miR21-5p靶向转录激活蛋白STAT3减轻高氧性急性肺损伤

miR21-5p targets transcriptional activator protein STAT3 to alleviate hyperoxia-induced acute lung injury

目的探讨miR21-5p调控转录激活蛋白STAT3在高氧性急性肺损伤(HALI)中的作用及机制。方法选择40只C57BL/6J小鼠随机分为常氧组、高氧组、高氧+miR21-5p组及高氧+空载体组,吸入90%以上浓度氧气建立HALI小鼠模型。常氧组饲养于正常空气中;高氧+miR21-5p组及高氧+空载体组分别经气管导管滴入miR21-5p AAV6或空载体,饲养3周后建立HALI小鼠模型。高氧暴露48 h后,取肺组织采用ELISA试剂盒检测炎症因子(TNF-α、IL-6、IL-1β)和氧化应激标志物(SOD、MDA、ROS)水平;计算肺水含量;行HE染色观察形态学变化并行病理学评分;Tunel法检测肺组织细胞凋亡率;RT-PCR检测miR21-5p表达水平;Western blot检测STAT3、p-STAT3、Bax及Bcl-2蛋白相对表达水平。结果与常氧组比较,高氧组肺损伤病理评分、肺水含量及肺组织细胞凋亡率升高(P<0.05),炎症因子TNF-α、IL-6及IL-1β水平升高(P<0.05),MDA和ROS水平升高(P<0.05),SOD水平降低(P<0.05),STAT3、p-STAT3及Bax蛋白表达升高(P<0.05),Bcl-2蛋白表达下降(P<0.05);与高氧组比较,高氧+miR21-5p组肺损伤病理评分、肺水含量及肺组织细胞凋亡率降低(P<0.05),炎症因子TNF-α、IL-6及IL-1β水平降低(P<0.05),MDA和ROS水平降低(P<0.05),SOD水平升高(P<0.05),STAT3、p-STAT3及Bax蛋白表达降低(P<0.05),高Bcl-2蛋白表达升高(P<0.05)。高氧组HE染色可见肺泡结构紊乱、间隔增厚,大量炎症细胞浸润,透明膜形成及肺泡萎陷,高氧+miR21-5p组HE染色结果显示肺损伤程度明显减轻。结论miR21-5p靶向转录激活蛋白STAT3活性抑制炎症反应及凋亡并维持氧化还原平衡减轻HALI。

Objective To investigate the role and mechanism of miR21-5p regulating transcriptional activator protein STAT3 in hyperoxia-induced acute lung injury(HALI).Methods Forty C57BL/6J mice were randomly divided into normoxia group,hyperoxia group,hyperoxia+miR21-5p group,hyperoxia+empty carrier group,and the HALI mouse model was established by inhaling more than 90%oxygen concentration.The normoxia group was raised in normal air;the hyperoxia+miR21-5p group and the hyperoxia+empty vector group were instilled with miR21-5p AAV6 or empty vector through the tracheal tube,respectively,and the HALI mouse model was established after three weeks of feeding.After 48 hours of hyperoxia exposure,lung tissue was collected,and ELISA kits were used to detect the levels of inflammatory factors(TNF-α,IL-6,IL-β)and oxidative stress markers(SOD,MDA,ROS);the lung water content was calculated;Morphological changes were observed by HE staining and pathological scoring;Tunel method to detect apoptosis rate of lung tissue cells;RT-PCR was used to detect the expression level of miR21-5p;Western blotting was used to detect the relative expression levels of STAT3,p-STAT3,Bax and Bcl-2.Results Compared with the normox group,the pathological score,lung water content and apoptosis rate of lung tissue cells in the hyperoxia group were increase(P<0.05),and the inflammatory factor TNF-α,IL-6 and IL-1βThe level of MDA and ROS increased(P<0.05),the level of SOD decreased(P<0.05),the expression of STAT3,p-STAT3 and Bax protein increased(P<0.05),and the expression of Bcl-2 protein decreased(P<0.05);Compared with the hyperoxia group,the pathological score,lung water content and apoptosis rate of lung tissue cells in the hyperoxia+miR21-5p group were reduced(P<0.05),and the inflammatory factor TNF-α、IL-6 and IL-1βThe level of MDA and ROS decreased(P<0.05),the level of SOD increased(P<0.05),the expression of STAT3,p-STAT3 and Bax decreased(P<0.05),and the expression of high Bcl-2protein increased(P<0.05).HE staining in the hyperoxia group showed disordered alveolar structure,septal thickening,extensive infiltration of inflammatory cells,formation of transparent membrane,and alveolar collapse.HE staining results in hyperoxia+miR21-5p group showed a significant reduction of lung injury.Conclusion miR21-5p targets transcriptional activator protein STAT3 activity to inhibit inflammatory response and apoptosis and maintain redox balance to alleviate HALI.