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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 study the effects of endothelial progenitor cells (EPC)-derived exosomes on hyperoxia-induced injury in type Ⅱ alveolar epithelial cell (AECⅡ) in neonatal rats.Methods:EPCs of rats were cultured and exosomes were collected using Total Exosome Isolation kit. Primary cultured AECⅡof neonatal rats were randomly assigned into three groups: the control group, the hyperoxia group and the exosome group. The control group was cultured in room air with 5%CO 2, the hyperoxia group was cultured in 95%O 2 with 5%CO 2 and the exosome group was cultured with 0.1 mg/ml EPC-derived exosomes in 95%O 2 with 5%CO 2. Cell viability was detected using cell counting kit-8 (CCK-8) and apoptosis was detected using flow cytometry on d2, d4, and d6. Results:EPC-derived exosomes isolated from EPC culture supernatant were confirmed morphologically using transmission electron microscopy. After co-incubation of Dil-labeled EPC-derived exosomes with AEC Ⅱ for 24 h, Dil fluorescence was detected in the cytoplasm of AEC Ⅱ, indicating exosomes were uptaken by AEC Ⅱ. Compared with the control group, hyperoxia decreased cell viability and increased apoptosis of AEC Ⅱ and the injury was aggravated with the prolongation of hyperoxia duration ( P<0.001). Cell injury in the exosome group was milder than the hyperoxia group ( P<0.001). Compared with the control group, cell viability on d4 and d6 of hyperoxia was lower ( P=0.029 and 0.005 respectively) and cell apoptosis at d6 of hyperoxia was higher in the exosome group ( P=0.007). Conclusions:EPC-derived exosomes may partially attenuate hyperoxia-induced cell injury in neonatal rat AEC Ⅱ.
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Objective To investigate the damage mechanism of typeⅡalveolar epithelial cells (AECⅡ) after hyperoxia exposure by proteomics. Methods The primary AECⅡ of preterm Sprague-Dawley (SD) rats were divided into normoxia and hyperoxia groups, and cultured in room air (21% O2) or hyperoxia (95% O2) condition, respectively. The cell morphology change was observed under an inverted contrast microscope; the protein expressions of Bcl-2 and caspase-3 were detected by Western Blot to ensure a successful model. Total protein in AECⅡ was collected, and mass spectrometry-based tandem mass tag (TMT)-labeled quantitative proteomics were used to detect the change of protein profile. Proteins with changes greater than 1.5-fold and P < 0.05 were considered differentially expressed, and bioinformatics analysis was performed. According to the proteomic results, AECⅡ were divided into three groups:normoxia group, hyperoxia group and hyperoxia+MW167 group (γ-secretase inhibitor MW167 was added to culture medium 30 minutes before they were placed into the chamber). The cell viability was detected by the cell proliferation and toxicity kit (CCK-8), and the expressions of Hes1, Bax mRNA were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results ① The cells in the normoxia group proliferated and prolonged significantly, and the cytoplasmic particulate matter was abundant. In the hyperoxia group, nucleus pyknosis and cytoplasmic particulate matter decreased significantly. Compared with the normoxia group, the expression of caspase-3 in the hyperoxia group was significantly increased, and the expression of Bcl-2 was significantly decreased (caspase-3/GAPDH: 1.352±0.086 vs. 0.769±0.080, Bcl-2/GAPDH: 0.614±0.060 vs. 1.361±0.078, both P < 0.01).② A total of 162 differentially expressed proteins were identified between normoxia and hyperoxia groups, the proteins up-regulated by hyperoxia were commonly associated with response processes to various stimuli, and located in the extracellular region; the proteins down-regulated by hyperoxia were commonly associated with synthesis of substances, and located in the cellular matrix. KEGG Pathway analyses suggested that metabolism by cytochrome P450, oxidative phosphorylation, and Notch signaling pathway were associated with the mechanism of hyperoxia injury on AECⅡ.③Compared with the normoxia group, the viability of cells in the hyperoxia group was significantly decreased, and the expressions of Hes1 and Bax mRNA were significantly increased [cell viability (A value): 0.060±0.003 vs. 1.058± 0.017, Hes1 mRNA (2-ΔΔCt): 2.235±0.606 vs. 1.144±0.107, Bax mRNA (2-ΔΔCt): 2.210±0.240 vs. 1.084±0.096, all P < 0.05]. Compared with the hyperoxia group, the viability of cells in the hyperoxia+MW167 group was significantly increased, and the expressions of Hes1 and Bax mRNA were significantly decreased [cell viability (A value): 0.271±0.025 vs. 0.060±0.003, Hes1 mRNA (2-ΔΔCt): 0.489±0.046 vs. 2.235±0.606, Bax mRNA (2-ΔΔCt): 1.289±0.041 vs. 2.210±0.240, all P < 0.05]. Conclusion The mechanism of hyperoxia injury on AECⅡ may be related to the metabolism by cytochrome P450, oxidative phosphorylation and activation of Notch signaling pathway.
