MiR-138-5p通过抑制SIRT1表达增强了海水吸入性肺损伤中炎症反应

doi:10.3877/cma.j.issn.1674-6902.2023.06.001

目的

观察miR-138-5p在海水吸入性肺损伤(acute lung injury, ALI)中的作用及机制。

方法

分别构建海水吸入性肺损伤大鼠及大鼠肺血管内皮细胞(rat pulmonary vascular endothelial cells, RPMVECs)细胞模型，分为空白对照组、海水处理组、miR-138-5p antagomir预处理组和antag NC预处理组。观察肺组织湿干比、伊文思蓝法检测肺微血管通透性，用ELISA法检测肺组织及细胞中肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白介素-1β(interleukin-1β，IL-1β)和白介素-6(interleukin-6, IL-6)的含量，western blot检测SIRT1及乙酰化(Acetyl)-核因子-κB(nuclear factor kappa-B, NF-κB)的表达变化，采用双荧光素酶报告基因实验验证SIRT1和miR-138-5p的作用关系。

结果

海水干预4 h后，大鼠肺组织湿干比及通透性明显增加，组织及细胞中TNF-α、IL-1β和IL-6的含量增高，miR-138-5p表达明显增高，SIRT1表达出现了下降，Acetyl-NF-κB的表达明显升高。而抑制miR-138-5p的表达后，炎症反应得到了明显的减轻，SIRT表达出现了回升，Acetyl-NF-κB的表达出现了下降。

结论

增加表达的miR-138-5p在海水吸入性肺损伤炎症的发展中起到了关键作用，这种作用是通过调节SIRT1通路形成的。

关键词: 急性肺损伤, 海水, SIRT1通路, miR-138-5p

Objective

To observe the role and mechanism of miR-138-5p in seawater aspiration-induced acute lung injury(ALI).

Methods

Rats and rat pulmonary vascular endothelial cells(RPMVECs) seawater aspiration induced ALI models were established and were divided into 4 groups: normal control group, seawater group, miR-138-5p antagomir group and antag NC pre-treated group. W/D ratio and Evans blue were carried out after modeling. The contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) were measured by enzyme linked immunosorbent serologic assay(ELISA) and the expression of SIRT1 and Acetyl-nuclear factor kappa-B(NF-κB) were measured by western blot. Doubleluciferase reporter gene assay was used to verify the functional relationship between SIRT1 and miR-138-5p.

Results

After 4 h seawater stimulation, W/D ratio and Evans blue were obvious and the contents of TNF-α, IL-1β and IL-6 were increased. The expression of miR-138-5p, Acetyl-NF-κB were increased and SIRT1 expression was inhibited. Inhibition of miR-138-5p decreased the inflammation, oxidative stress and Acetyl-NF-κB and increased the expression of SIRT1.

Conclusion

These results suggest that increased expression of miR-138-5p was an important cause in seawater-induced ALI and this phenomenon was through SIRT1 pathway.

Key words: Acute lung injury, Seawater, SIRT1 pathway, miR-138-5p

图1 组织及细胞中miR-138-5p表达的改变。注：A：空白对照组；B：海水刺激组；C：miR-138-5p antagomir预处理组；D：antag NC预处理组***P<0.001 vs.空白对照组；###P<0.001 vs.海水刺激组

图2 肺组织湿干比及通透性的改变。注：A：空白对照组；B：海水刺激组；C：miR-138-5p antagomir预处理组；D：antag NC预处理组***P<0.001 vs.空白对照组；#P<0.05 vs.海水刺激组

图3 炎症因子的改变。注：A：空白对照组；B：海水刺激组；C：miR-138-5p antagomir预处理组；D：antag NC预处理组***P<0.001 vs.空白对照组；##P<0.01，###P<0.001 vs.海水刺激组

图4 miR-138-5p直接靶向作用于SIRT1。A: TargetScan网站预测SIRT1为miR-138-5p的下游靶点；B：采用双荧光素酶报告基因法验证SIRT1和miR-138-5p之间的相互作用

图5 大鼠肺组织SIRT1及NF-κB乙酰化的改变。注：A：空白对照组；B：海水刺激组；C：miR-138-5p antagomir预处理组；D：antag NC预处理组***P<0.001 vs.空白对照组；###P<0.001 vs.海水刺激组

图6 RPMVECs细胞中SIRT1及NF-κB乙酰化的改变。注：A：空白对照组；B：海水刺激组；C：miR-138-5p antagomir预处理组；D：antag NC预处理组***P<0.001 vs.空白对照组；##P<0.01，###P<0.001 vs.海水刺激组

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MiR-138-5p enhanced the inflammation in seawater aspiration-induced acute lung injury via inhibition the expression of SIRT1

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MiR-138-5p enhanced the inflammation in seawater aspiration-induced acute lung injury via inhibition the expression of SIRT1