Mechanical ventilation (MV) is currently widely used in the treatment of respiratory failure and anesthesia surgery, and is a commonly used respiratory support method for critically ill patients; however, improper usage of MV can lead to ventilator-induced lung injury (VILI), which poses a significant threat to patient life. Alveolar epithelial cell (AEC) has the functions of mechanosensation and mechanotransduction. Physiological mechanical stretching is beneficial for maintaining the lineage homeostasis and normal physiological functions of AEC cells, while excessive mechanical stretching can cause damage to AEC cells. Damage to AEC cells is an important aspect in the occurrence and development of VILI. Understanding the effects of mechanical stretching on AEC cells is crucial for developing safe and effective MV strategies, preventing the occurrence of VILI, and improving the clinical prognosis of VILI patients. From the perspective of cell mechanics, this paper aims to briefly elucidate the mechanical properties of AEC cells, mechanosensation and mechanotransduction of mechanical stretching in AEC cells, and the injury and repair of AEC cells under mechanical stretch stimulation, and potential mechanisms with the goal of helping clinical doctors better understand the pathophysiological mechanism of VILI caused by MV, improve their understanding of VILI, provide safer and more effective strategies for the use of clinical MV, and provide theoretical basis for the prevention and treatment of VILI.
Humans ; Mechanotransduction, Cellular ; Ventilator-Induced Lung Injury ; Stress, Mechanical ; Alveolar Epithelial Cells ; Respiration, Artificial/adverse effects* ; Epithelial Cells ; Pulmonary Alveoli/cytology* ; Animals

OBJECTIVE: To elucidate the role and mechanism of microRNA-145-5p (miR-145-5p) in hypoxia-induced pyroptosis of human alveolar epithelial cells. METHODS: In vitro, human alveolar epithelial cell line BEAS-2B was cultured. Cells in the logarithmic growth phase were cultured to 80% confluence and then used for the experiment. (1) BEAS-2B cells were cultured under 1% O₂ hypoxic condition, with a normoxic control group. Western blotting was employed to detect the expressions of pyroptosis marker proteins [NOD-like receptor protein 3 (NLRP3), Gasdermin D N-terminal domain (GSDMD-N), and caspase-1] in cells cultured for 24 hours. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-145-5p in cells cultured for 6 hours and 12 hours. (2) Cells were transfected with 30 nmol/L miR-145-5p mimic to overexpress miR-145-5p expression under normoxic condition or 30 nmol/L miR-145-5p inhibitor to suppress miR-145-5p expression under hypoxic condition. Control group and negative control group were respectively set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of pyroptosis marker proteins and nuclear factor-E2-related factor 2 (Nrf2) in cells. Flow cytometry was applied to detect the level of reactive oxygen species (ROS) in cells. The target genes of miR-145-5p were predicted by miR target gene prediction software miRWalk and verified by Western blotting. (3) Under hypoxic condition, cells were transfected with 6.94 ng/μL silent information regulator 5 (Sirt5) overexpression plasmid or pretreated with 12.5 mmol/L N-acetyl-L-cysteine (NAC) as an ROS inhibitor. The empty plasmid group and control group were set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of Sirt5, Nrf2, and pyroptosis marker proteins in cells. Flow cytometry was used to detect the level of ROS in cells. RESULTS: (1) Compared with the normoxic control group, the expression levels of pyroptosis marker proteins in the 24-hour hypoxia group was significantly increased, indicating that hypoxia could induce pyroptosis in BEAS-2B cells. The expression level of miR-145-5p in cells gradually increased with the extension of hypoxia induction time, indicating that hypoxia could cause the increase of miR-145-5p expression level. (2) The expression levels of pyroptosis marker proteins in cells of miR-145-5p mimic group significantly increased under normoxic condition as compared with the control and negative control groups [NLRP3 protein (NLRP3/β-actin): 1.58±0.07 vs. 1.00±0.01, 0.98±0.07, GSDMD-N protein (GSDMD-N/β-actin): 1.71±0.03 vs. 1.01±0.01, 0.85±0.03, caspase-1 protein (caspase-1/β-actin): 2.33±0.04 vs. 1.01±0.01, 1.05±0.04, all P < 0.05], Nrf2 protein expression level was significantly decreased (Nrf2/β-actin: 0.79±0.03 vs. 1.00±0.01, 1.03±0.04, both P < 0.05), ROS level was significantly up-regulated (fluorescence intensity: 1.74±0.03 vs. 1.00±0.01, 0.92±0.03, both P < 0.05). Under hypoxia condition, compared with control group and negative control group, the expression levels of pyroptosis marker proteins in miR-145-5p inhibitor group were significantly decreased [NLRP3 protein (NLRP3/β-actin): 0.21±0.04 vs. 1.70±0.02, 1.63±0.04; GSDMD-N protein (GSDMD-N/β-actin): 1.32±0.02 vs. 2.51±0.02, 2.72±0.03; caspase-1 protein (caspase-1/β-actin): 0.56±0.01 vs. 2.77±0.02, 3.12±0.03; all P < 0.05], Nrf2 protein expression level was significantly increased (Nrf2/β-actin: 1.57±0.04 vs. 1.22±0.01, 1.28±0.04, both P < 0.05), ROS level was significantly down-regulated (fluorescence intensity: 0.64±0.05 vs. 1.87±0.04, 1.70±0.07, both P < 0.05). The results indicated that miR-145-5p could promote cell pyrodeath. The predictive result of miRWalk showed that the 3' untranslated region (3'UTR) of Sirt5 had complementary base binding sites with miR-145-5p. The expression level of Sirt5 protein in cells of miR-145-5p mimic group was significantly lower than that of control group and negative control group under normoxic condition (Sirt5/β-actin: 0.59±0.03 vs. 1.00±0.01, 1.01±0.03, both P < 0.05), which verified that Sirt5 was the target gene of miR-145-5p. (3) The occurrence of pyrodeath could be partially reversed by transfection with Sirt5 overexpression plasmid or adding ROS inhibitor NAC into cells, and Sirt5 overexpression could also up-regulate Nrf2 expression and eliminate intracellular ROS. CONCLUSION In human alveolar epithelial cells, miR-145-5p can down-regulate Nrf2 by targeting Sirt5, thereby increasing ROS expression and inducing pyrodeath.
Humans ; MicroRNAs ; Pyroptosis ; Cell Hypoxia ; Alveolar Epithelial Cells/cytology* ; Cell Line ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Epithelial Cells/metabolism* ; Gasdermins ; Phosphate-Binding Proteins