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Objective To observe the morphological and functional changes of different lung cells in hyperoxia environment.Methods Type Ⅱ alveolar epithelial cells (AECⅡ) and lung fibroblasts (LFs) of fetal rats with 18 days old were isolated and culturedin vitro, and divided into air group (placed in an atmospheric incubator, and culturing with oxygen volume fraction of 0.21) and hyperoxia group (placed in a high oxygen culture chamber, and culturing with oxygen volume fraction of 0.90). Morphological changes of two kinds of cells were observed under microscope. Cell migration was observed by scratch test. The levels of reactive oxygen species (ROS) and apoptosis in cells were detected by flow cytometry.Results After 8 hours of hyperoxia, the volume of AECⅡincreased and the cells were loosely arranged; the clearance of LFs cells was increased and arranged in disorder. Scratch test showed that, compared with air group, the immigration rate of AECⅡ was inhibited at 6 hours hyperoxia [migration rate: (38.67±1.15)％ vs. (58.67±2.31)％,P < 0.01], the immigration rate of LFs was promoted at 12 hours after hyperoxia [migration rate:(55.37±1.50)％ vs. (46.90±1.20)％,P < 0.01]. With the increase of hyperoxia time, intracellular ROS contents of two cells were gradually increased, which were significantly higher than those of the air group (fluorescence intensity:130.67±4.04 vs. 54.67±2.51, 85.00±2.00 vs. 60.33±1.52, bothP < 0.01). Both two kinds of cells showed apoptosis after exposure to high oxygen, the apoptosis rate of AECⅡ at 2 hour exposure were significantly higher than that of air group [(1.93±0.28)％ vs. (1.07±0.11)％,P < 0.05], the apoptosis rate of LFs at 6 hour exposure was significantly higher than that of air group [(1.66±0.09)％ vs. (1.46±0.09)％,P < 0.05].Conclusion High concentration of oxygen can cause poor growth of lung cells, reduce AEC Ⅱ migration level and increase LFs migration, and the production of intracellular ROS eventually leads to apoptosis of lung cells.
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Objective To evaluate the effect of mechanical stretch preconditioning on pathological stretch-induced activation of γ-aminobutyric acid (GABA) signaling pathway in human type Ⅱ alveolar epithelial cells (AEC Ⅱ).Methods AEC Ⅱ cell line (A549 cells) culturedin vitro were divided into control group (group C), pathological stretch group (group P1) and mechanical stretch preconditioning group (group P2). In group C, A549 cells were cultured routinely. In group P1, A549 cells were exposed to 20% cyclic stretch for 6 hours. In group P2, A549 cells were exposed to 5% cyclic stretch for 60 minutes, and then exposed to 20% cyclic stretch for 6 hours. The cells were harvested for determination of the cell viability by methyl thiazolyl tetrazolium assay, lactate dehydrogeuase (LDH) release was determined by colorimetric method, the levels of interleukin (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA), the mRNA expressions of IL-1β, IL-6 and TNF-α were determined by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expressions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid A receptor (GABAAR) were determined by Western Blot.Results Compared with group C, the cell viability of group P1 was significantlydecreased (A value: 0.196± 0.071 vs. 0.886±0.107), the release rate of LDH was significantly increased [(12.3±2.4)% vs. (1.9±0.5)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly increased [IL-1β (ng/L): 138.6±19.7 vs. 32.7±7.4, IL-6 (ng/L): 196.5±31.7 vs. 55.4±13.8, TNF-α (ng/L): 111.3±21.8 vs. 20.8±7.6; IL-1β mRNA (2-ΔΔCT): 2.79±0.44 vs. 0.83±0.12, IL-6 mRNA (2-ΔΔCT): 1.99±0.25 vs. 0.56±0.11, TNF-α mRNA (2-ΔΔCT): 2.54±0.37 vs. 0.72±0.09]; the protein expressions of GAD and GABAAR were significantly decreased [GAD (gray value): 0.38±0.12 vs. 1.75±0.45, GABAAR (gray value): 0.29±0.09 vs. 1.68±0.39; allP < 0.05]. Compared with group P1, the cell viability of group P2 was significantly increased (A value: 0.523±0.132 vs. 0.196±0.071),the release rate of LDH was significantly decreased [(6.9±1.7)% vs. (12.3±2.4)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly decreased [IL-1β (ng/L): 79.2±11.6 vs. 138.6±19.7, IL-6 (ng/L): 89.6±15.6 vs. 196.5±31.7, TNF-α (ng/L): 55.9±11.4 vs. 111.3±21.8; IL-1β mRNA (2-ΔΔCT): 1.92±0.36 vs. 2.79±0.44, IL-6 mRNA (2-ΔΔCT): 1.09±0.18 vs. 1.99±0.25, TNF-α mRNA (2-ΔΔCT): 1.77±0.25 vs. 2.54±0.37]; the protein expressions of GAD and GABAAR were significantly increased [GAD (gray value): 1.26±0.33 vs. 0.38±0.12, GABAAR (gray value): 1.04±0.15 vs. 0.29±0.09; allP < 0.05]. Conclusion The mechanism by which mechanical stretch preconditioning attenuates pathological stretch-induced injury in human AECⅡ is related to the activation of GABA signaling pathway.
