The purpose of this study was to establish a three-dimensional (3D) organoid culture system for type 2 alveolar epithelial (AT2) cells in mice. AT2 cells were isolated from ICR mouse lung and purified by enzymatic digestion and MicroBeads sorting. The purity of AT2 cells was determined by immunofluorescence (IF) staining using an antibody against proSPC. The AT2 differentiation was examined by IF staining with proSPC/HopX and proSPC/T1α antibodies, and proliferation of AT2 cells was assessed by EdU incorporation assays after two-dimensional (2D) culture for 8 days. In addition, AT2 cells were co-cultured with mouse lung fibroblasts (Mlg) in three-dimensional (3D) culture system. After 13 days of co-culture, the organoids were fixed in 2% paraformaldehyde for histological analysis and IF staining. The results showed that the purity of the AT2 cells was over 95%, as assessed by proSPC staining. 2D cultured AT2 cells were negative for EdU staining, which indicates that no proliferation occurs. proSPC expression was gradually disappeared, whereas T1α and HopX expression was gradually increased after 3, 5 and 8 days of culture. In 3D culture system, the alveolar organoids were formed after co-culturing AT2 cells with Mlg for 4 days. Histological analysis showed that alveolar organoids displayed a hollow morphology. proSPC was highly expressed in the peripheral cells, whereas type 1 alveolar epithelial (AT1) cells transdifferentiated from AT2 cells expressing HopX were mainly located in the interior of organoid bodies after 13 days. Some of the proSPC-positive AT2 cells located in the outer circle of alveolar organoids were stained positive for both proSPC and EdU, indicating that the AT2 cells in the alveolar organoids were proliferative. These results showed that the 3D organoid culture system of mouse AT2 cells was successfully established.
Alveolar Epithelial Cells/metabolism* ; Animals ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; Lung ; Mice ; Mice, Inbred ICR ; Organoids

Congenital cystic adenomatoid malformation (CCAM), which is classified into five types according to size and bronchial invasion, is a rare type of developmental anomaly of the lung. CCAM is occasionally accompanied by malignancy, such as bronchioloalveolar carcinoma (BAC) or rhabdomyosarcoma. As defined by the WHO, atypical adenomatous hyperplasia (AAH) is a non-invasive spread of atypical epithelial cells in single rows along the alveolar wall, within a lesion that is usually less than 5 mm in diameter. AAH was also regarded as a pre-invasive neoplasia, especially associated with BAC and adenocarcinoma. We report a case of type II CCAM with AAH in adults, with a review of the references.
Adenocarcinoma ; Adenocarcinoma, Bronchiolo-Alveolar ; Adult ; Cystic Adenomatoid Malformation of Lung, Congenital ; Epithelial Cells ; Humans ; Hyperplasia ; Lung ; Rhabdomyosarcoma

There have been recent extensive studies and rapid advancement on the pathogenesis underlying idiopathic pulmonary fibrosis (IPF), and intricate pathogenesis of IPF has been suggested. The purpose of this study was to clarify the logical relationship between these mechanisms. An extensive search was undertaken of the PubMed using the following keywords: "etiology," "pathogenesis," "alveolar epithelial cell (AEC)," "fibroblast," "lymphocyte," "macrophage," "epigenomics," "histone," acetylation," "methylation," "endoplasmic reticulum stress," "mitochondrial dysfunction," "telomerase," "proteases," "plasminogen," "epithelial-mesenchymal transition," "oxidative stress," "inflammation," "apoptosis," and "idiopathic pulmonary fibrosis." This search covered relevant research articles published up to April 30, 2020. Original articles, reviews, and other articles were searched and reviewed for content; 240 highly relevant studies were obtained after screening. IPF is likely the result of complex interactions between environmental, genetic, and epigenetic factors: environmental exposures affect epigenetic marks; epigenetic processes translate environmental exposures into the regulation of chromatin; epigenetic processes shape gene expression profiles; in turn, an individual's genetic background determines epigenetic marks; finally, these genetic and epigenetic factors act in concert to dysregulate gene expression in IPF lung tissue. The pathogenesis of IPF involves various imbalances including endoplasmic reticulum, telomere length homeostasis, mitochondrial dysfunction, oxidant/antioxidant imbalance, Th1/Th2 imbalance, M1-M2 polarization of macrophages, protease/antiprotease imbalance, and plasminogen activation/inhibition imbalance. These affect each other, promote each other, and ultimately promote AEC/fibroblast apoptosis imbalance directly or indirectly. Excessive AEC apoptosis and impaired apoptosis of fibroblasts contribute to fibrosis. IPF is likely the result of complex interactions between environmental, genetic, and epigenetic factors. The pathogenesis of IPF involves various imbalances centered on AEC/fibroblast apoptosis imbalance.
Alveolar Epithelial Cells ; Apoptosis ; Endoplasmic Reticulum Stress ; Fibroblasts ; Humans ; Idiopathic Pulmonary Fibrosis/genetics*

