Pulmonary Alveolar Epithelial Cells Research Articles

Metformin: one of the possible options to reduce the mortality of severe coronavirus disease 2019?

According to the world epidemic report, the mortality of patients with severe coronavirus disease 2019 (COVID-19) is high. Diabetic patients are more susceptible to COVID-19. Since the mortality of COVID-19 patients with diabetes is on the top of list, hyperglycemia is considered an independent risk factor for severe COVID-19. Up to now, there is few effective treatment for severe patients infected with 2019 novel coronavirus (2019-nCoV). Clinical studies observed that cytokine storms existed in patients with severe COVID-19. Sustained high levels of cytokines cause diffuse damage to pulmonary capillary endothelial cells and alveolar epithelial cells, resulting in acute respiratory distress syndrome (ARDS). ARDS is the main cause of death in COVID-19 patients. Host-directed therapy (HDT) is an emerging therapeutic method in the field of anti-infection, which can activate the self-protective immune response, suppress excessive inflammatory response, and be used to assist the treatment of traditional drugs to shorten the course of disease. Metformin has been shown to be effective in HDT and can assist in the treatment of the viral and bacterial infectious disease. This paper discusses the rationality and potential therapeutic mechanism of metformin in the treatment of severe COVID-19. It was speculated that the use of metformin for controlling blood glucose in severe COVID-19 patients with diabetes may prevent or inhibit the occurrence of ARDS, thereby reducing the mortality of COVID-19 patients. The possible mechanism is that metformin could inhibit cytokine storm via suppressing interleukin-6 (IL-6) signaling, prevent the process of lung fibrosis, suppress endocytosis, thereby elevating angiotensin converting enzyme 2 (ACE2) expression.

Inhibition of miR-21 ameliorates LPS-induced acute lung injury through increasing B cell lymphoma-2 expression.

The aberrant expression of microRNAs (miRNAs) is associated with the pathogenesis of inflammation-related diseases. However, the biological functions of miR-21 in acute lung injury (ALI) remain largely unknown. In this study, the level of miR-21 was obviously increased, but B cell lymphoma-2 (Bcl-2) expression was markedly decreased in LPS-treated human pulmonary alveolar epithelial cells (HPAEpiC). Suppression of miR-21 attenuated LPS-induced apoptosis and inflammation in HPAEpiC and promoted the survival of mice with ALI by decreasing the inflammatory cell count, release of cytokines and permeability in lung tissues. Importantly, Bcl-2 was a direct target of miR-21, and its expression was significantly inhibited by miR-21 mimics at a post-transcriptional level. Besides, Bcl-2 over-expression reversed miR-21-induced apoptosis and inflammation status and showed synergic effects with miR-21 inhibitor in LPS-treated HPAEpiC. In conclusion, inhibition of miR-21 could ameliorate apoptosis and inflammation by restoring the expression of Bcl-2 in LPS-induced HPAEpiC and mice, which might provide therapeutic strategies for the treatment of ALI.

Proteinase‐activated receptor 1: A target for repurposing in the treatment of COVID‐19?

In the search to rapidly identify effective therapies that will mitigate the morbidity and mortality of COVID‐19, attention has been directed towards the repurposing of existing drugs. Candidates for repurposing include drugs that target COVID‐19 pathobiology, including agents that alter angiotensin signalling. Recent data indicate that key findings in COVID‐19 patients include thrombosis and endotheliitis. Activation of proteinase‐activated receptor 1 (PAR1), in particular by the serine protease thrombin, is a critical element in platelet aggregation and coagulation. PAR1 activation also impacts on the actions of other cell types involved in COVID‐19 pathobiology, including endothelial cells, fibroblasts and pulmonary alveolar epithelial cells. Vorapaxar is an approved inhibitor of PAR1, used for treatment of patients with myocardial infarction or peripheral arterial disease. We discuss evidence for a possible beneficial role for vorapaxar in the treatment of COVID‐19 patients and other as‐yet non‐approved antagonists of PAR1 and proteinase‐activated receptor 4 (PAR4).Linked ArticlesThis article is part of a themed issue on The Pharmacology of COVID‐19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc

Friedelin Protects Against Alveolar Epithelial Cells Apoptosis in Lps-Induced Acute Pneumonia in Neonatal Rats by Suppressing NF-κB Activation

Neonatal pneumonia is caused by inflammation mediated by lipopolysaccharide from gram negative bacteria. This type of pneumonia is characterized by inflammatory and apoptotic responses. In this study, we have examined the effect of friedelin on lipopolysaccharide-induced pneumonia and the role of nuclear factor kappa B in this process. Also, using the human pulmonary alveolar epithelial cells as a model we examined the effect of lipopolysaccharide on the cell apoptosis and its protection by friedelin. The results show that friedelin prevented lipopolysaccharide-induced acute pneumonia in neonatal rats and cellular apoptosis by suppressing the nuclear factor kappa B signaling pathway. In summary, this study shows that the friedelin exhibits a remarkable protective effect on lung tissues exposed to lipopolysaccharides.

MiR-143-3p impacts on pulmonary inflammatory factors and cell apoptosis in mice with mycoplasmal pneumonia by regulating TLR4/MyD88/NF-κB pathway.

miR-143-3p is correlated with inflammatory pain responses, such as hsa-miR-143-3p expression reduction in fibromyalgia. The present study aimed to explore the effects of miR-143-3p and Toll-like receptor (TLR) 4/myeloid differentiation factor 88 (MyD88)/NF-κB signaling pathway on pulmonary inflammatory factors levels and alveolar epithelial cell apoptosis in mycoplasmal pneumonia mice. Twenty mice were selected as normal group. The 120 successfully modeled Mycoplasma pneumoniae (MP) infection mice were randomly divided into model group (without any treatment), negative control (NC) group (injected with NC mimic), miR-143-3p mimic group (injected with miR-143-3p mimic), miR-143-3p inhibitor group (injected with miR-143-3p inhibitor), TAK-242 group (treatment with TAK-242), and miR-143-3p inhibitor + TAK-242 group (treatment with miR-143-3p inhibitor + TAK-242). Compared with model group, model mice had up-regulated miR-143-3p expression and decreased MyD88 and p-NF-κB p50 protein expressions (all P<0.05); Model mice treated with miR-143-3p mimic and TAK-242 had reduced interleukin (IL)-2 and tumor necrosis factor (TNF)-α contents and protein expressions of MyD88, p-NF-κB p50, increased IL-10 content, fewer alveolar epithelial cell apoptosis, lower Bax expression and higher Bcl-2 expression (all P<0.05); however, mice with miR-143-3p inhibitor treatment showed opposite trends in terms of above indicators. The exacerbation of mycoplasmal pneumonia caused by miR-143-3p inhibitor was partly improved by miR-143-3p inhibitor + TAK-242 combination treatment (all P<0.05). Therefore, up-regulation of miR-143-3p expression may ameliorate pulmonary inflammatory factors levels and reduce alveolar epithelial cell apoptosis in mycoplasmal pneumonia mice by inhibiting TLR4/MyD88/NF-κB signaling pathway.

MicroRNA-92a serves as a risk factor in sepsis-induced ARDS and regulates apoptosis and cell migration in lipopolysaccharide-induced HPMEC and A549 cell injury