PURPOSE: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. METHODS: Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. RESULTS: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. CONCLUSION AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.
Alveolar Epithelial Cells/cytology* ; Animals ; Cell Culture Techniques ; Cell Separation/methods* ; Immunoglobulin G/metabolism* ; Lung ; Magnetic Phenomena ; Rats ; Rats, Sprague-Dawley

Alveolar Epithelial Cells/cytology* ; Animals ; Bleomycin ; Epithelial-Mesenchymal Transition ; Lung ; Pulmonary Fibrosis/chemically induced* ; Rats ; Receptor, Notch1/metabolism* ; Signal Transduction ; Transforming Growth Factor beta1 ; Twist-Related Protein 1/metabolism*

OBJECTIVETo investigate the effect of silicon dioxide (SiO₂) on the expression of E-cadherin, α-smooth muscle actin (α-SMA), and transforming growth factor β₁(TGF-β₁) in human pulmonary epithelial cells (A549) with epithelial-mesenchymal transition (EMT), and to study the roles of epidermal growth factor receptor (EGFR) signaling pathway in SiO₂-induced EMT in A549 cells in vitro.

METHODSAlveolar macrophages (AMs) were stimulated with 50 µg/ml SiO₂for 3, 6, 12, 18, 24, or 36 h, and the supernatants were collected to measure the expression of TGF-β₁protein by ELISA. The AM supernatant in which TGF-β₁reached the highest expression (T=18 h) was used as AM-conditioned supernatant. A549 cells were cultured in AM-conditioned supernatant and stimulated with indicated doses of SiO₂(0, 50, 100, and 200 µg/ml) for 48 h. The cell morphological changes were observed using an inverted microscope. The cells were collected at different times, and the mRNA and protein expression levels of E-cadherin, α-SMA, and EGFR were measured by RT-PCR and immunocytofluorescence, respectively.

RESULTSAfter stimulation by SiO₂, the expression level of TGF-β₁protein at each time point was significantly higher in the presence of AM supernatants than in the absence of AM supernatants (P<0.05). With the action time, the expression level of TGF-β₁protein increased at first and then decreased, and the highest level was reached at 18 h. After exposure to SiO₂, A549 cells exhibited mesenchymal characteristics, such as a spindle shape, pseudopodia change, and fibroblast-like morphology, as observed by inverted microscope, especially in the 200 µg/ml group. With increased concentration of SiO₂, the mRNA and protein expression of E-cadherin was down-regulated gradually, especially in the 200 µg/ml group, whereas the mRNA and protein expression of α-SMA and EGFR was up-regulated gradually, especially in the 200 µg/m1 group. There were significant differences between the SiO₂-treated groups (50, 100, and 200 µg/ml SiO₂) and the control group (P<0.05).

CONCLUSIONAfter being stimulated by SiO₂in vitro, AMs have significantly increased expression level of TGF-β₁protein. The AM supernatant together with SiO₂can induce the transition of pulmonary epithelial cells to mesenchymal cells, and its mechanism may be related to the EGFR signaling pathway.

