In the study, the effects of Panax notoginseng saponins (PNS) on alveolar epithelial to mesenchymal transition (EMT) and extracellular matrix degradation were observed in a type of human alveolar epithelial cell, A549 cells, stimulated by TGF-beta1. Firstly, MTT method was applied to evaluation of cellular proliferation and found that PNS from 12.5 mg x L(-1) to 200 mg x L(-1) dosage could not inhibit significantly cellular proliferation. Then, cells were divided into five groups, normal group, TGF-beta1 group, TGF-beta1 + 50 mg x L(-1) PNS group, TGF-beta1 + 100 mg x L(-1) PNS group and TGF-beta1 + 200 mg x L(-1) PNS group. Normal cells were not stimulatec by TGF-beta1; TGF-beta1 cells were only stimulated by TGF-beta1 and the other cells were stimulated by TGF-beta1 with different doses of PNS, respectively. After stimulation, cells and supernatants were collected for assays. Cellular roundness was applied to quantitative evaluation of morphological change. Immunocytochemistry was applied to examine E-cadherion, a-SMA and FN proteins expression in the cells. Enzyme linked-immunosorbent assay was applied to MMP-9 and TIMP-1 levels. The results showed that EMT of A549 cells was induced by TGF-beta1, showing significant change of roundness, E-cadherion, alpha-SMA and FN (P < 0.05, P < 0.01). Compared to TGF-beta1, PNS significantly inhibited the changes of roundness (P < 0.05), FN and alpha-SMA (P < 0.05, P < 0.01) and not significantly inhibited the change of E-cadherion. Furthermore, MMP-9 levels were significantly increased by TGFbeta1 stimulation (P < 0.05), without significant change of TIMP-1. Compared with TGF-beta1, PNS could significantly increase MMP-9 level (P < 0.05) and decrease TIMP-1 levels (P < 0.05, P < 0.01). In conclusion, PNS could inhibit alveolar epithelial cell EMT induced by TGF-beta1, with increase of extracellular matrix degradation ability, which showed anti-fibrosis of lung ability.
Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Panax notoginseng ; chemistry ; Pulmonary Alveoli ; cytology ; drug effects ; metabolism ; Saponins ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.
Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Dependovirus ; genetics ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Ganciclovir ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Humans ; In Situ Nick-End Labeling ; Luciferases ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; Pulmonary Alveoli ; cytology ; metabolism ; Pulmonary Surfactant-Associated Protein A ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism

This study is to investigate the effect of artesunate on transforming growth factor-beta1 (TGF-beta1) induced epithelial-mesenchymal transition (EMT) and its possible mechanism. After the in vitro cultured RLE-6TN cells were treated with TGF-beta1 then artesunate intervened on it, after 24 h, expression of the markers of mesenchymal cell was assayed using Western blotting and real-time PCR analysis. Western blotting was also used to detect the effect of TGF-beta1 on the Smad3 and Smad7 expressions of RLE-6TN cells. Morphological alterations were examined by phase-contrast microscope, and ultrastructure changes by electron microscope. Incubation of RLE-6TN cells with TGF-beta1 resulted in the up-regulation of the expression of the mesenchymal cell markers, after artesunate intervened on it, resulted in the down-regulation of the expression. Meanwhile, incubation with artesunate intervened on RLE-6TN cells could lead to the apparent down-regulation of the expression of Smad3 and up-regulation of Samd7 and the transition of RLE-6TN cells to mesenchymal-like by TGF-beta1 induction, after artesunate intervened on it, RLE-6TN cells to epithelial-like. TGF-beta1 induced epithelial-mesenchymal transition process; artesunate can inhibit TGF-beta1-induced epithelial-mesenchymal transition process, the possible mechanism is up-regulation of the expression of Smad7 and down-regulation of the expression of Smad3, meanwhile inhibits phosphorylation of Smad3.
Actins ; genetics ; metabolism ; Animals ; Artemisia ; chemistry ; Artemisinins ; isolation & purification ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Idiopathic Pulmonary Fibrosis ; pathology ; Plants, Medicinal ; chemistry ; Pulmonary Alveoli ; cytology ; RNA, Messenger ; metabolism ; Rats ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; pharmacology ; Vimentin ; genetics ; metabolism

OBJECTIVETo investigate the protective effect of hydrogen against hyperoxia-induced oxidative stress injury in premature rat type II alveolar epithelial cells (AECs).

METHODSThe type II AECs isolated from premature rats were randomly divided into air (21% oxygen) control group, hyperoxia (95% oxygen) control group, air + hydrogen group, and hyperoxia+ hydrogen group. The cells with hydrogen treatment were cultured in the presence of rich hydrogen. After the corresponding exposure for 24 h, the cell morphology was observed microscopically. MTT assay was used to evaluated the cell proliferation ability, and JC-1 fluorescence probe was used to detect the mitochondrial membrane potential (δφ) changes of the type II AECs. The concentration of maleic dialdehyde (MDA) and superoxide dismutase (SOD) activity in the cell supernatant were detected using colorimetric method.