AimsSepsis-induced acute respiratory distress syndrome (ARDS) is a common, high mortality complication in intensive care unit (ICU) patients. MicroRNA-92a (miR-92a) plays a role in many diseases, but its association with sepsis-induced ARDS is unclear. Materials and methodsWe enrolled 53 patients, including 17 with sepsis only, and 36 with sepsis-induced ARDS. Lipopolysaccharide (LPS) was used to stimulate pulmonary microvascular endothelial cells (HPMEC) and alveolar epithelial A549 cells, which were used to investigate the miR-92a roles in ARDS. MiR-92a expression levels in patient serum and cells were quantified using quantitative reverse transcription-polymerase chain reaction (RT-PCR), and protein expression was examined using Western blotting. The effect of miR-92a on apoptosis was examined using flow cytometry. Wound healing and transwell migration assays were used to evaluate cell migration. Key findingsSerum miR-92a expression was higher in patients with sepsis-induced ARDS, when compared to patients with sepsis only. After LPS treatment in cells, miR-92a expression was higher when compared with control group, cell apoptosis and inflammatory responses were increased and cell migration was inhibited. However, cell apoptosis and inflammatory responses were decreased and cell migration was enhanced after miR-92a downregulation, when compared with inhibitor negative control (NC) group. Moreover, phosphorylated-Akt and phosphorylated-mTOR expression were increased after miR-92a inhibition. SignificanceOur study provides evidence that circulating serum miR-92a could act as a risk factor for sepsis-induced ARDS. MiR-92a inhibition attenuated the adverse effects of LPS on ARDS through the Akt/mTOR signaling pathway.

Modeling Pharmacokinetic Profiles for Assessment of Anti-Cancer Drug on a Microfluidic System.

The pharmacokinetic (PK) properties of drug, which include drug absorption and excretion, play an important role in determining the in vivo pharmaceutical activity. However, current in vitro systems that model PK profiles are often limited by the in vivo-like concentration profile of a drug. Herein, we present a perfused and multi-layered microfluidic chip system to model the PK profile of anti-cancer drug 5-FU in vitro. The chip device contains two layers of culture channels sandwiched by a porous membrane, which allows for drug exposure and diffusion between the two channels. The integration of upper intestine cells (Caco-2) and bottom targeted cells within the device enables the generation of loading and clearance portions of a PK curve under peristaltic flow. Fluorescein as a test molecule was initially used to generate a concentration-time curve, investigating the effects of parameters of flow rate, administration time, and initial concentration on dynamic drug concentration profiles. Furthermore, anti-cancer drug 5-FU was performed to assess its pharmaceutical activity on target cells (human lung adenocarcinoma cells or human pulmonary alveolar epithelial cells) using different drug administration regimens. A dynamic, in vivo-like 5-FU exposure refers to PK profile regimen, led to generate a lower drug concentration (dynamically fluctuate from 0 to 1 μg/mL affected by absorption) compared to the constant exposure. Moreover, the PK profile regimen alleviates the drug-induced cytotoxicity on target cells. These results demonstrate the feasibility of determining the PK profiles using this microfluidic system with in vivo-like drug administration regimens. This established system may provide a powerful platform for the prediction of drug safety and effectiveness in the pharmaceutical research.

Liquid biopsy of lung cancer by deep learning and spectroscopic analysis of circulating exosomes.

e15532 Background: Lung cancer has a high mortality rate because of belated diagnosis at advanced stages beyond the treatable condition. Early detection of lung cancer can improve the survival rate. A liquid biopsy that detects tumor-related biomarkers in body fluids has a great potential for the purpose. Particularly, tumor-derived exosomes in blood have been proposed as a promising biomarker. The tumor-derived exosomes carry molecules of their parental cells; thus, they provide information about the tumor in the body. Unfortunately, exosomal markers conducive to the early detection of lung cancer are still obscure. Therefore, using the molecular fingerprint of exosomes markers can be useful to detect the tumor exosomes. Raman spectroscopy is one of the representative methods for the purpose. However, because the exosomes have a heterogeneous composition in blood, interpreting their spectroscopic signals is hard. Thus, we utilized a deep learning approach to analyze the spectroscopic signal of the exosomes for liquid biopsy of lung cancer. Methods: The basic concept was to evaluate how much the exosomes in human plasma resemble cancer cell exosomes. As a proof of concept, exosomes of 43 non-small cell lung cancer (NSCLC) adenocarcinoma patients and 20 healthy controls were isolated from plasma of peripheral blood. Also, cell exosomes were isolated from culture media of adenocarcinoma cell lines and a human pulmonary alveolar epithelial cell line. Then, the spectroscopic signals were detected using surface-enhanced Raman spectroscopy (SERS). Further, the deep learning algorithm was employed to classify the signals. Then, we calculated the relative similarity to cancerous exosomes against human plasma exosomes. Results: Our method was able to classify cancer and normal cell exosomes with 95% accuracy. Also, Raman signals of cancer patients’ exosomes were more similar to the cancer cell exosomes than those of healthy controls. Notably, the similarity was proportional to cancer stages. Importantly, our method even detected stage I patients. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was 0.912 for stage I and II, and 0.910 for stage I. Conclusions: We reported a novel diagnostic method using deep learning analysis against spectroscopic signals of circulating exosomes. Our method that evaluates the similarity to cancer exosomes accurately identified lung cancer patients, even stage I with high accuracy.

