Lei Shi1, Ying He, Bing Bai, Miao Chen. 1. 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: chenmiao64@163.com.
Abstract
OBJECTIVE: 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). METHODS: 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). RESULTS: (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-ΔΔCt: 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-ΔΔCt: 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-ΔΔCt: 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. CONCLUSIONS: Inhibition of miR-21 expression in vivo could aggravate the injury of lung tissue in HALI rats, and increase the apoptosis of AEC II.
OBJECTIVE: 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). METHODS: 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). RESULTS: (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-ΔΔCt: 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-ΔΔCt: 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-ΔΔCt: 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. CONCLUSIONS: Inhibition of miR-21 expression in vivo could aggravate the injury of lung tissue in HALIrats, and increase the apoptosis of AEC II.