Actins ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Epithelial Cells ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Lung ; cytology ; Macrophages, Alveolar ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Silicon Dioxide ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the role of p38 MAPK on ischemic postconditioning (IPO) attenuating pneumocyte apoptosis after lung ischemia/reperfusion injury (LIRI).

METHODSForty adult male SD rats were randomly divided into 5 groups based upon the intervention (n = 8): control group (C), LIR group (I/R), LIR + IPO group (IPO), IPO + solution control group (D), IPO + SB203580 group (SB). Left lung tissue was isolated after the 2 hours of reperfusion, the ratio of wet lung weight to dry lung weight (W/D), and total lung water content (TLW) were measured. The histological structure of the left lung was observed under light and electron transmission microscopes, and scored by alveolar damage index of quantitative assessment (IQA). Apoptosis index (AI) of lung tissue was determined by terminal deoxynuleotidyl transferase mediated dUTP nick end and labeling (TUNEL) method. The mRNA expression and protein levels of and Bax were measured by RT-PCR and quantitative immunohistochemistry (IHC).

RESULTSCompared with C group, W/D, TLW, IQA, AI and the expression of Bax of I/R were significantly increased, the expression of Bcl-2 and Bcl-2/Bax were significantly decreased (P < 0.05, P < 0.01), and was obviously morphological abnormality in lung tissue. Compared with I/R group, all the indexes of IPO except for the expression of Bcl-2 and Bcl-2/ Bax were obviously reduced, the expression of Bcl-2 and Bcl-2/Bax were increased (P < 0.05, P < 0.01). All the indexes between D and IPO were little or not significant( P > 0.05). The expression of Bcl-2 and Bcl-2/Bax of SB were significantly increased and other indexes were reduced than those of IPO (P < 0.05, P < 0.01).

CONCLUSIONIPO may attenuate pneumocyte apoptosis in LIRI by inactivation of p38 MAPK, up-regulating expression of Bcl-2/Bax ratio.

Alveolar Epithelial Cells ; cytology ; Animals ; Apoptosis ; Disease Models, Animal ; Ischemic Postconditioning ; Lung ; blood supply ; enzymology ; pathology ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; enzymology ; pathology ; prevention & control ; bcl-2-Associated X Protein ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effects of ischemic postconditioning (IPostC) on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats.

METHODSAdult male SD rats were randomly divided into 3 groups based upon the intervention (n = 8): control group (C), lung ischemic reperfusion group (LIR), LIR+ IPostC group (IPostC). At the end of the experiment, blood specimens drawn from the arteria carotis were tested for the content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO); the pneumocyte apoptosis index (AI) was achieved by tennrminal deoxynucleotidyl transferase mediated dUTP nick end abeling (TUNEL); the expression of Bcl-2, Bax protein in lung tissue was accessed by quantitative immunohistochemistry (MHC) and Bcl-2, Bax mRNA by RT-PCR.

RESULTSIPostC could significantly attenuate the MDA level, MPO activity and improve SOD activity in blood serum which was comparable to I/R and significantly reduced the number of TUNEL-positive cells compared with I/R group, expressed as Al (% total nuclei) from (39.0 +/- 3.46) to (8.0 +/- 0.88) (P < 0.01). The protein and mRNA expression of Bcl-2 and Bax showed that IPO significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein but improved the expression of Bcl-2 that improved the Bcl-2/Bax ratio (P < 0.01) .

CONCLUSIONIPostC may attenuate pneumocyte apoptosis in LIRI by up-regulating expression of Bcl-2/Bax ratio and by inhibiting oxidant generation and neutrophils filtration.

Alveolar Epithelial Cells ; cytology ; Animals ; Apoptosis ; Ischemic Postconditioning ; Lung ; metabolism ; pathology ; Lung Injury ; physiopathology ; Male ; Malondialdehyde ; metabolism ; Peroxidase ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; physiopathology ; Superoxide Dismutase ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the effects of Matrigel on expression of focal adhesion kinase and on proliferation and apoptosis of alveolar epithelial cell II of premature rat exposed to hyperoxia.

METHODSThe primary premature rat AECII (gestation 19 d) were cultured in vitro. For establishing hyperoxia-exposed cell model, purified AECII were cultured for 12 hours after culture flasks were filled with 95% oxygen-5% CO2 at 5 L/min, and then sealed for 12 hours. DNA content, phosphor and total protein of FAK were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively after 12 hours of air or hyperoxia exposure in the presence or absence of Matrigel. To investigate the relationship between FAK activated and proliferation or apoptosis of type II alveolar epithelial cells, levels of proliferation and apoptosis of AECII were measured by immunohistochemical assay of proliferating cell nuclear antigen (PCNA) and TUNEL method respectively.

