微小RNA-125b在小鼠呼吸机相关性肺损伤中的作用

Role of microRNA-125b in ventilator-induced lung injury in mice

目的评价微小RNA-125b(miR-125b)在小鼠呼吸机相关性肺损伤(VILI)中的作用。方法健康雄性C57BL/6小鼠40只,体重25~30 g,2~3月龄,采用随机数字表法分为4组(n=10):假手术组(Sham组)、VILI组、VILI+miR-125b阴性对照组(VILI+NC组)和VILI+miR-125b过表达组(VILI+miR-125b agomir组)。VILI+NC组和VILI+miR-125b agomir组分别经气管内滴入miR-125b阴性对照或miR-125b agomir转染复合物50μl,48 h后制备VILI模型。采用大潮气量(40 ml/kg)机械通气4 h制备VILI。机械通气4 h时采集股动脉血样测定PaO2,然后处死小鼠,取肺组织,测定湿重/干重(W/D)比值,光镜下观察病理学结果,并行肺损伤评分。VILI+NC组和VILI+miR-125b agomir组收集支气管肺泡灌洗液(BALF),采用ELISA法测定TNF-α和IL-6的浓度;采用TUNEL法测定肺组织细胞凋亡情况,计算细胞凋亡指数,采用Western blot检测肺组织caspase-3表达水平,采用RT-PCR法检测肺组织miR-125b表达水平。结果与Sham组比较,VILI组、VILI+NC组和VILI+miR-125b agomir组PaO2降低,肺组织W/D比值和肺损伤评分升高,VILI组和VILI+NC组肺组织miR-125b表达下调(P<0.05);与VILI组比较,VILI+miR-125b agomir组PaO2升高,肺组织W/D比值和肺损伤评分降低,miR-125b表达上调(P<0.05),肺组织病理学损伤减轻,VILI+NC组上述各指标差异无统计学意义(P>0.05);与VILI+NC组比较,VILI+miR-125b agomir组BALF中TNF-α及IL-6浓度和细胞凋亡指数、肺组织caspase-3表达水平均降低(P<0.05)。结论miR-125b参与了VILI的内源性保护机制。

Objective To evaluate the role of microRNA-125b(miR-125b)on ventilator-induced lung injury(VILI)in mice.Methods Forty healthy male C57BL/6 male mice,weighing 25-30 g,aged 2-3 months,were divided into 4 groups(n=10 each)using a random number table method:sham operation group(group Sham),group VILI,VILI plus miR-125b negative control group(group VILI+NC),and VILI plus miR-125b overexpression group(group VILI+miR-125b agomir).In VILI+NC and VILI+miR-125b agomir groups,miR-125b negative control and miR-125b agomir transfection complex 50μl were intratracheally instilled,respectively,and 48 h later VILI model was established.The animals were mechanically ventilated for 4 h with high tidal volume(40 ml/kg)to induce VILI.Blood samples were obtained from the femoral artery at 4 h of mechanical ventilation for detection of PaO2,then animals were sacrificed,lungs were removed for determination of wet to dry weight ratio(W/D ratio)and for examination of pathological changes(with a light microscope),and lung injury was scored.In VILI+NC and VILI+miR-125b agomir groups,bronchoalveolar lavage fluid(BALF)was collected to measure the concentrations of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)by enzyme-linked immunosorbent assay.The cell apoptosis of lung tissues was measured using TUNEL,apoptosis index was calculated,the caspase-3 expression was detected by Western blot,and the miR-125b expression was detected by real-time polymerase chain reaction.Results Compared with Sham group,PaO2 was significantly decreased,and W/D ratio and lung injury score were increased in VILI,VILI+NC and VILI+miR-125b agomir groups,and the expression of miR-125b was down-regulated in VILI and VILI+NC groups(P<0.05).Compared with VILI group,PaO2 was significantly increased,W/D ratio and lung injury score were decreased,the expression of miR-125b was up-regulated(P<0.05),the pathological changes of lung tissues were significantly attenuated in group VILI+miR-125b agomir,and no significant change was found in the parameters mentioned above in group VILI+NC(P<0.05).Compared with group VILI+NC,the concentrations of TNF-αand IL-6 in BALF and apoptotic index were significantly decreased,and the expression of caspase-3 was down regulated in group VILI+miR-125b agomir(P<0.05).ConclusionMiR-125b is involved in the endogenous protective mechanism of VILI in mice.