通过NF-κB调控蛋白激酶SPAK通路促进NR8383细胞炎症介质释放及氧化应激发生

doi:10.3877/cma.j.issn.1674-6902.2022.02.002

目的

分析SPAK在高渗透压应激所致大鼠肺泡世噬细胞系(NR8383)细胞炎症介质释放及氧化应激发生中的作用。

方法

在细胞培养液中加入核因子κB(nuclear factor κappa-B, NF-κB)的抑制剂吡咯烷二硫代氨基甲盐酸(1-Pyrrolidinecarbodithioic acid, PDTC)阻断其磷酸化，检测其对高渗刺激引起的SPAK表达升高的抑制作用。通过siRNA干扰技术下调SPAK的表达，检测NR8383细胞炎症介质TNF-α和IL-1的分泌以及MPO活性和LDH释放，阐明SPAK在细胞应对高渗透压刺激时的调控作用。

结果

高渗透压刺激可以导致NR8383细胞中的NF-κB的磷酸化并上调SPAK蛋白表达，而通过对NR8383细胞进行PDTC预处理，NF-κB的磷酸化及SPAK蛋白表达水平可以显著受到抑制。而SPAK表达下调可以减轻高渗刺激所致的炎症因子释放增加，降低MPO活性以及H₂O₂浓度。

结论

磷酸化NF-κB可以调控SPAK转录及翻译，而SPAK可以通过促进炎性介质释放、诱导氧化应激参与到高渗透压刺激的调控中。

关键词: 大鼠肺泡世噬细胞, 核因子-κB, 高渗透压刺激, 蛋白激酶SPAK, 氧化应激

Objective

To investigate the role of STE20/SPS1-related proline/alanine-rich kinase (SPAK) in the release of inflammatory mediators and occurrence of oxidative stress in rat alveolar macrophage cells(NR8383) cells exposed hyperosmotic stress.

Methods

1-Pyrrolidinecarbodithioic acid (PDTC), a specific inhibitor of NF-κB, was added to inhibit its phosphorylating, and the increase of SPAK expression induced by hypertonic stimulation was also detected. After down regulating the expression of SPAK in NR8383 cells by siRNA interference technology, the secretion of TNF-α and IL-1, MPO activity and H₂O₂ concentration were detected, which further elucidated the role of SPAK in hyperosmotic stress regulation.

Results

PDTC pretreatment significantly inhibited the phosphorylation of NF-κB and the expression of SPAK protein in NR8383 cells. However, the down-regulation of SPAK expression could reduce the increased release of inflammatory factors, MPO activity and H₂O₂ concentration caused by hypertonic stimulation.

Conclusion

The transcription of SPAK is regulated by the activation of NF-κB, and plays an important role in promoting the inflammatory response, the release of inflammatory mediators and occurrence of oxidative stress.

Key words: Rat alveolar macrophage cells, NF-κB, Hyperosmotic stress, STE20/SPS1-related proline/alanine-rich kinase, Oxidative stress

图1 海水刺激及PDTC预处理对NR8383细胞NF-κB磷酸化及SPAK表达的影响。注：A：海水刺激及PDTC预处理对NF-κB磷酸化的影响；B：PDTC预处理及海水高渗透压刺激对SPAK转录的影响；C：海水刺激及PDTC预处理对SPAK蛋白表达的影响；N为正常对照组，SW为高渗透压海水刺激组，SW+PDTC为PDTC预处理+高渗透压海水刺激组

图2 SPAK特异性siRNA可以抑制蛋白激酶SPAK在NR8383细胞中的转录和翻译。注：A：转染SPAK特异性siRNA可抑制SPAK的转录；B：转染SPAK特异性siRNA可降低SPAK蛋白的表达。Con为对照组，Scram为非特异性干扰组，siSPAK为SPAK特异性干扰组

图3 抑制NR8383细胞SPAK的转录、翻译可以减少海水刺激所致炎症因子TNF-α及IL-1β释放。注：N为对照组，SW为高渗海水刺激组，SW+siSPAK为SPAK干扰+高渗海水刺激组；*P<0.05 vs. N，***P<0.001 vs. N，#P<0.05 vs. SW

图4 抑制NR8383细胞SPAK的转录、翻译可以减少海水刺激所致MPO活性增加以及H₂O₂浓度升高。注：N为正常对照组，SW为高渗海水刺激组，SW+siSPAK为SPAK干扰+高渗海水刺激组；*P<0.05 vs. N, ***P<0.001 vs. N，#P<0.05 vs. SW

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NF-κB regulates the release of inflammatory mediators and occurrence of oxidative stress in NR8383 cells through SPAK pathway

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NF-κB regulates the release of inflammatory mediators and occurrence of oxidative stress in NR8383 cells through SPAK pathway