Shi Lei, He Ying, Bai Bing, Chen Miao
Second Ward of Department Critical Care Medicine, Affiliated Hospital of Zunyi Medical College, Zunyi 563000, Guizhou, China (Shi L, He Y, Chen M); Department of Urinary Medicine, Affiliated Hospital of Zunyi Medical College, Zunyi 563000, Guizhou, China (Bai B). Corresponding author: Chen Miao, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2017 Mar;29(3):244-248. doi: 10.3760/cma.j.issn.2095-4352.2017.03.010.
To observe the effects of microRNA-21 (miR-21) inhibitor on apoptosis of type II alveolar epithelial cells (AEC II) in rats with hyperoxia-induced acute lung injury (HALI).
Eighty Sprague-Dawley (SD) rats were divided into air-control group, hyperoxia injury group, empty-virus control group (200 μL solution with lentivirus was dropped into the nasal) and miR-21 inhibitor pretreatment group (200 μL solution with lentivirus contained miR-21 inhibitor was dropped through the nasal) by random number table. After treatment, the rats in all groups were fed in the hyperoxia incubator with oxygen concentration exceeding 90% for production of HALI model, and the rats in air-control group were fed normally without any treatment. Ten rats were selected at 0, 24, 48 and 72 hours after exposure in hyperoxia environment respectively, and the general changes of lung tissues were observed in light microscope. The right lung tissues were harvested to observe the pathological changes under light microscopy. The left lung tissues of other 10 rats in each group were harvested at 48 hours after execution, the miR-21 expression was determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), the protein expression of cysteinyl aspartate-specific proteinase-3 (caspase-3) was determined by Western Bolt, and apoptosis of AEC II was detected by TdT-mediated dUTP nick end labeling (TUNEL).
(1) No abnormal appearance in lung tissues was observed at all time points in the air-control group. In hyperoxia injury group, the lung injury would be more severe if the exposure time was longer, and lung tissues turned dark red after exposure for 72 hours, with patchy hemorrhage in several places; the structure of lung tissues was disordered, the alveolar wall was broken, the alveolar septum was significantly edematous and broadened, and there was plenty of inflammatory cell infiltration and edema fluid appeared inside the alveolar space. In miR-21 inhibitor pretreatment group, the degree of lung tissue injury was more severe than that of the hyperoxia injury group, and there was no significant change in empty-virus control group. (2) Compared with air-control group, miR-21 expression of the hyperoxia injury group was significantly decreased (2: 0.021±0.005 vs. 0.037±0.006), and the protein expression of caspase-3 was significantly increased (A value: 0.423±0.081 vs. 0.123±0.023, both P < 0.05). After pretreatment with miR-21 inhibitor, the expression of miR-21 was further decreased (2: 0.014±0.003 vs. 0.021±0.005), while the protein expression of caspase-3 was further increased (A value: 0.691±0.085 vs. 0.423±0.081, both P < 0.05). There were no statistically significant differences in the expression of miR-21 (2: 0.025±0.007 vs. 0.021±0.005) and caspase-3 (A value: 0.475±0.062 vs. 0.423±0.081) between empty-virus control group and hyperoxia injury group (both P > 0.05). (3) Compared with air-control group, the apoptosis cells in hyperoxia injury group were increased, which was further increased after pretreatment of miR-21 inhibitor, but no changes were found in empty-virus control group.
Inhibition of miR-21 expression in vivo could aggravate the injury of lung tissue in HALI rats, and increase the apoptosis of AEC II.
观察微小RNA-21（miR-21）抑制剂对高氧诱导的急性肺损伤（HALI）大鼠Ⅱ型肺泡上皮细胞（AEC II）凋亡的影响。
将80只Sprague-Dawley（SD）大鼠通过随机数字表法分为空气对照组、高氧损伤组、空病毒对照组（经鼻滴入200 μL含慢病毒的溶液）和miR-21抑制剂预处理组（经鼻滴入200 μL含miR-21抑制剂的慢病毒溶液）。处理后，将所有组的大鼠置于氧浓度超过90%的高氧培养箱中饲养以建立HALI模型，空气对照组大鼠正常饲养不做任何处理。分别在高氧环境暴露后0、24、48和72小时各选取10只大鼠，光镜下观察肺组织的大体变化。取右肺组织，光镜下观察病理变化。每组另外10只大鼠在处死48小时后取左肺组织，采用实时荧光定量逆转录-聚合酶链反应（RT-qPCR）检测miR-21表达，采用蛋白质免疫印迹法检测半胱氨酸天冬氨酸特异性蛋白酶-3（caspase-3）蛋白表达，采用TdT介导的dUTP缺口末端标记法（TUNEL）检测AEC II凋亡情况。
（1）空气对照组各时间点肺组织均未见异常外观。高氧损伤组，暴露时间越长肺损伤越严重，暴露72小时后肺组织呈暗红色，多处有片状出血；肺组织结构紊乱，肺泡壁破裂，肺泡间隔明显水肿增宽，肺泡腔内有大量炎性细胞浸润及水肿液。miR-21抑制剂预处理组肺组织损伤程度比高氧损伤组更严重，空病毒对照组无明显变化。（2）与空气对照组比较，高氧损伤组miR-21表达显著降低（2：0.021±0.005 vs. 0.037±0.006），caspase-3蛋白表达显著增加（A值：0.423±0.081 vs. 0.123±0.023，均P<0.05）。用miR-21抑制剂预处理后，miR-21表达进一步降低（2：0.014±0.003 vs. 0.021±0.005），而caspase-3蛋白表达进一步增加（A值：0.691±0.085 vs. 0.423±0.081，均P<0.05）。空病毒对照组与高氧损伤组之间miR-21表达（2：0.025±0.007 vs.
