Corrigendum: Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation.

[This corrects the article DOI: 10.3389/fcell.2023.1165581.].

Cigarette Smoke Extract Induces MUC5AC Expression Through the ROS/ IP3R/Ca2+ Pathway in Calu-3 Cells.

Background: Chronic obstructive pulmonary disease (COPD) is caused by exposure to noxious external particles, air pollution, and the inhalation of cigarette smoke. Airway mucus hypersecretion particularly mucin5AC (MUC5AC), is a crucial pathological feature of COPD and is associated with its initiation and progression. In this study, we aimed to investigate the effects of cigarette smoke extract (CSE) on MUC5AC expression, particularly the mechanisms by which reactive oxygen species (ROS) induce MUC5AC expression. Methods: The effects of CSE on the expression of MUC5AC and mucin5B (MUC5B) were investigated in vitro in Calu-3 cells. MUC5AC and MUC5B expression levels were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Total cellular levels of ROS and Ca2+ were determined using DCFH-DA and Fluo-4 AM. Subsequently, the expression levels of IP3R, IRE1α, p-IRE1α and XBP1s were measured by Western blotting. Gene silencing was achieved by using small-interfering RNAs. Results: Our findings revealed that exposure to CSE increased MUC5AC levels and upregulated ROS, IP3R/Ca2+ and unfolded protein response (UPR)-associated factors. In addition, knockdown of IP3R using siRNA decreased CSE-induced Ca2+ production, UPR-associated factors, and MUC5AC expression. Furthermore, 10 mM N-acetyl-l-cysteine (NAC) treatment suppressed the effects of CSE, including ROS generation, IP3R/ Ca2+, UPR activation, and MUC5AC overexpression. Conclusion: Our results suggest that ROS regulates CSE-induced UPR and MUC5AC overexpression through IP3R/ Ca2+ signaling. Additionally, we identified NAC as a promising therapeutic agent for mitigating CSE-induced MUC5AC overexpression.

Protective effect of the total alkaloid extract from Bulbus Fritillariae Pallidiflorae on cigarette smoke-induced Beas-2B cell injury model and transcriptomic analysis.

Background: Bulbus Fritillariae Pallidiflorae (BFP) is a traditional Chinese medicine that has long been used to treat lung diseases, but the active components and mechanism are still unclear. Objective: This study aimed to investigate the effect and mechanism of the total alkaloid extract from BFP (BFP-TA) on cigarette smoke extract (CSE)-induced Beas-2B cells injury. Design: The Beas-2B cells injury model was induced by 2% CSE, then the effect of BFP-TA on the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) was detected according to the instructions of the T-AOC assay kit, the SOD detection kit and the MDA detection kit, and the production of ROS was detected by fluorescence microscopy. The effect of BFP-TA on Beas-2B cells apoptosis was detected by flow cytometry, and the effect of BFP-TA on related protein expression was detected by western blot. Subsequently, the effect of BFP-TA on differentially expressed genes (DEGs) in CSE-induced Beas-2B cells was studied by transcriptomic sequencing, and the expression of DEGs was verified by quantitative real-time polymerase chain reaction (qPCR). Results: The results showed that BFP-TA could attenuate CSE-induced oxidative damage in Beas-2B cells by elevating T-AOC and SOD levels while inhibiting ROS and MDA levels, and the mechanism was potentially related to the SIRT1/Nrf2/Keap1 signaling pathway. Furthermore, BFP-TA could inhibit CSE-induced apoptosis by inhibiting the protein expression of Bax, MST1 and FOXO3a, and exert anti-inflammatory effect by inhibiting the activation of MAPK signaling pathway. Subsequently, transcriptome analysis and qPCR validation showed that BFP-TA could alleviate inflammation, oxidative stress, apoptosis and lipid metabolism disorders by regulating the expression of DEGs in PPAR and PI3K-Akt signaling pathways, thereby exerting a protective effect against CSE-induced Beas-2B cell injury. Conclusion: This study is the first to demonstrate that BFP-TA could exert a protective effect on CSE-induced Beas-2B cell injury by exerting anti-inflammatory, antioxidant, anti-apoptotic and regulate lipid metabolism disorders.

Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

MiRNA-210 is involved in cigarette smoke extract-induced apoptosis of MLE-12 via the Shh signaling pathway.

INTRODUCTION: The aim of the study is the regulatory effect of MicroRNA-210 (MiR-210) on cigarette smoke extract (CSE)-induced mouse lung epithelial type II cells (MLE-12) apoptosis and determine whether the MiR-210 is involved in cigarette smoke extract-induced apoptosis of MLE-12 via Shh signaling pathway. METHODS: Expression of MiR-210 in CSE-induced MLE-12 was assessed by qRT-PCR. The emphysema mouse model and MiR-210 knockdown mice were each established by inhaling cigarette smoke or intratracheal lentiviral vector instillation. The Sonic hedgehog (Shh), Ptch1, Gli1, B-cell lymphoma-2 (Bcl-2), and Caspase 3 protein expressions were detected by Western blotting. mRNA expressions of MiR-210, Shh, Ptch1, and Gli1 were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Apoptotic ratios in mice and CSE-induced HPVEC were assessed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays and flow cytometry. RESULTS: Our results showed that MiR-210 mRNA levels were significantly down-regulated in the CSE-induced MLE 12. MLE 12 apoptosis with down-regulated Shh, Ptch1, Gli1, and Bcl-2 expression, increased Caspase 3 expression in the emphysema mouse model and CSE-induced MLE 12. Knockdown MiR-210 can facilitate cell apoptosis and emphysema via the Shh signaling pathway in mice. In vitro, MiR-210 can attenuate the apoptosis of CSE-exposed MLE 12. Moreover, MiR-210 regulated the Shh pathway and promoted its expression. CONCLUSIONS: MiRNA-210 is involved in cigarette smoke extract-induced apoptosis of MLE-12 via the Shh signaling pathway. The present study reveals that MiRNA-210 may be a key regulator of cellular apoptosis and could be explored as a potential therapeutic target in the future.

The role and mechanism of macrophage autophagy in the experimental model of chronic obstructive pulmonary disease.

INTRODUCTION: Macrophages play an important role in chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) impairs autophagy in alveolar macrophages from COPD patients, and autophagic impairment leads to reduced clearance of protein aggregates, dysfunctional mitochondria, and defective bacterial delivery to lysosomes. However, the exact function of lung macrophage autophagy in the pathogenesis of CS-induced COPD remains largely unknown. METHODS: Western blot detected the expression of autophagy-related proteins induced by CSE. The model of COPD mice was established by CS exposure combined with CSE intraperitoneal injection. Double immunofluorescence was used to measure the CD206+LC3B+ cells. The morphological changes and effects on lung function were observed. Masson staining detected the changes in collagen fibers in lung tissue. The expression levels of E-cadherinb and N-cadherinb were detected by immunohistochemistry. Western blot detected the expression of ATP6V1E1 in lung tissue. RESULTS: At 24 hours of exposure to CSE, the expression levels of LC3B (microtubule-associated protein 1A/1B-light chain 3B) and P62 (nucleoporin 62) were highest at 1% CSE and AGT5 (nucleoporin 62) at 2.5% CSE; at 48 hours, the expression levels of LC3B, P62 and AGT5 were highest at 2.5% CSE, and as the intervention time increased.CD206+LC3B+ cells were significantly higher in the COPD group. Enhanced macrophage autophagy may promote emphysema formation and aggravate lung function damage. The expression of E-cadherinb in lung tissue of the COPD group was decreased, and N-cadherinb expression was increased; the expression of E-cadherinb was increased, and N-cadherinb expression was decreased in ATG5myeΔ COPD mice. The expression of ATP6V1E1 in the lung tissue was increased in the COPD group; ATP6V1E1 expression was decreased in the lung tissues of ATG5myeΔ COPD mice. CONCLUSIONS: CSE enhanced macrophage autophagy, leads to increased lung function impairment and collagenous fiber in lung tissue, as well as promotes epithelial-mesenchymal transition, and eventually leads to small airway remodeling, which may be achieved through the ATG5/ATP6V1E1 pathway.