RESULTSNo significant differences were found in cell growth or measurements between air control and air + hydrogen groups. Compared with air control group, the cells exposed to hyperoxia showed significantly suppressed proliferation, reduced mitochondrial membrane potential, increased MDA content, and decreased SOD activity. Intervention with hydrogen resulted in significantly increased cell proliferation and SOD activity and lowered MDA content, and restored the mitochondrial membrane potential in the cells with hyperoxia exposure (P<0.05).

CONCLUSIONHydrogen can significantly reduce hyperoxia-induced oxidative stress injury in premature rat type II AECs, improve the cellular antioxidant capacity, stabilize the mitochondrial membrane potential, and reduce the inhibitory effect of hyperoxia on cell proliferation.

Animals ; Animals, Newborn ; Antioxidants ; metabolism ; Cell Proliferation ; Cells, Cultured ; Epithelial Cells ; drug effects ; Female ; Hydrogen ; pharmacology ; Male ; Malondialdehyde ; metabolism ; Membrane Potential, Mitochondrial ; Oxidative Stress ; drug effects ; Oxygen ; adverse effects ; Pulmonary Alveoli ; cytology ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; 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

BACKGROUNDToll-like receptor-4 (TLR-4) is integrally involved in lipopolysaccharide (LPS) signaling and has a requisite role in the activation of nuclear factor-κB (NF-κB). The exact mechanisms that lend perfluorocarbon (PFC) liquids a cytoprotective effect have yet to be elucidated. Therefore we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated alveolar epithelial cellls (AECs).

METHODSAECs (A549 cells, human lung adenocarcinoma cell line) were divided into four groups: control, PFC, LPS and LPS + PFC (coculture group) groups. Intercellular adhesion molecule-1 (ICAM-1) was detected by ELISA, tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) were detected by radioimmunological methods. The expression of TLR-4 mRNA and protein was detected by real time PCR and Western blotting, respectively. The activation of NF-κB was detected by Western blotting (proteins of I-κBa and NF-κB p65).

RESULTSICAM-1, TNF-α and IL-8 were significantly increased in LPS-stimulated AECs groups. The expression of TLR-4 mRNA and protein in LPS-stimulated groups was markedly increased. Meanwhile, NF-κB was activated as indicated by the significant degradation of IκB-α and the significant release of NF-κB P65 and its subsequent translocation into the nucleus. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-α and IL-8, the expression of TLR-4 mRNA and the activity of NF-κB.

CONCLUSIONSTaken together, our results demonstrate that LPS can induce AEC-related inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect AECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway, which is indicated by the significant decrease of TLR-4 expression and NF-κB activation.

Blotting, Western ; Cell Line, Tumor ; Epithelial Cells ; drug effects ; immunology ; Fluorocarbons ; pharmacology ; Humans ; Inflammation ; chemically induced ; immunology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Interleukin-8 ; genetics ; metabolism ; Lipopolysaccharides ; pharmacology ; NF-kappa B ; genetics ; metabolism ; Pulmonary Alveoli ; cytology ; Real-Time Polymerase Chain Reaction ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

BACKGROUNDIt is widely accepted that tumor necrosis factor-α (TNF-α) plays an important role in the pathogenesis of emphysema. This study aimed at investigating the protective effects of anti-TNF-α antibody, infliximab, in the development of emphysema induced by passive smoking in rats.

METHODSThirty-nine rats were randomly divided into a normal control group (group 1), an emphysema group (group 2), and an infliximab-intervention group (group 3). Rat models of emphysema were established by exposure to cigarette smoking daily for 74 days. After 1 month, the infliximab intervention group was treated with infliximab via subcutaneous injection. The levels of TNF-α, IL-8 and vascular endothelial growth factor (VEGF) in bronchoalveolar lavage fluid (BALF) were measured with enzyme linked immunosorbent assay (ELISA). The number and classification of cells in the BALF were measured. Lung tissue sections stained by hematoxylin and eosin (HE) were observed, and mean linear intercept (MLI) and mean alveolar numbers (MAN) were measured. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods were used to examine the percentage of positive cells and distribution of apoptotic cells.

RESULTSThe levels of TNF-α and IL-8 in BALF were higher in group 2 than in group 1 and group 3. The MLI was greater in group 2 than that in group 1 and group 3 while MAN was decreased. The concentration of VEGF in BALF of group 2 was significantly decreased as compared with group 1. The total cells and neutrophils number was significantly increased in group 2 as compared with group 1 and group 3, so was the percentage of neutrophils. The number of TUNEL positive cells in the alveolar septa was significantly increased in group 2 as compared with group 1 and group 3.

