Lung Alveolar Epithelial Cells Research Articles

Protective Effect of Remdesivir Against Pulmonary Fibrosis in Mice.

Pulmonary fibrosis is a known sequela of severe or persistent lung damage. Existing clinical, imaging and autopsy studies have shown that the lungs exhibit a pathological pulmonary fibrosis phenotype after infection with coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary fibrosis may be one of the most serious sequelae associated with coronavirus disease 2019 (COVID-19). In this study, we aimed to examine the preventative effects of the antiviral drug remdesivir on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of remdesivir on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of remdesivir in lung fibroblasts and alveolar epithelial cells in vitro. The preventive remdesivir treatment was started on the day of bleomycin installation, and the results showed that remdesivir significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro experiments showed that remdesivir dose-dependently suppressed TGF-β1-induced lung fibroblast activation and improved TGF-β1-induced alveolar epithelial to mesenchymal transition. Our results indicate that remdesivir can preventatively alleviate the severity of pulmonary fibrosis and provide some reference for the prevention of pulmonary fibrosis in patients with COVID-19.

Mangiferin inhibits hypoxia/reoxygenation-induced alveolar epithelial cell injury via the SIRT1/AMPK signaling pathway.

Lung ischemia-reperfusion injury (LIRI) is one of the complications that can occur after lung transplantation and may lead to morbidity and mortality. Mangiferin (MAF) is a naturally occurring glucosyl xanthone that has been documented to possess anti-inflammatory, immunomodulatory and potent antioxidant effects. The purpose of the present study was to investigate the effect of MAF on LIRI using a hypoxia-reoxygenation (H/R) cell model. In the present study, the viability of lung alveolar epithelial cells (A549) and H/R-A549 were detected by MTT assay. ELISA was used to evaluate the expression levels of IL-6 and IL-1β. TUNEL assay and western blotting were used to evaluate the apoptosis. In addition, H/R-A549 cells were treated with sirtinol, which is known inhibitor of sirtuin 1 (SIRT1) activity, to determine the effects of MAF on proteins associated with the SIRT1/5'AMP-activate protein kinase (AMPK) signaling pathway using western blotting. The results showed that 20 µM MAF exerted a protective effect on A549 cells against H/R mediating no clear cytotoxic effects. In terms of inflammation, MAF reduced IL-6, IL-1β, cyclooxygenase-2 and inducible nitric oxide synthase expression, which was accompanied by activation of the SIRT1/AMPK signaling pathway. In addition, compared with those in the group treated with sirtinol, expression of SIRT1, Bcl-2 and AMPK activity were elevated in MAF-treated H/R-A549 cells, whereas the expression of Bax, cleaved caspase-3 and cleaved caspase-9 was suppressed. TUNEL analysis of H/R-A549 cells treated with MAF in combination with sirtinol revealed that treatment with sirtinol blocked the SIRT1/AMPK signaling pathway and increased the apoptosis rate compared with the MAF group. Taken together, results of the present study revealed that MAF could inhibit lung H/R cell injury through the SIRT1/AMPK signaling pathway.

H7N9 virus infection triggers lethal cytokine storm by activating gasdermin E-mediated pyroptosis of lung alveolar epithelial cells.

The H7N9 influenza virus emerged in China in 2013, causing more than 1560 human infections, 39% of which were fatal. A ‘cytokine storm’ in the lungs of H7N9 patients has been linked to a poor prognosis and death; however, the underlying mechanism that triggers the cytokine storm is unknown. Here, we found that efficient replication of the H7N9 virus in mouse lungs activates gasdermin E (GSDME)-mediated pyroptosis in alveolar epithelial cells, and that the released cytosolic contents then trigger a cytokine storm. Knockout of Gsdme switched the manner of death of A549 and human primary alveolar epithelial cells from pyroptosis to apoptosis upon H7N9 virus infection, and Gsdme knockout mice survived H7N9 virus lethal infection. Our findings reveal that GSDME activation is a key and unique mechanism for the pulmonary cytokine storm and lethal outcome of H7N9 virus infection and thus opens a new door for the development of antivirals against the H7N9 virus.