Tobacco toxins trigger bone marrow mesenchymal stem cells aging by inhibiting mitophagy.

Smoking disrupts bone homeostasis and serves as an independent risk factor for the development and progression of osteoporosis. Tobacco toxins inhibit the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), promote BMSCs aging and exhaustion, but the specific mechanisms are not yet fully understood. Herein, we successfully established a smoking-related osteoporosis (SROP) model in rats and mice through intraperitoneal injection of cigarette smoke extract (CSE), which significantly reduced bone density and induced aging and inhibited osteogenic differentiation of BMSCs both in vivo and in vitro. Bioinformatics analysis and in vitro experiments confirmed that CSE disrupts mitochondrial homeostasis through oxidative stress and inhibition of mitophagy. Furthermore, we discovered that CSE induced BMSCs aging by upregulating phosphorylated AKT, which in turn inhibited the expression of FOXO3a and the Pink1/Parkin pathway, leading to the suppression of mitophagy and the accumulation of damaged mitochondria. MitoQ, a mitochondrial-targeted antioxidant and mitophagy agonist, was effective in reducing CSE-induced mitochondrial oxidative stress, promoting mitophagy, significantly downregulating the expression of aging markers in BMSCs, restoring osteogenic differentiation, and alleviating bone loss and autophagy levels in CSE-exposed mice. In summary, our results suggest that BMSCs aging caused by the inhibition of mitophagy through the AKT/FOXO3a/Pink1/Parkin axis is a key mechanism in smoking-related osteoporosis.

Codonopsis Radix Inhibits the Inflammatory Response and Oxidative Stress in Chronic Obstructive Pulmonary Disease Mice through Regulation of the Nrf2/NF-κB Signaling Pathway.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a nonspecific chronic inflammatory lung disease with no known cure. Codonopsis Radix (CR) has been shown to exhibit anti-inflammatory and antioxidant effects. Therefore, this study aimed to investigate the potential anti-inflammatory effects of different CR varieties on COPD mice. METHODS: Sixty male-specified pathogen-free grade C57BL/6J mice were randomly divided into 6 groups, 10 mice in each group. The COPD mice model was induced by cigarette smoke extract combined with lipopolysaccharide, and the mice in each group were given corresponding drugs. Lung function was assessed in all mice. Lung tissues were stained with hematoxylin-eosin, Masson, and periodic acid-Schiff stains, and serum levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α were detected using an ELISA. Further, serum and lung tissue levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by colorimetric assay. Network pharmacology and molecular docking were used to predict signaling pathways, which were validated by Western blot analysis. RESULTS: Compared with the COPD group, the mice in each dosing group of CR exhibited significant reductions in serum IL-8 and TNF-α levels, serum and lung tissue MDA levels, and pathological lung tissue damage, alongside elevations in lung function and SOD levels (p < 0.01). Western blot analysis also indicated significant downregulation of p-p65/p65 and p-IκB-α/IκB-α protein expression, alongside significant upregulation of Nrf2 protein expression in the lung tissues of mice treated with CR (p < 0.01). CONCLUSION: In summary, CR effectively enhances lung function, minimizes lung tissue damage, and inhibits inflammation and oxidative stress in mice with COPD. Additionally, these findings suggest that inhibition of the Nrf2/NF-κB axis may be a key mechanism of action of CR in the alleviation of COPD.

Cigarette smoke extract exacerbates progression of osteoarthritic-like changes in cartilage explant cultures.

