BACKGROUND: Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust. METHODS: Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R₂₀₀ cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method. RESULTS: Both the cells in the R group and R₂₀₀ group express leptin receptor OB-R. Compared to the R₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R₂₀₀ group (R₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R₂₀₀ (R₂₀₀LU) group. Additionally, the cells in the R₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R₂₀₀LU group and R₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R₂₀₀L group. However, when the cells in the R₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased. CONCLUSIONS Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.
Rats ; Animals ; Alveolar Epithelial Cells/pathology* ; Dust ; Tin/adverse effects* ; Lung Neoplasms/pathology* ; Leptin/adverse effects* ; Receptors, Leptin ; China ; Signal Transduction ; Epithelial Cells/pathology* ; Mitogen-Activated Protein Kinase Kinases/adverse effects*

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by diffuse alveolar injury primarily caused by an excessive inflammatory response. Regrettably, the lack of effective pharmacotherapy currently available contributes to the high mortality rate in patients with this condition. Xuebijing (XBJ), a traditional Chinese medicine recognized for its potent anti-inflammatory properties, exhibits promise as a potential therapeutic agent for ALI/ARDS. This study aimed to explore the preventive effects of XBJ on ALI and its underlying mechanism. To this end, we established an LPS-induced ALI model and treated ALI mice with XBJ. Our results demonstrated that pre-treatment with XBJ significantly alleviated lung inflammation and increased the survival rate of ALI mice by 37.5%. Moreover, XBJ substantially suppressed the production of TNF-α, IL-6, and IL-1β in the lung tissue. Subsequently, we performed a network pharmacology analysis and identified identified 109 potential target genes of XBJ that were mainly involved in multiple signaling pathways related to programmed cell death and anti-inflammatory responses. Furthermore, we found that XBJ exerted its inhibitory effect on gasdermin-E-mediated pyroptosis of lung cells by suppressing TNF-α production. Therefore, this study not only establishes the preventive efficacy of XBJ in ALI but also reveals its role in protecting alveolar epithelial cells against gasdermin-E-mediated pyroptosis by reducing TNF-α release.
Animals ; Mice ; Alveolar Epithelial Cells ; Pyroptosis ; Gasdermins ; Lipopolysaccharides/adverse effects* ; Tumor Necrosis Factor-alpha ; Acute Lung Injury/drug therapy* ; Respiratory Distress Syndrome

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*

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

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

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*

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*

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.
Adenosine Monophosphate/therapeutic use* ; Alanine/therapeutic use* ; Alveolar Epithelial Cells/virology* ; Antibodies, Neutralizing/therapeutic use* ; COVID-19/virology* ; Down-Regulation ; Drug Discovery ; Human Embryonic Stem Cells/metabolism* ; Humans ; Immunity ; Lipid Metabolism ; Lung/virology* ; RNA, Viral/metabolism* ; SARS-CoV-2/physiology* ; Virus Replication/drug effects*

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*

BACKGROUND: We previously demonstrated that continuous exposure to nitrous acid gas (HONO) for 4 weeks, at a concentration of 3.6 parts per million (ppm), induced pulmonary emphysema-like alterations in guinea pigs. In addition, we found that HONO affected asthma symptoms, based on the measurement of respiratory function in rats exposed to 5.8 ppm HONO. This study aimed to investigate the dose-response effects of HONO exposure on the histopathological alterations in the respiratory tract of guinea pigs to determine the lowest observed adverse effect level (LOAEL) of HONO. METHODS: We continuously exposed male Hartley guinea pigs (n = 5) to four different concentrations of HONO (0.0, 0.1, 0.4, and 1.7 ppm) for 4 weeks (24 h/day). We performed histopathological analysis by observing lung tissue samples. We examined samples from three guinea pigs in each group under a light microscope and measured the alveolar mean linear intercept (Lm) and the thickness of the bronchial smooth muscle layer. We further examined samples from two guinea pigs in each group under a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: We observed the following dose-dependent changes: pulmonary emphysema-like alterations in the centriacinar regions of alveolar ducts, significant increase in Lm in the 1.7 ppm HONO-exposure group, tendency for hyperplasia and pseudostratification of bronchial epithelial cells, and extension of the bronchial epithelial cells and smooth muscle cells in the alveolar duct regions. CONCLUSIONS These histopathological findings suggest that the LOAEL of HONO is < 0.1 ppm.
Alveolar Epithelial Cells ; drug effects ; Animals ; Bronchi ; drug effects ; Dose-Response Relationship, Drug ; Emphysema ; chemically induced ; Epithelial Cells ; drug effects ; Guinea Pigs ; Hyperplasia ; chemically induced ; Inhalation Exposure ; adverse effects ; Lung ; drug effects ; pathology ; ultrastructure ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Myocytes, Smooth Muscle ; drug effects ; Nitrous Acid ; toxicity