PURPOSE: This study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. METHODS: AECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis. RESULTS: The successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11. CONCLUSION EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.
Humans ; Rats ; Animals ; Alveolar Epithelial Cells ; Lung ; Lung Injury ; Cell Movement/physiology*

Alveolar Epithelial Cells ; metabolism ; pathology ; Animals ; Apoptosis ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; physiology ; Genes, bcl-2 ; genetics ; Genes, p53 ; genetics ; Oxidative Stress ; genetics ; Rats

In order to find potential therapeutic agents on lung inflammatory conditions, the extracts of Acanthopanax divaricatus var. albeofructus were prepared and its constituents were isolated. They include lignans such as (+)-syringaresinol (1), acanthoside B (2), salvadoraside (3) and acanthoside D (4), lariciresinol-9-O-beta-D-glucopyranoside (5) and phenylpropanoids such as 4-[(1E)-3-methoxy-1-propenyl]phenol (6), coniferin (7), and methyl caffeate (8). The extracts and several constituents such as compound 1, 6 and 8 inhibited the production of inflammatory markers, IL-6 and nitric oxide, from IL-1beta-treated lung epithelial cells and lipopolysaccharide (LPS)-treated alveolar macrophages. Furthermore, the extracts and compound 4 significantly inhibited lung inflammation in lipolysaccharide-treated acute lung injury in mice by oral administration. Thus it is suggested that A. divaricatus var. albeofructus and its several constituents may be effective against lung inflammation.
Eleutherococcus* ; Acute Lung Injury ; Administration, Oral ; Animals ; Epithelial Cells ; Interleukin-6 ; Lignans ; Lung* ; Macrophages ; Macrophages, Alveolar ; Mice ; Nitric Oxide ; Pneumonia*

OBJECTIVETo establish the integrated discrete multiple organ cell culture (IdMOC) system.

METHODSRat primary cell of hepatocyte, nephrocyte, cardiomyocytes, alveolar macrophage, dermal fibroblasts were isolated by collagenase digestion, separation of bronchial lavage, two-step digestion method and cultured respectively, with monolayer culture. To establish the integrated discrete multiple organ cell culture (IdMOC) system, glass slides of five different cells were used to the same dish with 10% FBS DMEM medium cultured 7d, using MTT comparison primary cells cultured alone and cocultured when growth.

RESULTSEstablished rat hepatocytes, renal cell, cardiomyocyte, alveolar macrophages, dermal fibroblasts separation method was stable, cell separation survival rate was about 90.0%. Hepatocytes separation survival rate 90.3% ,renal cell separation survival rate 91.9%, cardiomyocyte separation survival rate 93.0% and beating rate indifference curve among 3d-15d, alveolar macrophages cell separation survival rate 90.8%, dermal fibroblasts cell separation survival rate 92.7%. Five primary cells multiple organ cells coculture showed cocultured cell growth proliferation well, cultured alone and cocultured cells growth curve basic coincide.

CONCLUSIONEstablished rat multiple organ cell co-culture is successful.

