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Objective:To investigate whether microRNA-21-5p (miR-21-5p) alleviates hyperoxia-induced acute lung injury (HALI) through activating the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway by regulating apoptosis of type Ⅱ alveolar epithelial cell (AECⅡ).Methods:Seventy-two male Sprague-Dawley (SD) rats were divided into normozone-controlled group, HALI group, PI3K/Akt signaling pathway inhibitor LY294002+HALI group (LY+HALI group), miR-21-5p overexpression+LY294002+HALI group (miR-21-5p+LY+HALI group), miR-21-5p overexpression+HALI group (miR-21-5p+HALI group), and dimethyl sulfoxide (DMSO)+HALI group by random number table method with 12 rats in each group. Animal models of HALI were prepared using 95% concentrations of oxygen. The animals in the normozone-controlled group were fed normally under normoxia. Transfection of lung tissue by miR-21-5p adeno-associated viral vector AAV6-miR-21-5p was performed by instillation of 200 μL titer (1×10 12 TU/mL) through a tracheal catheter 3 weeks prior to modeling. DMSO and LY294002 were administered via the tail vein at 0.3 mg/kg 1 hour before modeling. After 48 hours of modeling, carotid artery blood was collected to detect oxygenation index (OI) and respiratory index (RI), and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect miR-21-5p expression. Lung tissue was collected, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA), and the ratio of pulmonary wet/dry weight (W/D) was determined, and the pathological changes of lung histopathology were observed under the light microscopy after hematoxylin-eosin (HE) staining. Each group was purified AECⅡ cells from 6 rats, the apoptosis rate was detected by flow cytometry, and the expression levels of phosphatase and tensin homologous gene (PTEN), and proteins from the PI3K/Akt signaling pathway were detected by Western blotting. Results:Compared with the normozone-controlled group, alveolar septal thickening and massive inflammatory cell infiltration were found after hyperoxia exposure, RI, inflammatory factors, lung W/D ratio, pathological score, AECⅡ cells early apoptosis rate, PTEN protein expression and phosphorylation level of Akt were increased, while OI and miR-21-5p expression were decreased, indicating the successful preparation of the model. After pretreatment, LY294002 could aggravate the pathological injury of lung tissue in HALI rats, RI, inflammatory factors and lung W/D ratio were further increased, and OI was further reduced compared with HALI group. At the same time, it could promote the AECⅡ cell apoptosis, further up-regulate the expression of PTEN, and reduce the phosphorylation of Akt. However, miR-21-5p pretreatment could negatively regulate PTEN, activate PI3K/Akt signal pathway, inhibit AECⅡ cell apoptosis, and reduce HALI, which was shown by the decreased level of inflammatory factors in miR-21-5p+LY+HALI group compared with LY+HALI group [TNF-α (μg/L): 100.33±3.48 vs. 116.55±2.53, IL-6 (ng/L): 141.06±3.70 vs. 161.31±3.59, IL-1β (μg/L): 90.82±3.69 vs. 112.23±2.87, all P < 0.05], RI, lung injury pathology score, lung W/D ratio, and AECⅡ cell early apoptosis rate were significantly decreased [RI: 0.81±0.02 vs. 1.05±0.07, pathology score: 0.304±0.008 vs. 0.359±0.007, lung W/D ratio: 5.29±0.03 vs. 5.52±0.08, apoptosis rate: (27.20±2.34)% vs. (34.17±1.49)%, all P < 0.05], OI and expressions of miR-21-5p were significantly increased [OI (mmHg, 1 mmHg≈0.133 kPa): 266.71±2.75 vs. 230.12±4.04, miR-21-5p (2 -ΔΔCt): 2.21±0.13 vs. 0.33±0.03, both P < 0.05], and PTEN protein expression in AECⅡ cell was significantly reduced (PTEN/GAPDH: 0.50±0.06 vs. 0.93±0.06, P < 0.05), and phosphorylation level of Akt was significantly increased [phosphorylated Akt (p-Akt) protein (p-Akt/GAPDH): 0.86±0.05 vs. 0.56±0.06, P < 0.05]. Conclusion:miR-21-5p attenuates HALI by inhibiting AECⅡ cell apoptosis, possibly through negative regulation of PTEN to activate PI3K/Akt signaling pathway.