Mevastatin-Induced AP-1-Dependent HO-1 Expression Suppresses Vascular Cell Adhesion Molecule-1 Expression and Monocyte Adhesion on Human Pulmonary Alveolar Epithelial Cells Challenged with TNF-α

Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFRα/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor α (TNFα) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-κB p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNFα-induced VCAM-1 expression was attenuated by an NF-κB inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKCα (Gö6983), Pyk2 (PF431396), p38α MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKCα, Pyk2, p38α MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKCα/Pyk2/p38α MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNFα-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation.

Ginkgolide B protects human pulmonary alveolar epithelial A549 cells from lipopolysaccharide-induced inflammatory responses by reducing TRIM37-mediated NF-κB activation.

The treatment options for acute stroke combined with pulmonary infection are limited. Clinically, there are several therapies to promote blood circulation and dissipate blood stasis; these treatment options include ginkgolide B (GB), which has PAF (platelet activating factor)-inhibiting effects. PAF-receptor (PAF-R) antagonists are used to treat a variety of inflammatory diseases; however, the potential of PAF-R antagonists as a treatment for lung infections remains unclear. The aim of the present study is to investigate the protective effect of GB on lipopolysaccharide-induced inflammatory responses in A549 human pulmonary alveolar epithelial cells (HPAEpiC) in vitro. Cell viability and apoptosis were measured by CCK-8 and flow cytometry. TRIM37, Caspase-3, and NF-κBp65 expression levels were measured by real-time PCR and Western blotting. The release of tumor necrosis factor-α and interleukin-1β was measured by ELISA. The data indicates that GB may reduce TRIM37 expression by antagonizing the PAF-R pathway, thereby inhibiting the activation of nuclear factor-κB and alleviating the inflammatory response of alveolar epithelial cells. This study is the first to provide insight into the therapeutic potential of GB and suggests that clinical application of GB in acute stroke combined with pulmonary inflammation may be efficacious.

Titanium Dioxide Nanoparticles Alter the Cellular Phosphoproteome in A549 Cells.

TiO2 nanoparticles (NPs) are one of the most produced NPs worldwide and are used in many consumer products. Their impact on human health, especially through inhalation, has been studied for more than two decades. TiO2 is known for its strong affinity towards phosphates, and consequently interaction with cellular phosphates may be one of the mechanisms driving its toxicity. In the present study, we used a phosphoproteomics approach to document the interaction of TiO2-NP with phosphoproteins from A549 human pulmonary alveolar epithelial cells. Cells were exposed to 21 nm anatase/rutile TiO2-NPs, then their phosphopeptides were extracted and analyzed using shotgun proteomics. By comparing the phosphoprotein content, phosphorylation status and phosphorylation sites of exposed cells with that of control cells, our results show that by affecting the phosphoproteome, TiO2-NPs affect cellular processes such as apoptosis, linked with cell cycle and the DNA damage response, TP53 being central to these pathways. Other pathways including inflammation and molecular transport are also affected. These molecular mechanisms of TiO2-NP toxicity have been reported previously, our study shows for the first time that they may derive from phosphoproteome modulation, which could be one of their upstream regulators.