The cell cycle inhibitor P21 promotes the development of pulmonary fibrosis by suppressing lung alveolar regeneration

The cell cycle inhibitor P21 has been implicated in cell senescence and plays an important role in the injury–repair process following lung injury. Pulmonary fibrosis (PF) is a fibrotic lung disorder characterized by cell senescence in lung alveolar epithelial cells. In this study, we report that P21 expression was increased in alveolar epithelial type 2 cells (AEC2s) in a time-dependent manner following multiple bleomycin-induced PF. Repeated injury of AEC2s resulted in telomere shortening and triggered P21-dependent cell senescence. AEC2s with elevated expression of P21 lost their self-renewal and differentiation abilities. In particular, elevated P21 not only induced cell cycle arrest in AEC2s but also bound to P300 and β-catenin and inhibited AEC2 differentiation by disturbing the P300–β-catenin interaction. Meanwhile, senescent AEC2s triggered myofibroblast activation by releasing profibrotic cytokines. Knockdown of P21 restored AEC2-mediated lung alveolar regeneration in mice with chronic PF. The results of our study reveal a mechanism of P21-mediated lung regeneration failure during PF development, which suggests a potential strategy for the treatment of fibrotic lung diseases.

Desertifilum sp. EAZ03 cell extract as a novel natural source for the biosynthesis of zinc oxide nanoparticles and antibacterial, anticancer and antibiofilm characteristics of synthesized zinc oxide nanoparticles.

The use of cyanobacterial cell extracts for the synthesis of zinc oxide nanoparticles (ZnO NPs) seems to be superior to other methods of synthesis because of its a green, environmentally friendly and low-cost approach. In this study, the cell extract of a newly characterized cyanobacterial strain Desertifilum sp. EAZ03 was used for the biosynthesis of ZnO NPs. The antimicrobial, antibiofilm and anticancer activities of the biosynthesized ZnO NPs (hereinafter referred to as CED-ZnO NPs) were examined as well. UV-Vis spectroscopy analysis of CED-ZnO NPs showed an absorbance band at 364nm, and powder X-ray diffraction analysis confirmed the purity of the synthesized nanoparticles. The analyses of scanning electron microscopy and transmission electron microscopy images revealed that CED-ZnO NPs were rod-shaped with a size of 88nm. The study of the biological features of CED-ZnO NPs showed a significant antimicrobial potential against the bacterial strains tested. CED-ZnO NPs were able to impede the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa up to 80%, 89% and 85%, respectively. The nanoparticles also showed 69%, 70% and 62% degrading activity against S. aureus, E. coli and P. aeruginosa 1-day-old biofilms, respectively. The antibiofilm activity of the synthesized nanoparticles was investigated by confocal laser scanning microscopy. The MTT assay showed that CED-ZnO NPs, at a concentration of 100μg/ml, had less cytotoxicity towards normal lung (MRC-5) cells, at the half, compared to cancerous lung alveolar epithelial (A549) cells. The minimum inhibitory concentration and minimum bactericidal concentration values of CED-ZnO NPs against E. coli, P. aeruginosa and S. aureus were 1500, 2000 and 32μg/ml, and 2500, 3500 and 64μg/ml, respectively. The multifunctional CED-ZnO NPs seem to be promising for possible applications in the therapeutic and pharmaceutical industries. This study proposes a new approach for the biosynthesis of zinc oxide nanoparticles using a newly characterized cyanobacterial strain Desertifilum sp. EAZ03. The considerable antimicrobial, antibiofilm and anticancer activities of the biosynthesized zinc oxide nanoparticles further emphasize the emerging role of microbial systems in the green synthesis of metal oxide nanoparticles.

M2 macrophages have unique transcriptomes but conditioned media does not promote profibrotic responses in lung fibroblasts or alveolar epithelial cells in vitro.

Macrophages are critical regulators of pulmonary fibrosis. Their plasticity, proximity, and ability to cross talk with structural cells of the lung make them a key cell type of interest in the regulation of lung fibrosis. Macrophages can express a variety of phenotypes, which have been historically represented through an "M1-like" to "M2-like" delineation. In this classification, M1-like macrophages are proinflammatory and have increased phagocytic capacity compared with alternatively activated M2-like macrophages that are profibrotic and are associated with wound healing. Extensive evidence in the field in both patients and animal models aligns pulmonary fibrosis with M2 macrophages. In this study, we performed RNA sequencing (RNAseq) to fully characterize M1- vs. M2-skewed bone marrow-derived macrophages (BMDMs) and investigated the profibrotic abilities of M2 BMDM conditioned media (CM) to promote fibroblast migration and proliferation, alveolar epithelial cell (AEC) apoptosis, and mRNA expression of key fibrotic genes in both fibroblasts and AECs. Although M2 CM-treated fibroblasts had increased migration and M2 CM-treated fibroblasts and AECs had increased expression of profibrotic proteins over M1 CM-treated cells, all differences can be attributed to M2 polarization reagents IL-4 and IL-13 also present in the CM. Collectively, these data suggest that the profibrotic effects associated with M2 macrophage CM in vitro are attributable to effects of polarization cytokines rather than additional factors secreted in response to those polarizing cytokines.