Animals ; Cell Culture Techniques ; methods ; Epithelial Cells ; cytology ; Hepatocytes ; cytology ; Macrophages, Alveolar ; cytology ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Sprague-Dawley

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P<0.05 or P<0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P<0.05 or P<0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P<0.05 or P<0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P<0.05 or P<0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P<0.05 or P<0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P<0.05 or P<0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.
Alveolar Epithelial Cells/metabolism* ; Animals ; Epithelial-Mesenchymal Transition ; Flavonoids ; NF-kappa B/metabolism* ; Rats ; Signal Transduction ; Transforming Growth Factor beta1/genetics*

Bronchial asthma is a chronic airway inflammation disorder involving lymphocyte activation and various cytokines secretion by lymphocyte. The inflammatory response results from a complex network of interactions between inflammatory cells (mast cells, eosinophils, macrophages) and resident cells belonging to the lung structure itself like EC, fibroblasts, or bronchial epithelial cells. IL-6 which is known to up-regulate the endothelial cell expression of adhesion molecules participating in the development of the inflammatory reaction in bronchial asthma is produced by alveolar macrophage. ICAM-1 is produced by bronchial epithelial cell and expression by endothelial cell, which is known to enhance of the influx of various cells. RANTES which is known to a potent chemoattractant for eosinophils, lymphocytes, and monocytes, a member of the CC chemokine family, is expressed by bronchial epithelial cell. To evaluate whether markers of lymphocyte activation are useful markers of disease activity in bronchial asthma, we measured sIL-6, sICAM-1, sRANTES in 42 patients with mild to moderate bronchial asthma and in 26 normal controls and compared the result with other disease activity markers in asthma (pulmonary function, blood eosinophil counts). The mean level of sIL-6 was higher than that of normal control and correlated significantly with sICAM-1, FEV1% to predicted value. The mean level of sICAM-1 was higher than that of normal control and correlated significantly with FEV1%, FEV1% to predicted value. The mean level of sRANTES showed the tendency to be higher than that of normal control, but not significant statistically, and did not correlated with sIL-6, sICAM-1, FEV1%, FEV1% to predicted value, blood eosinophil counts. It appeared that sIL-6 and sICAM-1 could be a disease marker in bronchial asthma. But, clinical application of the measurement of these markers needs to be studied further.
Asthma* ; Chemokine CCL5 ; Cytokines ; Endothelial Cells ; Eosinophils ; Epithelial Cells ; Fibroblasts ; Humans ; Inflammation ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Lung ; Lymphocyte Activation ; Lymphocytes ; Macrophages, Alveolar ; Monocytes

OBJECTIVETo access rat lung toxicity of nano silica particles.

METHODSTransmission electron microscope was used to observe size, shape and dispersibility of two silica particles; Size of two particles in water and RPMI 1640 containing 1% FBS were measured using Zeta Potential Analyzer. LDH activities of rat type I-like alveolar epithelial cell line R3/1 cells after 6, 24 and 48 h incubation with 2.5, 5.0, 10.0, 20.0 microg/ml of two silica nano particles were detected by spectrophotometric method; protein carbonylation and MIP-2 release of R3/1 cells after 24 h incubation with 2.5, 5.0, 10.0, 20.0 microg/ml of two silica nano particles were measured using ELISA kits.

RESULTSTEM image showed nano silica particles were round and dispersed evenly; the average sizes of the two silica particles were (43+/-4.2) and (68+/-5.7) nm. Two silica particles had similar size in water and RPMI 1640 containing 1% FBS, respectively. Both nano silica particles in 2.5 approximately 20.0 microg/ml dose range did not cause significant increase of LDH activities (P>0.05), did not elevate protein carbonylation and MIP-2 levels in R3/1 cells (P>0.05).

CONCLUSIONTwo nano silica particles do not have lung toxicity in 2.5 approximately 20.0 microg/ml dose range.

Alveolar Epithelial Cells ; drug effects ; metabolism ; Animals ; Cells, Cultured ; L-Lactate Dehydrogenase ; metabolism ; Nanoparticles ; toxicity ; Rats ; Silicon Dioxide ; toxicity ; Toxicity Tests