Established risk factors for osteoarthritis (OA) include obesity, joint injury, age, race, and genetics. However, the relationship between cigarette smoking and OA has yet to be established. In the present study, we have employed the use of cigarette smoke extract (CSE), the water-soluble vapor phase of cigarette smoke, with porcine cartilage explants to investigate the effects of cigarette smoking on cartilage catabolism at the tissue level. Articular cartilage explants were first exposed to 2.5%, 5%, and 10% CSE to assess its effects on cartilage homeostasis. Following, the effects of CSE on OA-like inflammation was observed by culturing explants with a combined treatment of IL-1ß and TNF-α and 10% CSE (CSE + OA). Cartilage explants were assessed for changes in viability, biochemical composition, extracellular matrix (ECM) integrity, and equilibrium mechanical properties (aggregate modulus and hydraulic permeability). CSE alone leads to both a time- and dose-dependent decrease in chondrocyte viability but does not significantly affect sGAG content, percent sGAG loss, or the ECM integrity of cartilage explants. When IL-1ß and TNF-α were combined with 10% CSE, this led to a synergistic effect with more significant losses in viability, significantly more sGAG loss, and significantly higher production of ROS than OA-like inflammation only. Cartilage explant equilibrium mechanical properties were unaffected. Within the timeframe of this study, CSE alone does not cause OA but when combined with OA-like inflammation leads to worsened articular cartilage degeneration as measured by chondrocyte viability, sGAG loss, proteoglycan staining, and ROS production.

Integrated mRNA- and miRNA-sequencing analyses unveil the underlying mechanism of tobacco pollutant-induced developmental toxicity in zebrafish embryos.

Tobacco pollutants are prevalent in the environment, leading to inadvertent exposure of pregnant females. Studies of these pollutants' toxic effects on embryonic development have not fully elucidated the potential underlying mechanisms. Therefore, in this study, we aimed to investigate the developmental toxicity induced by cigarette smoke extract (CSE) at concentrations of 0.25, 1, and 2.5% using a zebrafish embryo toxicity test and integrated transcriptomic analysis of microRNA (miRNA) and messenger RNA (mRNA). The findings revealed that CSE caused developmental toxicity, including increased mortality and decreased incubation rate, in a dose-dependent manner. Moreover, CSE induced malformations and apoptosis, specifically in the head and heart of zebrafish larvae. We used mRNA and miRNA sequencing analyses to compare changes in the expression of genes and miRNAs in zebrafish larvae. The bioinformatics analysis indicates that the mechanism underlying CSE-induced developmental toxicity was associated with compromised genetic material damage repair, deregulated apoptosis, and disturbed lipid metabolism. The enrichment analysis and RT-qPCR show that the ctsba gene plays a crucial function in embryo developmental apoptosis, and the fads2 gene mainly regulates lipid metabolic toxicity. The results of this study improve the understanding of CSE-induced developmental toxicity in zebrafish embryos and contribute insights into the formulation of novel preventive strategies against tobacco pollutants during early embryonic development.

Anti-inflammatory actions of aspirin-triggered resolvin D1 (AT-RvD1) in bronchial epithelial cells stimulated by cigarette smoke extract.

Smoking causes several diseases such as chronic obstructive pulmonary disease (COPD). Aspirin-triggered-resolvin D1 (AT-RvD1) is a lipid mediator produced during the resolution of inflammation and demonstrates anti-inflammatory and pro-resolution effects in several inflammatory experimental models including in the airways. Here we evaluated the role of AT-RvD1 (100 nM) in bronchial epithelial cells (BEAS-2B) stimulated by cigarette smoke extract (CSE; 1%; 1 cigarette) for 24 h. CSE induced the productions of IL-1ß, TNF-α, IL-10, IL-4 and IFN-γ as well as the activations of NF-κB and STAT3 and the expression of ALX/FPR2 receptor. AT-RvD1 reduced the IL-1ß and TNF-α production and increased the production of IFN-γ. These effects were reversed BOC2, an antagonist of ALX/FPR2 receptor for AT-RvD1. The production of IL-4 and IL-10 were not altered by AT-RvD1. In addition, AT-RvD1 reduced the phosphorylation of NF-κB and STAT3 when compared to CSE-stimulated BEAS-2B cells. No alteration of ALX/FPR2 expression was observed by AT-RvD1 when compared to CSE group. In the human monocytic leukemia cell line, the relative number of copies of IL-1ß and IL-4 was significantly higher in CSE + AT-RvD1 group compared CSE group, however, the expression of M1 cytokine was more pronounced than M2 profile. AT-RvD1 could be an important target for the reduction of inflammation in the airways associated with smoking.