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Objective:To investigate whether signal transducer and activator of transcription (STAT1/3/5) have a protective effect on hyperoxia-induced acute lung injury (HALI) and its mechanism.Methods:Seventy C57BL/6J mice were randomly divided into five groups: normoxia control group, HALI group, and STAT1/3/5 inhibitor groups, with 14 mice in each group. The HALI model was established by exposure to more than 90% hyperoxia for 48 hours; three STAT inhibitor groups were pretreated by intraperitoneal injection of STAT1 inhibitor 40 mg/kg and STAT3 inhibitor 5 mg/kg, and STAT5 inhibitor 10 mg/kg for 1 week. Six blood samples were randomly collected from each group, and microRNA-21 (miR-21) expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Lung tissue of the sacrificed mice was obtained, and enzyme linked immunosorbent assay (ELISA) was used to detect the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β), superoxide dismutase (SOD), malonic dialdehyde (MDA), and matrix metalloproteinase 9 (MMP9). The water content of lung tissue was calculated. The pathological changes in lung tissue were observed under the light microscope, and the pathological score of lung injury was performed. Western blotting was used to detect the expression of phosphorylated STAT (p-STAT1, p-STAT3, p-STAT5) in lung tissue. The 7-day cumulative survival rates of the remaining 8 mice in each group were analyzed using Kaplan-Meier survival curves.Results:Under the light microscope, the alveolar structures in the HALI group and the STAT1 inhibitor group were destroyed, a large number of neutrophils (NEU) infiltrated in the alveoli and lung interstitium, which were thickened. The pathological score of lung injury and the water content of the lung tissue was significantly increased. In STAT3 inhibitor and STAT5 inhibitor groups, the alveolar cavity was clear, the degree of NEU infiltration and the thickness of lung interstitium were lower than those in HALI group, the pathological score of lung injury and the water content of lung tissue were significantly decreased, especially in STAT3 inhibitor group. Compared with the normoxia control group, the contents of TNF-α, IL-6, IL-1β, MDA, and MMP9, and the expression levels of p-STAT3 and p-STAT5 in the HALI group were significantly increased. In contrast, the content of SOD and the expression of miR-21 were significantly decreased. Compared with the HALI group, the contents of TNF-α, IL-6, IL-1β, MDA, and MMP9 in the STAT3 inhibitor group and STAT5 inhibitor group were significantly decreased. At the same time, the content of SOD and the expression of miR-21 were significantly increased, especially in STAT3 inhibitor group [TNF-α (μg/L): 42.53±3.25 vs. 86.36±5.48, IL-6 (ng/L): 68.46±4.28 vs. 145.00±6.89, IL-1β (μg/L): 28.74±3.53 vs. 68.00±5.64, MDA (μmol/g): 20.33±2.74 vs. 42.58±3.45, and MMP9 (ng/L): 128.55±6.35 vs. 325.13±6.65, SOD (kU/g): 50.53±4.19 vs. 22.53±3.27, miR-21 (2 -ΔΔCt): 0.550±0.018 vs. 0.316±0.037, all P < 0.05]. Kaplan-Meier survival curve analysis showed that the 7-day cumulative survival rates of the STAT3 inhibitor group and STAT5 inhibitor group were significantly higher than those of the HALI group [62.5% (5/8), 37.5% (3/8) vs. 12.5% (1/8), both P < 0.05]. Conclusion:Inhibition of STAT3 hyperactivation may suppress the inflammatory response, regulate oxidative stress, improve lung permeability through regulating the expression of miR-21, which exert lung protection in HALI.
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Objective To investigate the effects of agmatine (AGM) on the apoptosis of type Ⅱ alveolar epithelial cells (AECⅡ) in rats with hyperoxia-induced acute lung injury (HALI) and provide a theoretical basis for the treatment of HALI. Methods A total of 24 Sprague-Dawley (SD) rats were randomly divided into three groups:normal control group (fed in air), HALI model group and AGM pretreatment group (400 mg/kg AGM was given before the hyperoxia treatment or HALI model establishment), each group with 8 rats. The rats were placed in a self-made high oxygen model box with oxygen concentration of > 90%, temperature of 25-27 ℃, humidity of 50%-70% and carbon dioxide concentration < 0.5% to replicate the HALI rat model; no any treatment was given to the normal control group. After the hyperoxia was treated for 48 hours, the arterial blood was taken from the rat carotid artery for blood gas analysis;under light microscope, the pathological changes of lung tissues were observed and the pathological evaluation scores were carried out; the contents of tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA); the apoptosis of AECⅡ of lung tissues was determined by flow cytometry, and the apoptotic rate was calculated; the expressions of the apoptosis related protein Bcl-2 and Bax were detected by Western Blot. Results Compared with the normal control group, the oxygenation index (OI) and Bcl-2 of HALI model group and AGM pretreatment group were significantly decreased [OI (mmHg, 1mmHg = 0.133 kPa): 135.04±16.82 vs. 463.74±22.04, Bcl-2 protein expression (A value): 0.35±0.18 vs. 0.89±0.08], while the respiratory index (RI), pathological scores of lung injury, TNF-α, IL-6, IL-1, the apoptosis rate of AECⅡ, Bax protein expression were all significantly increased [RI: 1.29±0.15 vs. 0.24±0.03, pathological score of lung tissue: 4.72±1.32 vs. 0, TNF-α (μg/L): 44.48±1.42 vs. 14.12±0.88, IL-6 (μg/L): 51.46±1.62 vs. 23.20±0.89, IL-1 (μg/L): 44.03±2.45 vs. 11.64±1.34, apoptosis rate of AECⅡ: (56.24±1.14)% vs. (22.64±0.58)%, Bax protein expression (A value): 2.37±0.34 vs. 1.41±0.48, all P < 0.05]. Compared with HALI model group, the OI and Bcl-2 of AGM pretreatment group were significantly increased [OI (mmHg): 364.72±14.56 vs. 135.04±16.82, Bcl-2 protein expression (A value): 0.68±0.10 vs. 0.35±0.18, all P < 0.05], while the RI, pathological scores of lung injury, TNF-α, IL-6, IL-1, apoptosis rate of AECⅡ, and Bax protein expression were significantly decreased [RI: 0.45±0.09 vs. 1.29±0.15, pathological score of lung tissue: 2.30±0.96 vs. 4.72±1.32, TNF-α (μg/L):22.98±0.72 vs. 44.48±1.42, IL-6 (μg/L): 35.79±0.86 vs. 51.46±1.62, IL-1 (μg/L): 24.06±0.86 vs. 44.03±2.45, apoptosis rate of AECⅡ: (28.58±1.21)% vs. (56.24±1.14)%, Bax protein expression (A value): 1.98±0.42 vs. 2.37±0.34, all P < 0.05]. Conclusion The apoptotic rate of AECⅡ in HALI rats is reduced by AGM, and the regulatory mechanism needs to be further studied.