CONCLUSIONInfliximab protects against cigarette smoking-induced emphysema by reducing airway inflammation, attenuating alveolar septa cell apoptosis and improving pathological changes.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Infliximab ; Interleukin-8 ; metabolism ; Male ; Pulmonary Alveoli ; cytology ; drug effects ; Pulmonary Emphysema ; chemically induced ; metabolism ; prevention & control ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tobacco Smoke Pollution ; adverse effects ; Tumor Necrosis Factor-alpha ; metabolism

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-beta-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-beta-dependent signaling in AECs.
Animals ; Epithelial Cells/cytology ; Epithelial-Mesenchymal Transition/*drug effects ; Glycosylation End Products, Advanced/genetics/*metabolism ; Idiopathic Pulmonary Fibrosis/metabolism ; Pulmonary Alveoli/cytology ; RNA, Small Interfering/genetics ; Rats ; Receptors, Immunologic/genetics/*metabolism ; Smad7 Protein/genetics/*metabolism ; Transforming Growth Factor beta/genetics/metabolism

OBJECTIVETo explore the effect of terbutaline on sodium transport in rat alveolar type I (ATI) and type II (ATII) cells of rats.

METHODSThe whole cell currents were recorded from ATII cells isolated from rat lungs perfused with or without amiloride (inhibitor of epithelial sodium channel) and ZnCl(2) (inhibitor of cyclic nucleotide-gated cation channel) in the whole cell recording mode using the patch-clamp technique. The effect of terbutaline on the currents was examined.

RESULTSThe main currents recorded from ATII cells were amiloride-sensitive and Zn(2+)-sensitive. The amiloride-sensitive and Zn(2+)-sensitive current shared a similar proportion (P>0.05). Both currents could be significantly increased by terbutaline (P<0.05), and the proportion of amiloride-sensitive current was 1.7 times that of Zn(2+)-sensitive current (P<0.05).

CONCLUSIONThere are functional epithelial sodium channels (ENaC) and cyclic nucleotide-gated cation channels (CNG) on freshly isolated ATII cells, both serving as the main channels for sodium transport. Terbutaline increases the absorption of alveolar fluid primarily by increasing sodium transport of ENaC and CNG on ATI and AT II cells.

Amiloride ; pharmacology ; Animals ; Chlorides ; pharmacology ; Cyclic Nucleotide-Gated Cation Channels ; antagonists & inhibitors ; drug effects ; Male ; Peptides ; pharmacology ; Pulmonary Alveoli ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism ; Sodium Channels ; drug effects ; Terbutaline ; pharmacology ; Zinc Compounds ; pharmacology

AIMTo study the effects of bone marrow MSCs transplantation on the apoptosis of alveolar wall cells and the expression of Bcl-2 and Bax of lung tissue in papain and Co60-induced pulmonary emphysema rats.

METHODSFemale Lewis rats were randomly divided into three groups: control group, emphysema group, emphysema + MSCs transplantation group. Rats were sacrificed at days 14 and 28 after treatment. Morphologic analysis of the lung tissue was performed. The apoptosis of the lung cells was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of Bcl-2 and Bax were determined by immunohistochemical staining.

RESULTSEmphysematous changes of the lung tissue were observed in emphysema group and emphysema + MSCs transplantation group. However, the emphysematous change in emphysema + MSCs transplantation group was improved compared with the emphysema group. There was significant difference in the number of alveolar counted per unit area (MAN), mean alveoli area (MAA) and mean linear interval(MLI) between emphysema group and emphysema + MSCs transplantation group. The apoptotic index of the alveolar wall cells in emphysema + MSCs transplantation group was less than that in the emphysema group. The percentage of Bcl-2 positive cells in emphysema + MSCs transplantation group was significantly higher than that in the emphysema group. The percentage of Bax positive cells in emphysema + MSCs transplantation group was significantly lower than that in the emphysema group. The ratio of Bcl-2/Bax of emphysema + MSCs transplantation group was significantly higher than that in the emphysema group.

CONCLUSIONBone marrow MSCs transplantation inhibits the apoptosis of alveolar wall cells, upregulates the expression of Bcl-2 and downregulates the expression of Bax. This may be part of the reason that bone marrow MSCs transplantation improves the papain and Co60-induced pulmonary emphysema.

Animals ; Apoptosis ; Bone Marrow Transplantation ; Cells, Cultured ; Cobalt Isotopes ; adverse effects ; Female ; Lung ; cytology ; Mesenchymal Stem Cell Transplantation ; Papain ; adverse effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pulmonary Alveoli ; drug effects ; pathology ; radiation effects ; Pulmonary Emphysema ; chemically induced ; metabolism ; pathology ; surgery ; Rats ; bcl-2-Associated X Protein ; metabolism