Rice Bran Oil Improves Emphysema in Cigarette Smoke Extract-Induced Mice through Anti-Inflammatory and Antioxidative Effects.

Lung inflammation and alveolar enlargement are the major pathological conditions of chronic obstructive pulmonary disease (COPD) patients. Rice bran oil (RBO), a natural anti-inflammatory and antioxidative agent, has been used for therapeutic purposes in several inflammatory diseases. This study aimed to investigate the anti-inflammatory and antioxidative effect of RBO on a cigarette smoke extract (CSE)-induced emphysema model in mice. The results indicated that CSE significantly induced airspace enlargement in mouse lung. Increased inflammatory cells, macrophage, and TNF-alpha levels in bronchoalveolar lavage fluid (BALF) were noticed in CSE-treated mice. RBO (low and high dose)-supplemented mice showed decreased total BALF inflammatory cell, macrophage, and neutrophil numbers and TNF-alpha levels (p < 0.05). Additionally, the administration of RBO decreased the mean linear alveolar intercept (MLI) in the CSE-treated group. Additionally, RBO treatment significantly increased the total antioxidant capacity in both mouse BALF and serum. However, RBO did not have an effect on the malondialdehyde (MDA) level. These findings suggested that RBO treatment ameliorates lung inflammation in a CSE-induced emphysema mice model through anti-inflammatory and antioxidant pathways. Therefore, the supplementation of RBO could be a new potential therapeutic to relieve the severity of COPD.

Circular RNA_0025843 Alleviated Cigarette Smoke Extract Induced Bronchoalveolar Epithelial Cells Ferroptosis.

Purpose: Circular RNA (circRNA) plays an important role in various biological processes. However, their functions in cigarette smoke extract (CSE) induced human normal lung epithelial cells (BEAS-2B) injury remain vague. The study aimed to explore circRNA expression profiles and reveal their potential roles in CSE-treated BEAS-2B cells. Methods: 5% CSE exposure for 24 hours were used to build the BEAS-2B cells ferroptosis model. Differentially expressed circRNAs (DECs) were identified by next-generation RNA sequencing. Six randomly selected DECs were validated via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis were conducted to clarify the potential functions of the DECs. Furthermore, the role of hsa_circ_0025843 in CSE-related BEAS-2B cells ferroptosis was confirmed. Results: 5% CSE exposure induced BEAS-2B cells ferroptosis. Fifty-one up-regulated cirRNAs and 80 down-regulated circRNAs were revealed in CSE-treated BEAS-2B cells. Hsa_circ_0003461, hsa_circ_0007548, hsa_circ_0025843, hsa_circ_0068896, hsa_circ_0005832, and hsa_circ_0053378 were selected randomly to validate the reliability of next-generation RNA sequencing by qRT-PCR. After KEGG pathway analysis, DECs were found to participate in the process of EGFR tyrosine kinase inhibitor resistance and glycerophospholipid metabolism. The knockdown of hsa_circ_0025843 significantly alleviated CSE-induced BEAS-2B cells ferroptosis. Conclusion: The study indicated the circRNA expression profiles in CSE-treated BEAS-2B cells. Hsa_circ_0025843 alleviated CSE induced BEAS-2B cells ferroptosis, which might be a potential therapeutic target of CSE related lung injury.

Oro-Respiratory Dysbiosis and Its Modulatory Effect on Lung Mucosal Toxicity during Exposure or Co-Exposure to Carbon Nanotubes and Cigarette Smoke.