Asymmetric distribution of dynamin-2 and β-catenin relative to tight junction spikes in alveolar epithelial cells

ABSTRACT Tight junctions between lung alveolar epithelial cells maintain an air–liquid barrier necessary for healthy lung function. Previously, we found that rearrangement of tight junctions from a linear, cortical orientation into perpendicular protrusions (tight junction spikes) is associated with a decrease in alveolar barrier function, especially in alcoholic lung syndrome. Using quantitative super-resolution microscopy, we found that spikes in control cells were enriched for claudin-18 as compared with alcohol-exposed cells. Moreover, using an in situ method to measure barrier function, tight junction spikes were not associated with localized increases in permeability. This suggests that tight junction spikes have a regulatory role as opposed to causing a physical weakening of the epithelial barrier. We found that tight junction spikes form at cell–cell junctions oriented away from pools of β-catenin associated with actin filaments, suggesting that adherens junctions determine the directionality of tight junction spikes. Dynamin-2 was associated with junctional claudin-18 and ZO-1, but showed little localization with β-catenin and tight junction spikes. Treatment with Dynasore decreased the number of tight junction spikes/cell, increased tight junction spike length, and stimulated actin to redistribute to cortical tight junctions. By contrast, Dynole 34–2 and MiTMAB altered β-catenin localization, and reduced tight junction spike length. These data suggest a novel role for dynamin-2 in tight junction spike formation by reorienting junction-associated actin. Moreover, the greater spatial separation of adherens and tight junctions in squamous alveolar epithelial cells as compared with columnar epithelial cells facilitates analysis of molecular regulation of the apical junctional complex.

Nuclear Sphingosine-1-phosphate Lyase Generated ∆2-hexadecenal is A Regulator of HDAC Activity and Chromatin Remodeling in Lung Epithelial Cells.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to ∆2-hexadecenal (∆2-HDE) and ethanolamine phosphate by S1P lyase (S1PL) in mammalian cells. We have recently demonstrated the activation of nuclear SPHK2 and the generation of S1P in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa. Here, we have investigated the nuclear localization of S1PL and the role of ∆2-HDE generated from S1P in the nucleus as a modulator of histone deacetylase (HDAC) activity and histone acetylation. Electron micrographs of the nuclear fractions isolated from MLE-12 cells showed nuclei free of ER contamination, and S1PL activity was detected in nuclear fractions isolated from primary lung bronchial epithelial cells and alveolar epithelial MLE-12 cells. Pseudomonas aeruginosa-mediated nuclear ∆2-HDE generation, and H3/H4 histone acetylation was attenuated by S1PL inhibitors in MLE-12 cells and human bronchial epithelial cells. In vitro, the addition of exogenous ∆2-HDE (100-10,000 nM) to lung epithelial cell nuclear preparations inhibited HDAC1/2 activity, and increased acetylation of Histone H3 and H4, whereas similar concentrations of S1P did not show a significant change. In addition, incubation of ∆2-HDE with rHDAC1 generated five different amino acid adducts as detected by LC-MS/MS; the predominant adduct being ∆2-HDE with lysine residues of HDAC1. Together, these data show an important role for the nuclear S1PL-derived ∆2-HDE in the modification of HDAC activity, histone acetylation, and chromatin remodeling in lung epithelial cells.

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1β, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1β and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1β and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

The Impact of Angiotensin Converting Enzyme Inhibition and/or Angiotensin Receptor Blockade on Tissue Expression of the SARS‐CoV‐2 Receptor ACE2 in Mice