RESULTSFAK and FAK-Tyr(397) activity of AECII on Matrigel-coated substrate increased: compared with air group, the expression of PCNA decreased and apoptotic index increased markedly in hyperoxia group (0.1498 ± 0.009 vs. 0.0953 ± 0.006, P < 0.05; 1.232 ± 0.6 vs. 13.40 ± 3.2, P < 0.01), but the expression of PCNA of AECII on Matrigel-coated substrate increased significantly (0.1498 ± 0.009 vs. 0.1921 ± 0.008, P < 0.01) and apoptotic index did not change. The expression of PCNA increased significantly (0.0953 ± 0.006 vs. 0.1125 ± 0.012, P < 0.05) and apoptotic index decreased markedly in hyperoxia + Matrigel group as compared with hyperoxia group (13.40 ± 3.2 vs. 7.641 ± 1.6, P < 0.05).

CONCLUSIONHyperoxia decreased the level of FAK and FAK-Tyr(397) in AECII, which may be a contributory mechanism of impaired proliferation and apoptosis of AECII in hyperoxia induced lung injury in premature rat. Matrigel could inhibit apoptosis and promote proliferation of AECII resulted from hyperoxia in vitro. Matrigel may play a protective role in hyperoxia-induced lung injury partly due to activated FAK.

Alveolar Epithelial Cells ; Animals ; Animals, Newborn ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Collagen ; pharmacology ; Drug Combinations ; Epithelial Cells ; drug effects ; enzymology ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Hyperoxia ; Laminin ; pharmacology ; Male ; Proteoglycans ; pharmacology ; Pulmonary Alveoli ; cytology ; enzymology ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the pathological changes of pulmonary fibrosis induced by SiO2 in rats and pigs.

METHODSThe silicosis models in rats and pigs were established by non-exposure method. The pathologic changes in lung tissues of rats and pigs were observed with HE staining under a light microscopy and under a transmission electron microscopy (TEM), the expression of cytokines was detected by immunohistochemistry.

RESULTS(1) The main pathologic changes of silicosis models in rats and pigs included: in 7 ∼ 15 days after treatment, silica dusts, dust cells, a lot of macrophages, lung epithelial cells, a few neutrophils, macrophage alveolar inflammation and nodules of stage I were found in alveolar space; in 30 ∼ 90 days after treatment, many nodules of stage I-III or IV with lymphocytes infiltration were observed in respiratory bronchioles, alveoli, interlobular septa, the subpleural and around blood vessels and bronchi. (2) The expression levels of CK protein, SP-A protein, CD68, b-FGF, TNF-α, IL-6, TGF-β1, NFKappa/P50, Kappa/P65 and VEGF reduced with exposure time, but still were higher than those of the control. (3) The shed alveolar type I cells, proliferation of alveolar type II cells or macrophages and activated cellular function induced by silica were observed under TEM.

CONCLUSIONThe development of pulmonary fibrosis in silicosis models corresponded with the process from macrophages alveolar inflammation to pulmonary fibrosis.

Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Epithelial Cells ; metabolism ; Female ; Lung ; cytology ; pathology ; Macrophages, Alveolar ; metabolism ; Male ; Neutrophils ; metabolism ; Rats ; Rats, Sprague-Dawley ; Silicosis ; pathology ; Swine

OBJECTIVETo investigate the effects of cyclosporine A (CsA), a powerful inhibitor of mitochondrial permeability transition pore (MPTP), on pneumocyte apoptosis, the release of cytochrome C and the activity of caspase-3 after lung ischemia/reperfusion, and explore the mechanisms.

METHODSSingle lung in situ ischemia/reperfusion animal model was used. 30 SD rats were randomly divided into three groups (n = 10): sham (S) group, ischemia/reperfusion (I/R) group and cyclosporine A (CsA) group. Apoptosis of pneumocyte was assessed by TUNEL method, cytochrome C (CytC) in cytoplasm was detected by immunohistochemistry techniques, and the activity of caspase-3 was measured with spectrophotometer.

RESULTSThe content of CytC in cytoplasm, the activity of caspase-3, and the value of apoptosis index (AI) in ischemia/reperfusion group were evidently higher than that in S group (P < 0.01). CsA suppressed apoptosis as well as CytC release and caspase-3 activity (P < 0.01).

CONCLUSIONCsA can prevent the release of cytochrome C, block the apoptosis of pneumocyte accordingly maybe by closing the MPTP.

Alveolar Epithelial Cells ; cytology ; drug effects ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cyclosporine ; pharmacology ; Cytochromes c ; metabolism ; Lung ; blood supply ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology