2. Department of Hepatobiliary Surgery, Hubei General Hospital, Wuhan 430060, China
    3. Department of Critical Care Medicine, the Second Hospital of Shanxi Medical University, Taiyuan 030001, China
  • Received:2020-12-03 Published:2021-10-31
  • Corresponding author: Wenkai Zhang
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赵希伟, 周佳伟, 刘凯, 侯林义, 张文凯. 连接蛋白43通过蛋白激酶A介导丝氨酸373调控脓毒症急性肺损伤肺泡Ⅱ型上皮细胞屏障功能的研究[J/OL]. 中华危重症医学杂志(电子版), 2021, 14(05): 355-361.

Xiwei Zhao, Jiawei Zhou, Kai Liu, Linyi Hou, Wenkai Zhang. Connexin 43 regulates the barrier function of alveolar type Ⅱ epithelial cells in sepsis-induced acute lung injury through serine 373 mediated by protein kinase A[J/OL]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2021, 14(05): 355-361.

目的

观察脓毒症急性肺损伤肺泡Ⅱ型上皮细胞中连接蛋白43（Cx43）表达水平与肺泡气血屏障通透性的关系，并探讨Cx43及蛋白激酶A（PKA）信号通路在脓毒症急性肺损伤中的作用。

方法

将A549细胞分为对照组、脓毒症组[脂多糖（LPS）组]、LPS + PKA抑制剂组（LPS + H89组）和8-Bromo-cAMP组（PKA激活剂组）。LPS组和LPS + H89组加入LPS 1 μg/mL，PKA激活剂组加入8-Bromo-cAMP 10 μmol/L，均处理24 h；其中，LPS + H89组予H89 10 μmol/L预处理1 h后弃去。采用Western-blotting法检测各组细胞磷酸化Cx43（p-Cx43）、PKA及磷酸化PKA（p-PKA）的蛋白表达水平，并用Transwell板进行单层细胞培养，用酶标仪测定A549单层细胞的通透性。

结果

4组细胞p-Cx43、PKA和p-PKA蛋白表达水平及细胞通透性比较，差异均有统计学意义（F = 8.961、249.729、7 526.430、3 661.755，P均< 0.05）。进一步两两比较发现，LPS组和PKA激活剂组p-Cx43和PKA蛋白表达水平及细胞通透性均较对照组显著升高，LPS组、LPS + H89组和PKA激活剂组p-PKA蛋白表达水平均较对照组显著升高；LPS + H89组p-Cx43和PKA蛋白表达水平及细胞通透性均较LPS组降低，p-PKA则较LPS组有所升高；PKA激活剂组较LPS组和LPS + H89组细胞通透性均显著增大（P均< 0.05）。

结论

Cx43通过PKA介导丝氨酸373调控在脓毒症引起的急性肺损伤中起着关键作用，其可增加肺泡Ⅱ型上皮细胞的通透性，破坏肺泡屏障功能及增加缝隙连接细胞间通讯。

关键词: 脓毒症, 急性肺损伤, 蛋白激酶A, 连接蛋白43, 肺泡Ⅱ型上皮细胞
Objective

To observe the relationship between connexin 43 (Cx43) of alveolar type Ⅱ epithelial cells and the permeability of alveolar air blood barrier in sepsis-induced acute lung injury, and to explore the role of Cx43 and protein kinase A (PKA) signaling pathway in sepsis-induced acute lung injury.

Methods

A549 cells were divided into a control group, a sepsis group [lipopolysaccharide (LPS) group], a LPS + PKA inhibitor group (LPS + H89 group) and a 8-Bromo-cAMP group (PKA activator group). Both the LPS group and LPS + H89 group were treated with LPS 1 μg/mL, while the PKA activator group was treated with 8-Bromo-cAMP 10 μmol/L, all for 24 h. In addition, the LPS + H89 group was pretreated with H89 10 μmol/L for 1 h. The protein expression levels of phosphorylated Cx43 (p-Cx43), PKA and phosphorylated PKA (p-PKA) in each group were determined by the Western-blotting assay. Monolayer cell cultures were performed with Transwell plates, and the permeability of A549 monolayers was determined by a microplate reader.

Results

The p-Cx43, PKA and p-PKA protein expression levels and the cell permeability were significantly different among the four groups (F = 8.961, 249.729, 7 526.430, 3 661.755; all P < 0.05). Further pairwise comparisons revealed that compared with the control group, the p-Cx43 and PKA protein expression levels and the cell permeability significantly increased in the LPS and PKA activator groups, and the p-PKA protein expression levels significantly increased in the LPS, LPS + H89 and PKA activator groups (all P < 0.05). The p-Cx43 and PKA protein expression levels and the cell permeability reduced, while the p-PKA protein expression levels increased in the LPS + H89 group compared with the LPS group (all P < 0.05). The cell permeability in the PKA activator group significantly increased compared with the LPS and LPS + H89 groups (both P < 0.05).

Conclusion

Cx43 plays a key role in sepsis-induced acute lung injury through regulating serine 373 mediated by PKA, which can increase permeability of alveolar type Ⅱ epithelial cells, disrupt alveolar barrier function and increase gap junction intercellular communication.

Key words: Sepsis, Acute lung injury, Protein kinase A, Connexin 43, Alveolar type Ⅱ epithelial cells
图1 4组A549细胞p-Cx43蛋白表达水平的比较（n = 3）
图2 4组A549细胞PKA蛋白表达水平的比较（n = 3）
图3 4组A549细胞p-PKA蛋白表达水平的比较（n = 3）
图4 4组A549细胞通透性的比较（n = 3）
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