Induction of HO-1 by Mevastatin Mediated via a Nox/ROS-Dependent c-Src/PDGFRα/PI3K/Akt/Nrf2/ARE Cascade Suppresses TNF-α-Induced Lung Inflammation.

Background: Mevastatin (MVS), a 3-hydroxy-3-methylglutaryl coenzyme, a reductase (HMG-CoA) inhibitor, has anti-inflammatory effects potentially via up-regulation of heme oxygenase-1 (HO-1). However, the mechanisms underlying MVS-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). Methods: HO-1 and intercellular adhesion molecule (ICAM)-1 expression were determined using real-time PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated using pharmacological inhibitors or specific small interfering RNA (siRNA)s. Interaction between Nrf2 and the antioxidant response element (ARE) binding site for the HO-1 promoter was determined by chromatin immunoprecipitation (ChIP) assay. Results: Upregulation of HO-1 by MVS attenuated the tumor necrosis factor (TNF)-α-stimulated ICAM-1 expression associated with THP-1 adhesion to HPAEpiCs. These inhibitory effects of HO-1 were reversed by tin protoporphyrin (SnPP)IX or by transfection with HO-1 siRNA. MVS-induced HO-1 expression was mediated via NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation. Activation of Nox2/ROS further stimulated the phosphorylation of p47phox, proto-oncogene tyrosine-protein kinase (c-Src), platelet-derived growth factor receptor (PDFGR)α, protein kinase B (Akt), and Nrf2, which were inhibited by siRNAs. Pretreatment with pharmacological inhibitors, including diphenyleneiodonium (DPI), apocynin (APO), N-acetyl-L-cysteine (NAC), PP1, AG1296, or LY294002, reduced the MVS-activated Nrf2 nuclear-translocation binding to the ARE on the HO-1 promoter. Conclusions: MVS-induced HO-1 is, at least in part, mediated through a p47phox/Nox2/ROS-dependent activation of c-Src/PDGFRα/PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-α-mediated inflammatory responses in HPAEpiCs.

Synergistic effect of halofuginone and dexamethasone on LPS‑induced acute lung injury in type II alveolar epithelial cells and a rat model.

Acute lung injury (ALI) is characterized by neutrophilic infiltration, uncontrolled oxidative stress and inflammatory processes. Despite various therapeutic regimes having been performed, there remains no effective pharmacotherapy available to treat ALI. Halofuginone (HF), a ketone isolated from Dichroafebrifuga, exhibits significant anti‑inflammatory and antifibrotic effects. Dexamethasone (DEX), a synthetic glucocorticoid, has been routinely used as an adjuvant therapy in treating inflammatory diseases, including ALI. The present study aimed to investigate the effects of the combination of HF and DEX in the treatment of ALI. The present results suggested that the simultaneous administration of HF and DEX markedly decreased the level of pro‑inflammatory cytokines and increased the level of anti‑inflammatory cytokines, as assessed by western blot analysis. In addition, HF and DEX effectively decreased nuclear factor‑κB activity via suppressing the phosphorylation of P65 in lipopolysaccharide (LPS)‑induced human pulmonary alveolar epithelial cells (HPAEpiC) and lung tissues extracted from ALI rats, as determined by immunofluorescence. Furthermore, invivo experiments demonstrated that the combination of HF and DEX in LPS‑induced ALI rats defended against lung fibrosis, perivascular inflammation, congestion and edema of pulmonary alveoli, as assessed by histopathological analysis, TUNEL staining and immunohistochemistry assay. Taken together, the present study indicated the synergistic effect of HF and DEX on LPS‑induced ALI in HPAEpiC cells and a rat model. These results offer a novel therapeutic approach for the treatment of ALI.

MicroRNA-205-5p Targets HMGB1 to Suppress Inflammatory Responses during Lung Injury after Hip Fracture.