The oro-respiratory microbiome is impacted by inhalable exposures such as smoking and has been associated with respiratory health conditions. However, the effect of emerging toxicants, particularly engineered nanoparticles, alone or in co-exposure with smoking, is poorly understood. Here, we investigated the impact of sub-chronic exposure to carbon nanotube (CNT) particles, cigarette smoke extract (CSE), and their combination. The oral, nasal, and lung microbiomes were characterized using 16S rRNA-based metagenomics. The exposures caused the following shifts in lung microbiota: CNT led to a change from Proteobacteria and Bacteroidetes to Firmicutes and Tenericutes; CSE caused a shift from Proteobacteria to Bacteroidetes; and co-exposure (CNT+CSE) had a mixed effect, maintaining higher numbers of Bacteroidetes (due to the CNT effect) and Tenericutes (due to the CSE effect) compared to the control group. Oral microbiome analysis revealed an abundance of the following genera: Acinetobacter (CNT), Staphylococcus, Aggregatibacter, Allobaculum, and Streptococcus (CSE), and Alkalibacterium (CNT+CSE). These proinflammatory microbial shifts correlated with changes in the relative expression of lung mucosal homeostasis/defense proteins, viz., aquaporin 1 (AQP-1), surfactant protein A (SP-A), mucin 5b (MUC5B), and IgA. Microbiota depletion reversed these perturbations, albeit to a varying extent, confirming the modulatory role of oro-respiratory dysbiosis in lung mucosal toxicity. This is the first demonstration of specific oro-respiratory microbiome constituents as potential modifiers of toxicant effects in exposed lungs.

PU.1 alleviates the inhibitory effects of cigarette smoke on endothelial progenitor cell function and lung-homing through Wnt/ß-catenin and CXCL12/CXCR4 pathways.

INTRODUCTION: Endothelial progenitor cells (EPCs) dysfunction is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The transcription factor PU.1 is essential for the maintenance of stem/progenitor cell homeostasis. However, the role of PU.1 in COPD and its effects on EPC function and lung-homing, remain unclear. This study aimed to explore the protective activity of PU.1 and the underlying mechanisms in a cigarette smoke extract (CSE)-induced emphysema mouse model. METHODS: C57BL/6 mice were treated with CSE to establish a murine emphysema model and injected with overexpressed PU.1 or negative control adeno-associated virus. Morphometry of lung slides, lung function, and apoptosis of lung tissues were evaluated. Immunofluorescence co-localization was used to analyze EPCs homing into the lung. Flow cytometry was performed to detect EPC count in lung tissues and bone marrow (BM). The angiogenic ability of BM-derived EPCs cultured in vitro was examined by tube formation assay. We determined the expression levels of PU.1, ß-catenin, C-X-C motif ligand 12 (CXCL12), C-X-C motif receptor 4 (CXCR4), stem cell antigen-1 (Sca-1), and stemness genes. RESULTS: CSE exposure significantly reduced the expression of PU.1 in mouse lung tissues, BM, and BM-derived EPCs. PU.1 overexpression attenuated CSE-induced emphysematous changes, lung function decline, and apoptosis. In emphysematous mice, PU.1 overexpression markedly reversed the decreased proportion of EPCs in BM and promoted the lung-homing of EPCs. The impaired angiogenic ability of BM-derived EPCs induced by CSE could be restored by the overexpression of PU.1. In addition, PU.1 upregulation evidently reversed the decreased expression of ß-catenin, CXCL12, CXCR4, Scal-1, and stemness genes in mouse lung tissues, BM, and BM-derived EPCs after CSE exposure. CONCLUSIONS: PU.1 alleviates the inhibitory effects of CSE on EPC function and lung-homing via activating the canonical Wnt/ß-catenin pathway and CXCL12/CXCR4 axis. While further research is needed, our research may indicate a potential therapeutic target for COPD patients.

Mechanism of cytotoxicity induced by the cigarette smoke extract (CSE) of heated tobacco products in vascular smooth muscle cells: A comparative study of the cytotoxic effects of CSE and the ferroptosis inducer, erastin.

