MicroRNA-34a在脂多糖诱导急性呼吸窘迫综合征中的表达及作用机制

Expression of microRNA-34a in lipopolysaccharide-induced acute respiratory distress syndrome and its mechanism of action

目的:探讨microRNA-34a(miR-34a)在脂多糖(lipopolysaccharides,LPS)诱导急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)中表达的意义及作用机制。方法:40只雄性SD大鼠随机分为5组:对照组,腹腔注射10 mg/kg LPS 3、6、12、24 h组,人肺泡Ⅱ型上皮细胞株(A549)分为5组:对照组,10μg/mL LPS刺激3、6、12、24 h组。在A549细胞中转染miR-34a inhibitor,24 h后加入10μg/mL LPS,再刺激24 h。测定肺湿/干重值,观察肺组织病理学改变,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测肺组织及细胞miR-34a表达,ELISA检测肺组织及细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)浓度,Western blot检测细胞磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase,PI3K)、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-AKT)的蛋白表达。结果:实验组肺湿/干重值较对照组明显升高。苏木素-伊红(HE)染色显示实验组肺组织肺泡间隔增厚,肺泡腔内有粉红色水肿液,肺间质炎性细胞浸润及出血。miR-34a表达在实验组的各个时间点较对照组升高,TNF-α、IL-1β浓度在实验组的各个时间点较对照组升高,miR-34a表达与TNF-α、IL-1β浓度呈正相关。PI3K、p-AKT蛋白表达在实验组的各个时间点较对照组升高。转染miR-34a inhibitor能够明显下调LPS作用于A549细胞后TNF-α、IL-1β浓度及PI3K、p-AKT蛋白表达。结论:miR-34a可能通过抑制PI3K/AKT通路减轻LPS诱导的肺组织损伤,为ARDS的治疗提供新的靶点。

Objective:To investigate the significance of the expression of microRNA-34 a(miR-34 a)in lipopolysaccharide(LPS)-induced acute respiratory distress syndrome(ARDS)and its mechanism of action.Methods:Forty male Sprague-Dawley rats were randomly divided into five groups:control group and 10 mg/kg LPS intraperitoneal injection groups(3,6,12,and 24 hours);the human alveolar typeⅡepithelial cell line(A549)was divided into five groups:control group and 10μg/m L LPS stimulation groups(3,6,12,and 24 hours).The A549 cells were transfected with a miR-34 a inhibitor and were treated with 10μg/m L LPS(added 24 hours later)for 24 hours.The wet/dry weight(W/D)ratio of the lungs was measured and the histopathological changes in the lung tissue were evaluated.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to determine the expression of miR-34 a in the lung tissue and cells;enzyme-linked immunosorbent assay was used to determine the concentrations of tumor necrosis factor-α(TNF-α)and interleukin-1 beta(IL-1β)in the lung tissue and cells;Western blot was used to determine the protein expression of phosphatidylinositol 3-kinase(PI3 K)and phosphorylated protein kinase B(p-AKT)in the cells.Results:The W/D ratio of the lungs was significantly higher in the experimental groups than in the control group.The hematoxylin-eosin staining indicated that the experimental groups showed the presence of thickened alveolar septa in the lung tissue,pink edema fluids in the alveolar cavities,and inflammatory cell infiltration and hemorrhage in the pulmonary interstitium.The expression of miR-34 a,the concentrations of TNF-αand IL-1β,and the protein expression of PI3 K and p-AKT were higher in the experimental groups than in the control group at each time point,with a positive correlation observed between the expression of miR-34 a and the concentrations of TNF-αand IL-1β.Transfection with the miR-34 a inhibitor significantly downregulated the concentrations of TNF-αand IL-1βand the protein expression of PI3 K and p-AKT in the LPS-treated A549 cells.Conclusion:miR-34 a can attenuate LPS-induced lung injury by inhibiting the PI3 K/AKT signaling pathway,which provides a new target for ARDS treatment.