Hip fracture is the most common type of injury in elderly people and is associated with a high incidence of complications and risk of mortality. In these patients, subsequent pulmonary infection can contribute to the development of an acute lung injury, a consequence of the systemic inflammatory response induced by hip fracture. Although the crucial role of microRNAs (miRNAs) in inflammatory responses has been established, the functions of miRNAs in the inflammatory responses associated with lung injury after hip fracture remain poorly understood. In this study, we explored the potential role of miR-205-5p in lung injury after hip fracture in an in vivo hip fracture model and in vitro cultures of human pulmonary alveolar epithelial cells (HPAEpiC). An analysis of clinical serum samples revealed increased levels of miR-205-5p and high mobility group box 1 (HMGB1) after hip fracture. A bioinformatics analysis and dual-luciferase reporter assay identified HMGB1 as a potential target of miR-205-5p. The overexpression of miR-205-5p clearly reduced the expression of HMGB1 and inhibited NF-κB signaling, apoptosis, and proinflammatory cytokine production while enabling continued cell proliferation. Our results demonstrate that the upregulation of miR-205-5p suppresses inflammatory responses and promotes cell viability and proliferation by selectively targeting HMGB1 in the context of lung injury after hip fracture. Therefore, miR-205-5p may be an alternative target of therapeutic strategies for lung injury after hip fracture.

Biochemical and cellular activity of chemically synthesized elastase inhibitor (S-AFUEI) from Aspergillus fumigatus

PurposeElastase, produced by Aspergillus fumigatus and A. flavus, is an important pathogenic factor in pulmonary aspergillosis. We investigated the possibility of using A. fumigatus-derived A. fumigatus elastase inhibitor (AFUEI) as a therapeutic agent. As native-AFUEI (N-AFUEI) has an extremely low yield, we generated a synthetic-AFUEI (S-AFUEI) and investigated whether S-AFUEI has a biological activity against A. fumigatus elastase (AFUE) and inhibits cytotoxicity. MethodologyA. fumigatus was cultured in Yeast Carbon Base (YCB) -elastin culture medium for 3–7 days, and AFUE was purified by chromatography using DE52 cellulose and Sephadex G-75 column. Elastolytic activity was examined using Glt-Ala-Ala-Pro-Leu-pNA (GAAPLNA) as the substrate. The hydrolytic activity of AFUE was determined using the characteristic substrates, fibrinogen and collagen (Type IV), and human cell cytotoxicity was measured colorimetrically. Furthermore, the inhibitory effect of S-AFUEI on these activities was examined. ResultsWe confirmed that S-AFUEI demonstrated elastase inhibitory activity and heat stability equivalent to that demonstrated by N-AFUEI, and inhibited human collagen hydrolytic activity and human fibrinogen hydrolytic activity. Further, S-AFUEI inhibited cytotoxicity in AFUE human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), and human pulmonary alveolar epithelial cells (HPAEpiC). ConclusionAs S-AFUEI strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.

Inhibitory Effect of Chebulic Acid on Alveolar Epithelial to Mesenchymal Transition in Response to Urban Particulate Matter Using Co-treatment and Post-treatment Exposure.

Urban particulate matter (UPM) is atmospheric particulate samples obtained from industrialized urban areas. It is known that pulmonary fibrosis can result directly or indirectly from particulate matter. In this study, the protective effect of chebulic acid (CA) against UPM-induced epithelial-mesenchymal transition (EMT) in the pulmonary alveolar epithelial (PAE) cells were investigated. Our findings revealed that PAE cells were changed from the epithelial phenotype to mesenchymal one after exposure to UPM. Furthermore, co-treatment and post-treatment of CA inhibited EMT progression. Especially the key epithelial marker, E-cadherin, was down-regulated by UPM and recovered by CA. Also, gelatin zymogram showed that the activity of matrix metalloproteinase (MMP)-2 and MMP-9 was decreased by co-treatment and post-treatment of CA. Further investigation revealed that CA attenuated UPM-stimulated PAE cells invasion ability. These data showed that UPM promoted PAE cells invasion, reactive oxygen species-mediated extracellular matrix degradation and CA reduced the potential health risks associated with UPM.