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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.
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ObjectiveTo study the effect of hydrogen peroxide (H2O2) in inducing apoptosis of typeⅡalveolar epithelial cell (AECⅡ) after overexpression by adenoviral transfection of micro RNA-21-5p (miR-21-5p), and to explore the mechanism of its anti-apoptosis.Methods Subculture AECⅡ were randomly divided into four groups: normal control group (normal saline), H2O2 challenge group ( 0.5 mmol/L H2O2), miR-21-5p overexpression group (miR-21-5p adenovirus+ 0.5 mmol/L H2O2), miR-21-5p negative transfection group (adenovirus void+0.5 mmol/L H2O2). Transmission electron microscopy and flow cytometry were used to detect apoptotic morphology and early apoptotic rate. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-21-5p in AECⅡ, and Western Blot was used to detect the protein expressions of Bcl-2, Bax, and caspase-3 at the highest transfection efficiency at different time points (6, 12, 24, 48 hours).Results ① AECⅡ identification: fluorescence microscopy showed the presence of characteristic structure of AECⅡ, i.e. microvilli and osmiophilic lamellar bodies.② Apoptotic morphology: transmission electron microscopy showed cytoplasmic retraction, chromatin condensation, margination, lack of cell surface microvilli, and emptying of osmiophilic lamellar bodies in AECⅡ.③ The expression of miR-21-5p in AECⅡ: the highest transfection efficiency was found at 48 hours. The expression of miR-21-5p in miR-21-5p overexpression group was significantly higher than that of the normal control group, H2O2 challenge group and miR-21-5p negative transfection group (A value: 1.54±0.02 vs. 1.02±0.02, 0.56±0.03, 0.58±0.02, allP< 0.05).④ The rate of early apoptosis: compared with normal control group, the early apoptotic rates in H2O2 challenge group, miR-21-5p negative transfection group and miR-21-5p overexpression group were gradually elevated with the prolongation of injury time. The early apoptotic rate in miR-21-5p overexpression group was significantly lower than that of the H2O2 challenge group and miR-21-5p negative transfection group at all time points except 6 hours [12 hours: (10.73±2.80)% vs. (16.26±0.59)%, (16.04±0.70)%; 24 hours:(16.00±3.44)% vs. (23.29±2.78)%, (23.58±2.31)%; 48 hours: (31.30±3.55)% vs. (50.53±2.17)%, (49.41±1.97)%, allP< 0.05]. There was no significant difference in early apoptotic rate between miR-21-5p negative transfection group and H2O2 challenge group at each time point.⑤ Protein expression: the expressions of Bax and caspase-3 in miR-21-5p overexpression group were significantly lower than those of the H2O2 challenge group and miR-21-5p negative transfection group [Bax (A value): 0.07±0.01 vs. 0.18±0.01, 0.13±0.01; caspase-3 (A value): 0.07±0.01 vs. 0.23±0.01, 0.12±0.01, allP< 0.05], and Bcl-2 protein expression was significantly higher than that of the H2O2 challenge group and miR-21-5p negative transfection group (A value: 0.26±0.01 vs. 0.06±0.01, 0.10±0.01, both P< 0.05).Conclusions① miR-21-5p has the function of anti-apoptosis of AECⅡ.② Adenoviral vector is a successful gene transfer vector when transfected with AECⅡ.③ The anti-apoptosis of AECⅡ by miR-21-5p may be associated with reduced Bax and caspase-3 protein levels and raised expression levels of Bcl-2 protein.
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Objective To observe the expression of mitochondrial encoding cytochrome oxidase subunitⅠ(COXⅠ) and subunitⅡ(COXⅡ) in type Ⅱalveolar epithelial cell(AEC Ⅱ) of rats exposed to hyperoxia and explore the role of COXⅠand COXⅡ in hyperoxia-induced lung injury.Methods AECⅡ were gained by primary culture from 19-days fetal rats lung.After purified,AECⅡ were randomly assigned to hyperoxia group and air group.Hyperoxia group was flushed the flake with 950 mL/L O2 and 50 mL/L CO2 at 3 L/min for 10 min,then sealed.Both groups were in CO2 culture chamber(37 ℃,50 mL/L CO2).After 6,12 and 24 hours of exposure,AECⅡ were harvested and extracted for total RNA.COXⅠand COXⅡ mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR).The results were analyzed by SPSS 12.0 softwore.Results Compared with air group,COXⅠmRNA expression in hyperoxia group increased significantly at 6 hours(t=3.832 P=0.019) and 12 hours(t=10.431 P=0),respectively,then decreased to the equivalent level in 24 hours(t=0.360 P=0.731).Compared with air group,COXⅡmRNA expression in hyperoxia group increased significantly at 6 hours(t=2.795 P=0.035),then decreased to the equivalent level at 12 hours(t=0.892 P=0.40) and 24 hours(t=2.018 P=0.071).Conclusions Exposure of hyperoxia up-regulate the expressions of COXⅠmRNA and COXⅡmRNA in AECⅡ,which may be a protective mechanism of hyperoxia-induced lung injury.