The angiotensin‐converting enzyme 2 (ACE2) is an established receptor and entry point for the novel SARS‐CoV‐2 coronavirus, as the spike protein on the viral envelope binds the ACE2 receptor. Published histological profiling revealed ACE2 to be highly expressed on human lung alveolar epithelial cells and on enterocytes of the small intestine, as well as on arterial and venous endothelial cells. Given the widespread abundance of ACE2 across tissues, there has been much speculation regarding whether ACE inhibitors (ACEi) and/or Angiotensin Receptor Blockers (ARB) might alter tissue ACE2 expression and thereby change the risk of transmission or development of severe complications. Therefore, the aim of this ongoing study is to measure the impact of ACEi and ARB singly and in combination versus placebo control on tissue distribution of ACE2 in male and female mice.A factorial design was employed with the following exposures: ACEi (lisinopril, 10 mg/kg/day), ARB (losartan, 10 mg/kg/day), both drugs, or water control. Drugs were delivered via drinking water. C57BL/6J Mice were 8 weeks of age at the start of the experiment. After 21 days of treatment, four mice (two males and two females) from each treatment group were euthanized, followed by perfusion and tissue collection. 12 more untreated mice were combined with the placebo group to increase the sample size of this reference group to 16 (8 males, 8 females). ACE2 protein abundance in brain, kidney, liver, lung, and small intestine was measured by ELISA (Abcam) and normalized to BCA‐measured total protein. Two‐way ANOVA was performed to test for differences in ACE2 levels by organ or sex among untreated mice; Mann‐Whitney tests were performed to compare each drug treatment group to the control group.Among untreated mice, ACE2 levels differed by organ (p < 0.01), but there were no differences between males and females for a given organ (p = 0.20). Males and females were combined for subsequent analyses. After 21 days of treatment, ACEi, ARB, and the combination therapy lowered ACE2 tissue abundance compared to untreated mice in the brain (ACEi p = 0.04; ARB p = 0.04; ACEi+ARB p = 0.01), kidney (ACEi p = 0.03; ARB p = 0.02; ACEi+ARB p = 0.02), and lung (ACEi p = 0.01; ARB p = 0.01; ACEi+ARB p = 0.01). ACEi, but not ARB or combination therapy, lowered ACE2 tissue abundance in the liver (p = 0.02). None of the treatments changed ACE2 tissue abundance in the small intestine.These results indicate that ACEi, ARB, or combination therapy reduces levels of ACE2, the SARS‐CoV‐2 receptor, in key tissues affected by the virus, including lung, kidney, and brain. An ongoing validation study will evaluate ACE2 protein and gene expression at earlier time points, as well as after discontinuation of treatment. These results will provide valuable insight into the risk or potential benefit of starting or stopping ACEi, ARB, or combination therapy during the COVID‐19 pandemic.

Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection.

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID‐19) binds to the angiotensin‐converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced‐stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID‐19, and because hyperglycemia has been linked to increased COVID‐19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1‐ and hyperglycemia‐induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID‐19.

Hermansky-Pudlak syndrome-2 alters mitochondrial homeostasis in the alveolar epithelium of the lung

BackgroundMitochondrial dysfunction has emerged as an important player in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a common cause of idiopathic interstitial lung disease in adults. Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder that causes a similar type of pulmonary fibrosis in younger adults, although the role of mitochondrial dysfunction in this condition is not understood.MethodsWe performed a detailed characterization of mitochondrial structure and function in lung tissues and alveolar epithelial cells deficient in the adaptor protein complex 3 beta 1 (Ap3b1) subunit, the gene responsible for causing subtype 2 of HPS (HPS-2).ResultsWe observed widespread changes in mitochondrial homeostasis in HPS-2 cells, including the acquisition of abnormally shaped mitochondria, with reduced number of cristae, and markedly reduced activity of the electron transport chain and the tricarboxylic acid cycle. We also found that mitochondrial redox imbalance and activity of the mitochondrial unfolded protein response were dysregulated in HPS-2 cells and this associated with various other changes that appeared to be compensatory to mitochondrial dysfunction. This included an increase in glycolytic activity, an upregulation in the expression of mitochondrial biogenesis factors and enhanced activation of the energy-conserving enzyme AMP-activated protein kinase.ConclusionIn summary, our findings indicate that mitochondrial function is dramatically altered in HPS-2 lung tissues, suggesting dysfunction of this organelle might be a driver of HPS lung disease.

Second-generation lung-on-a-chip with an array of stretchable alveoli made with a biological membrane

The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.

Inhibition of inflammation-induced injury and cell migration by coelonin and militarine in PM2.5-exposed human lung alveolar epithelial A549 cells

Accumulating studies suggest that fine particulate matter (PM2.5) pollutants in the air are easily enter into alveoli and even the bloodstream, resulting in an inflammatory response that not only triggers respiratory disorders but also causes permanent damage to various organs. Recent findings suggest that coelonin and militarine enriched in orchids can inhibit inflammation-induced injury against respiratory diseases. Here, we evaluated the anti-inflammatory properties of coelonin and militarine and examined their underlying molecular mechanisms in A549 cells exposed to PM2.5. PM2.5 induced significant intracellular reactive oxidative stress accumulation at a concentration of 250 μg/ml, as determined using the dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay. Cell viability was assessed via the MTS assay to determine the concentrations of compounds appropriate for use in subsequent experiments. Data from the enzyme-linked immunosorbent assay (ELISA) showed that both coelonin (10 and 20 μg/ml) and militarine (5 and 10 μg/ml) mitigated PM2.5-induced inflammation by reducing the generation of inflammatory factors, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Quantitative real-time PCR (qRT-PCR) analysis revealed a remarkable decrease in IL-6, TNF-α, cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β) mRNA levels in the coelonin and militarine-pretreated groups. In Western blot analysis, expression of inhibitor of NF-κB (IκBα) protein in the coelonin and militarine pretreatment groups was significantly increased compared with the PM2.5 (only) treatment group (P < 0.05), concomitant with a significant decrease in phospho-IκB kinase β/IκB kinase β (p-IKKβ/IKKβ), phospho-nuclear factor of kappa B p65/nuclear factor of kappa B p65 (p–NF–κBp65/NF-κBp65) and COX-2 proteins (P < 0.05). Both coelonin and militarine inhibited migration and inflammation by suppressing PM2.5-induced IKK phosphorylation, and followed by IκBα protein degradation and NF-κB activation. Our collective data strongly supported the utility of coelonin and militarine as novel sources for development of treatments for PM2.5-induced lung diseases.