Supplementation of Adiantum capillus-veneris Modulates Alveolar Apoptosis under Hypoxia Condition in Wistar Rats Exposed to Exercise.

Background and Objectives: Several studies have reported that some conditions such as exercise and hypoxia induce DNA damage and dysfunction and apoptosis. Some plant foods contain numerous bioactive compounds and anti-inflammatory properties that can help fight DNA damage. Therefore, the current study evaluated the effect of supplementation of Adiantum capillus-veneris (ACV) extract on Bax/B-cell lymphoma 2 (Bcl-2) ratio apoptotic index and remodeling of pulmonary alveolar epithelial cells in lung tissue of healthy Wistar rats during stressful conditions (hypoxia). Materials and Methods: Twenty-seven Wistar male rats (four-week old, 72 ± 9 g) were randomly assigned into three groups: normoxic, sedentary, and not-supplemented (NG, n = 9); exercise and hypoxia and not-supplemented (HE, n = 9); and exercise and hypoxia and supplemented group (HS, n = 9). The NG remained sedentary in the normoxia environment for nine weeks. The HE group participated in a high-intensity (IT) program for six weeks, then remained sedentary in the hypoxia environment for three weeks. The low-pressure chamber simulated a ~2800 M altitude 24 h/d. HS participated in IT, then entered and remained sedentary in the hypoxia environment for three weeks, and they consumed 500 mg per kg of body weight ACV extract. Results: The Bax/Bcl-2 ratio of the HE group increased significantly (+50.27%, p ≤ 0.05), the average number of type I pneumocytes was reduced significantly (−18.85%, p ≤ 0.05), and the average number of type II pneumocytes was increased significantly (+14.69%, p ≤ 0.05). Also, after three weeks of consuming the ACV extract, the HS group in comparison with the HE group had their Bax/Bcl-2 ratio reduced significantly (−24.27%, p ≤ 0.05), the average number of type I pneumocytes increased significantly (+10.15%, p ≤ 0.05), and the average number of type II pneumocytes reduced significantly (−7.18%, p ≤ 0.05). Conclusion: The findings show that after three weeks of hypoxia following six weeks of high-intensity interval training in Wistar rats, the Bax/Bcl-2 ratio and the number of type II pneumocytes were increased and the number of type I pneumocytes was reduced significantly. These results strongly suggest that an apoptosis state was induced in the lung parenchyma, and consuming ACV extract modulated this state.

Gene expression changes in human bronchial epithelial cells (BEAS-2B) and human pulmonary alveolar epithelial cells (HPAEpiC) after interaction with Cladosporium sphaerospermum.

Cladosporium is one of the most abundant spore. Fungi of this genus can cause respiratory allergy and intrabronchial lesion. We studied the differential expression of host genes after the interaction of Cladosporium sphaerospermum conidia with Human Bronchial Epithelial Cells (BEAS-2B) and Human Pulmonary Alveolar Epithelial Cells (HPAEpiC). C. sphaerospermum conidia were harvested and co-cultured with BEAS-2B cells or HPAEpiC cells for 48 hours respectively. This culture duration was chosen as it was associated with high germination rate. RNA was extracted from two biological replicates per treatment. RNA of BEAS-2B cells was used to assess changes in gene expression using AffymetrixGeneChip® Human Transcriptome Array 2.0. After co-culture with Cladosporium spores, 68 individual genes were found differentially expressed (P≤0.05) and up-regulated≥1.5 folds while 75 genes were found differentially expressed at≤-1.5 folds compared with controls. Reverse transcription and qPCR were performed on the RNA collected from both BEAS-2B cells and HPAEpiC cells to validate the microarray results with 7 genes. Based on the findings, infected pulmonary epithelial cells exhibited an increase in cell death-related genes and genes associated with innate immunity.