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Objective To investigate the effect of heme oxygenase-1 (HO-1) on the apoptosis of type Ⅱalveolar epithelial cells (AEC-Ⅱ) in rats with hyperoxia-induced acute lung injury (HALI). Methods Twenty-four healthy male Sprague-Dawley (SD) rats were randomly divided into 4 groups (n = 6): control group, HALI group, HO-1 group, and HO-1 inhibition group. The control group was fed in the room air; the HALI group was fed in the hyperoxia box (the oxygen concentration was more than 90%, the temperature was kept at 25-27 ℃, the humidity was maintained at 50%-70%, and the CO2concentration was less than 0.5%); the HO-1 group was fed in the hyperoxia box after HO-1 (0.2 μmol/L) treatment; and the HO-1 inhibition group was fed in the hyperoxia box after treatment with zinc protoporphyrin Ⅸ (20 μmol/L). After 48 hours of hyperoxia treatment, rats were sacrificed, left upper lung tissue was stained with hematoxylin-eosin (HE) and the pathological changes of lung tissue were observed under light microscope. The ratio of wet/dry weight (W/D) was measured in the lower left lung. AECⅡ was extracted from the right lung tissue, the apoptosis rate was detected by flow cytometry, and the expressions of apoptosis-related proteins Bcl-2 and caspase-3 were detected by Western Blot. Results ①It was shown by light microscopy that the lung tissue structure of the control group was clear. In HALI group and HO-1 inhibitor group, the lung tissue structure was disordered, alveolar wall was broken and fused into pulmonary alveoli, alveolar septum was obviously swollen and widened, a large number of inflammatory cells infiltrated, and edema fluid and inflammatory cells appeared in alveolar cavity. The pathological changes of lung tissue in HO-1 group were significantly less than those in HALI group. ② Compared with the control group, the lung W/D ratio, the apoptosis rate of AECⅡand the expression of Bcl-2 protein in the HALI group and the HO-1 inhibitor group were significantly increased, and the expression of caspase-3 was significantly decreased [lung W/D ratio: 4.61±0.41 vs. 3.68±0.45, apoptosis rate of AECⅡ: (42.44±0.93) % vs. (24.74±0.64) %, Bcl-2 (integral absorbance): 0.72±0.18 vs. 0.41±0.12, caspase-3 (integral absorbance): 1.32±0.32 vs. 1.81±0.69, all P < 0.05]. Compared with the HALI group, the lung W/D ratio, the apoptosis rate of AECⅡ, the expression of Bcl-2 protein in HO-1 group were significantly decreased, and the expression of caspase-3 was significantly increased [lung W/D ratio: 3.82±0.28 vs. 4.61±0.41, apoptosis rate of AECⅡ: (26.67±1.58) % vs. (42.44±0.93) %, Bcl-2 (integral absorbance): 0.39±0.08 vs. 0.72±0.18, caspase-3 (integral absorbance): 1.78±0.46 vs. 1.32±0.32, all P < 0.05]. There was no significant difference between HO-1 inhibitor group and HALI group. Conclusions HO-1 can reduce the apoptosis rate of AECⅡin rats with HALI, which may be related to the expressions of apoptosis related proteins Bcl-2 and caspase-3.
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Objective To observe the effects of microRNA-21 (miR-21) inhibitor on apoptosis of type Ⅱalveolar 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-△△Ct: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-△△Ct:0.014±0.003 vs.0.021 ±0.005),while the protein expression of caspase-3was 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-△ △ct:0.025 ± 0.007 vs.0.021 ± 0.005) and caspase-3 (A value:0.475 ± 0.062vs.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.Conclusion Inhibition of miR-21 expression in vivo could aggravate the injury of lung tissue in HALI rats,and increase the apoptosis of AEC Ⅱ.