Inhibition of ACE2 Expression by Ascorbic Acid Alone and its Combinations with Other Natural Compounds.

Background:Angiotensin-converting enzyme II or ACE2 is an integral membrane protein present on many types of cells, including vascular endothelial cells and lung alveolar epithelial cells. This receptor serves as the entry point for SARS-coronaviruses (SARS-CoVs), including a novel coronavirus 2019-nCoV. Limited availability of these receptors can thwart cellular entry of this virus.Methods:We tested the effects of ascorbic acid (vitamin C) on cellular expression of ACE2 at the protein and RNA levels in human small alveolar epithelial cells and microvascular endothelial cells. In addition, we investigated whether combinations of ascorbic acid with other natural compounds can affect ACE2 expression.Results:The results show that ascorbic acid itself has moderate but consistent lowering effects on ACE2 expression at the cellular, protein, and RNA levels. Some natural compounds were effective in lowering ACE2 cellular expression, with the highest inhibitory effects observed for baicalin (75%) and theaflavin (50%). Significantly, combinations of these and other test compounds with ascorbic acid further decreased ACE2 expression. The highest impact of ascorbate on ACE2 expression was noted when combined with theaflavin (decrease from 50% to 87%), zinc (decrease from 22% to 62%), and with 10-undecenoic acid (from 18% to 53%). Ascorbic acid showed moderate additional benefits in decreasing ACE2 expression when combined with N-acetylcysteine and baicalin.Conclusion:Our study provides valuable experimental confirmation of the efficacy of micronutrients in controlling ACE2 expression—the coronavirus cellular “entry” point. It further validates the importance of nutrient interactions in various aspects of cellular metabolism and in considering potential therapeutic applications of nutrient-based approaches. The study shows that ascorbic acid and its combination with some natural compounds could be included in developing preventive and therapeutic approaches toward the current pandemic.

Salidroside Suppresses the Proliferation and Migration of Human Lung Cancer Cells through AMPK-Dependent NLRP3 Inflammasome Regulation.

Inflammatory reactions mediated by the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome contributes to non-small-cell lung cancer (NSCLC) progression, particularly in patients with bacterial infections. Salidroside (SAL) has recently been shown to suppress lipopolysaccharide- (LPS-) induced NSCLC proliferation and migration, but its mechanism of action remains unclear. It has been shown that SAL improves metabolic inflammation in diabetic rodents through AMP-activated protein kinase- (AMPK-) dependent inhibition of the NLRP3 inflammasome. However, whether the NLRP3 inflammasome is regulated by SAL in NSCLC cells and how its underlying mechanism(s) can be determined require clarification. In this study, human lung alveolar basal carcinoma epithelial (A549) cells were treated with LPS, and the effects of SAL on cell proliferation, migration, AMPK activity, reactive oxygen species (ROS) production, and NLRP3 inflammasome activation were investigated. We found that LPS induction increases the proliferation and migration of A549 cells which was suppressed by SAL. Moreover, SAL protected A549 cells against LPS-induced AMPK inhibition, ROS production, and NLRP3 inflammasome activation. Blocking AMPK using Compound C almost completely suppressed the beneficial effects of SAL. In summary, these results indicate that SAL suppresses the proliferation and migration of human lung cancer cells through AMPK-dependent NLRP3 inflammasome regulation.

Evaluation of potential anti-COVID-19 therapies

Evaluation of potential anti-COVID-19 therapies

Immortalisation of primary human alveolar epithelial lung cells using a non-viral vector to study respiratory bioreactivity in vitro

To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology.

Biokinetic modeling of nanoparticle interactions with lung alveolar epithelial cells: uptake, intracellular processing, and egress.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.