MicroRNA Let-7f-1-3p attenuates smoke-induced apoptosis in bronchial and alveolar epithelial cells in vitro by targeting FOXO1

Bronchial and alveolar epithelial cell apoptosis is a vital step in smoke-induced lung injury. We investigated whether and how microRNA (miRNA) Let-7f-1-3p would regulate smoke-induced apoptosis in bronchial and alveolar epithelial cells. Human small airway epithelial cells (HSAEC) and human pulmonary alveolar epithelial cells (HPAEpiC) were cultured using an air-liquid interface cell culture system. These cells were treated with Let-7f-1-3p agomir or antagomir for 24 h before smoke exposure or sham operation, after which the cells were rinsed and cultured for 24 h before cell viability, apoptosis, cytolysis, Caspase-9/8/3 activity assays, quantitative real-time polymerase chain reaction and Western blot. Bioinformatic and luciferase reporter assays were performed to predict or verify the target gene of Let-7f-1-3p. We found that smoke exposure significantly reduced Let-7f-1-3p expression level in HSAEC and HPAEpiC. Let-7f-1-3p agomir significantly attenuated cell apoptosis, cytolysis and Caspase-3, -8 and -9 activation while rescuing cell viability of smoke-exposed HSAEC and HPAEpiC. Let-7f-1-3p agomir downregulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL) and B-cell lymphoma-2 (Bcl2)-like protein 11 (Bim) protein level in HSAEC and HPAEpiC. Forkhead box-O1 (FOXO1) was verified as a putative regulatory target of Let-7f-1-3p. Smoke exposure increased FOXO1 mRNA and protein level in HSAEC and HPAEpiC, which was attenuated by Let-7f-1-3p agomir treatment. FOXO1 inhibition by small-molecule drug partially attenuated the increase in smoke-exposed HSAEC and HPAEpiC apoptosis, cytolysis and the decrease in cell viability caused by Let-7f-1-3p antagomir treatment. We concluded Let-7f-1-3p attenuated smoke-induced apoptosis in HSAEC and HPAEpiC by targeting FOXO1.

USP9X promotes LPS-induced pulmonary epithelial barrier breakdown and hyperpermeability by activating an NF-κBp65 feedback loop.

NF-κB is a central regulator of inflammatory and immune responses and has been shown to regulate transcription of several inflammatory factors as well as promote acute lung injury. However, the regulation of NF-κB signaling in acute lung injury has yet to be investigated. Human pulmonary alveolar epithelial cells (HPAEpiC) were treated with LPS to establish an acute lung injury model in vitro in which LPS stimulation resulted in pulmonary epithelial barrier breakdown and hyperpermeability. Cell viability was measured by CCK-8, and the transepithelial permeability was examined by measurement of transepithelial electrical resistance (TEER) and the transepithelial flux. Expression of ubiquitin-specific peptidase 9 X-linked (USP9X), zonula occludens (ZO-1), occludin and NF-κBp65, and the secretion of TNF-α and IL-1β were measured by Western blotting and ELISA, respectively. For in vivo studies, mice were intraperitoneally injected with LPS and/or NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Lung tissues were harvested for hematoxylin-eosin staining and Western blotting, and bronchoalveolar lavage fluid (BALF) was harvested for ELISA. We found that treatment with LPS in HPAEpiC inhibited cell viability and induced the expression of USP9X. Interestingly, knockdown of USP9X and treatment with PDTC suppressed LPS-induced HPAEpiC injury. USP9X overexpression promoted NF-κB activation, while NF-κB inactivation inhibited USP9X transcription and HPAEpiC injury induced by USP9X overexpression. Furthermore, LPS also induced the expression of USP9X in lungs, which was inhibited by PDTC. Taken together, these results demonstrate a critical role of USP9X-NF-κBp65 loop in mediating LPS-induced acute lung injury and may serve as a potential therapeutic target in acute lung injury.