摘要
目的探讨微小RNA-181a(miR-181a)对慢性阻塞性肺疾病(COPD)模型大鼠肺组织中气道重塑的作用及其可能的机制。方法80只大鼠随机取65只建立COPD模型,建模成功56只,随机分为模型组、mimics组、inhibitors组、NC组各14只,剩余15只为假手术组。建模2 h后,mimics组、inhibitors组和NC组分别尾静脉注射miR-181a mimics、miR-181a inhibitors和control mimics,假手术组和模型组注射生理盐水。干预24 h后取材,RT-qPCR检测肺组织miR-181a、母亲信号蛋白同源物7(Smad7)mRNA相对表达水平,ELISA法检测肺组织匀浆白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平,荧光素酶报告基因分析miR-181a对Smad7的靶向性,HE染色观察大鼠肺组织病理学变化,测量支气管壁及支气管壁胶原纤维厚度,Western blot检测肺组织基质金属蛋白酶-9(MMP-9)、转化生长因子-β1(TGF-β1)、Smad7、p-Smad7蛋白相对表达水平。结果与假手术组比较,模型组、NC组和inhibitors组肺组织miR-181a相对表达水平降低,mimics组肺组织miR-181a相对表达水平升高,且mimics组>模型组和NC组>inhibitors组(P<0.05)。与假手术组比较,模型组、NC组、mimics组、inhibitors组肺组织匀浆IL-1β、TNF-α水平升高,且mimics组>模型组和NC组>mimics组(P<0.05)。双荧光素酶报告基因分析系统结果显示,miR-181a可显著抑制野生型Smad7相对荧光素酶活性(P<0.05),而对突变型Smad7相对荧光素酶活性无显著影响(P>0.05)。HE染色显示,假手术组大鼠肺泡细胞正常,模型组、NC组和inhibitors组大鼠肺实质破坏,有炎性细胞浸润和胶原纤维形成。mimics组肺泡细胞趋于正常,炎症减轻,胶原纤维减少,肺泡结构紊乱明显好转。与假手术组比较,模型组、NC组、mimics组、inhibitors组支气管壁和支气管壁胶原纤维厚度增加,且inhibitors组>模型组和NC组>mimics组(P<0.05)。与假手术组比较,模型组、NC组、mimics组和inhibitors组肺组织TGF-β1、MMP-9蛋白相对表达水平升高,p-Smad7蛋白相对表达水平降低,且TGF-β1、MMP-9蛋白相对表达水平inhibitors组>模型组和NC组>mimics组,p-Smad7蛋白相对表达mimics组>模型组和NC组>inhibitors组(P<0.05)。模型组和NC组之间的肺组织miR-181a相对表达水平,IL-1β、TNF-α水平,支气管壁和支气管壁胶原纤维厚度,MMP-9、p-Smad7、TGF-β1蛋白相对表达水平比较,差异无统计学意义(P>0.05)。结论miR-181a对COPD模型大鼠肺组织中气道重塑有改善作用,可能通过靶向调控Smad7表达发挥作用。
Objective To investigate the effect and possible mechanism of microRNA-181a(miR-181a)on airway remodeling in the lung tissue of rats with chronic obstructive pulmonary disease(COPD).Methods Sixty-five rats were randomly selected from 80 rats to establish COPD models,56 rats were successfully modeled.They were randomly divided into model group,NC group,mimics group and inhibitors group with 14 rats in each group,and the remaining 15 rats were sham operation group.Two hours after modeling,the mimics group,the inhibitors group and the NC group were injected with miR-181a mimics,miR-181a inhibitors and control mimics through the tail vein,and the sham operation group and the model group were injected with saline.After 24 hours of intervention,RT-qPCR was used to detect the relative expression levels of miR-181a and maternal signal protein homolog 7(Smad7)mRNA in lung tissue,ELISA method was used to detect the levels of interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)in lung tissue homogenate,luciferase reporter gene analysis miR-181a targeting Smad7,HE staining to observe rat lung tissue disease physiological changes,measure bronchial wall and bronchial wall collagen fiber thickness,Western blot was used to detecte lung tissue matrix metalloproteinase-9(MMP-9),transforming growth factorβ1(TGF-β1),Smad7,p-Smad7 protein relative expression level.Results Compared with the sham operation group,the relative expression level of miR-181a in lung tissues of the model group,NC group and inhibitors group decreased,and the relative expression level of miR-181a in lung tissue of the mimics group increased,and the mimics group>model group and NC group>inhibitors group(P<0.05).Compared with the sham operation group,the levels of IL-1βand TNF-αin lung tissue homogenate of the model group,NC group,mimics group,and inhibitors group increased,and the inhibitors group>model group and NC group>mimics group(P<0.05).The results of the dual luciferase reporter gene analysis system showed that miR-181a could significantly inhibit the relative luciferase activity of wild-type Smad7(P<0.05),but had no significant effect on the relative luciferase activity of mutant Smad7(P>0.05).HE staining showed that the alveolar cells of the rats in the sham operation group were normal,and the lung parenchyma of the rats in the model group,NC group and inhibitors group was destroyed,with inflammatory cell infiltration and collagen fiber formation.In the mimics group,alveolar cells tended to be normal,inflammation reduced,collagen fibers decreased,and alveolar structural disorder was significantly improved.Compared with the sham operation group,the bronchial wall and bronchial wall collagen fibers in the model group,NC group,mimics group,and inhibitors group were thickened,and the inhibitors group>model group and NC group>mimics group(P<0.05).Compared with the sham operation group,the relative expression levels of MMP-9 and TGF-β1 protein in the lung tissues of the model group,NC group,mimics group and inhibitors group increased,while the relative expression level of p-Smad7 protein decreased,and MMP-9,TGF-β1 protein relative expression level inhibitors group>model group and NC group>mimics group,relative expression of p-Smad7 protein mimics group>model group and NC group>inhibitors group(P<0.05).Comparison of the relative expression levels of miR-181a,IL-1β,TNF-αlevels,bronchial wall and bronchial wall collagen fiber thickness,MMP-9,p-Smad7,and TGF-β1 protein expression levels in the model group and NC group,there was no statistical significance(P>0.05).Conclusion miR-181a can improve the airway remodeling in the lung tissue of COPD model rats,and may play a role in the targeted regulation of Smad7 expression.
作者
王雅杰
王嘉
岳洪娟
窦亚平
李娜
Wang Yajie;Wang Jia;Yue Hongjuan(Dept of Pulmonary and Critical Care Medicine,The First Hospital of Hebei Medical University,Shijiazhuang 050000)
出处
《安徽医科大学学报》
CAS
北大核心
2021年第9期1379-1385,共7页
Acta Universitatis Medicinalis Anhui
基金
河北省卫健委医学科学研究项目(编号:20190042)。
作者简介
责任作者:王雅杰,女,副主任医师,E-mail:chu895litao@163.com。
