摘要
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4h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-KB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-KB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.
Abstract
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.
基金项目
解放军总医院第八医学中心国科金配套基金(QN202211004)
万方数据