Heated tobacco products (HTPs) are marketed worldwide as less harmful alternatives to combustible cigarettes; however, their cytotoxic mechanisms in vascular smooth muscle cells are poorly understood. Ferroptosis is defined as iron-dependent cell death caused by the accumulation of lipid peroxidation products. In this study, the cytotoxic effects of nicotine- and tar-free cigarette smoke extracts (CSE) derived from three types of HTPs and the ferroptosis inducer, erastin, on vascular smooth muscle A7r5 cells were compared. Cigarette smoke from all HTPs was generated according to the following puffing regime: 55 mL, puff volume; 30 s, puff interval; 2 s, puff duration; bell-shaped, puff profile; and no blocking of the ventilation holes. Erastin and CSE decreased mitochondrial metabolic activity and increased lactate dehydrogenase leakage. The cytotoxic effects of erastin were almost completely inhibited by the radical-trapping antioxidant, UAMC-3203; iron chelator, deferoxamine mesylate (DFO); 12/15-lipoxygenase (12/15-LOX) inhibitor, baicalein; and selective 15-LOX inhibitor, ML351. In contrast, CSE-induced cell damage was partially attenuated by UAMC-3203, baicalein, and ML351 but not by DFO. These results suggest that erastin induces ferroptosis via 15-LOX-mediated iron-dependent lipid peroxidation, whereas CSE causes iron-independent cell damage via 15-LOX-mediated lipid peroxidation-dependent and -independent mechanisms.

ß-escin activates ALDH and prevents cigarette smoke-induced cell death.

BACKGROUND: The tobacco use is one of the biggest public health threats worldwide. Cigarette smoke contains over 7000 chemicals among other aldehydes, regarded as priority toxicants. ß-escin (a mixture of triterpenoid saponins extracted from the Aesculus hippocastanum. L) is a potent activator of aldehyde dehydrogenase (ALDH) - an enzyme catalyzing oxidation of aldehydes to non-toxic carboxylic acids. PURPOSE: The aim of this study was to evaluate the effect of ß-escin on ALDH activity, ALDH isoforms mRNA expression and cytotoxicity in nasal epithelial cells exposed to cigarette smoke extract (CSE). METHODS: Nasal epithelial cells from healthy non-smokers were treated with ß-escin (1 µM) and exposed to 5% CSE. After 6- or 24-hours of stimulation cell viability, DNA damage, ALDH activity and mRNA expression of ALDH isoforms were examined. RESULTS: 24 h ß-escin stimulation revised CSE induced cytotoxicity and DNA damage. Cells cultured with ß-escin or exposed to CSE responded with strong increase in ALDH activity. This effect was more pronounced in cultures treated with combination of ß-escin and CSE. The strongest stimulatory effect on ALDH isoform mRNA expression was observed in cells cultured simultaneously with ß-escin and CSE: at 6 h for ALDH1A1 and ALDH3A1, and at 24 h for ALDH1A3, ALDH3A2, ALDH3B1, and ALDH18A1. Combined ß-escin and CSE treatment prevented the CSE-induced inhibition of ALDH2 expression at 24 h. CONCLUSIONS: ß-escin is an effective ALDH stimulatory and cytoprotective agent and might be useful in the prevention or supportive treatment of tobacco smoke-related diseases.

CSE triggers ferroptosis via SIRT4-mediated GNPAT deacetylation in the pathogenesis of COPD.

BACKGROUND: It is now understood that ferroptosis plays a significant role in the progression of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke extract (CSE). However, the mechanisms underlying this relationship remain largely unclear. METHODS: In this study, we established a COPD mouse model through exposure to cigarette smoke particulates, followed by H&E staining, analysis of bronchoalveolar lavage fluid, and immunohistochemistry assay. A549 cells were exposed to increasing concentrations of CSE, with the addition of the ferroptosis activator erastin or the inhibitor Fer-1. Cell viability, LDH (lactate dehydrogenase) release, inflammatory cytokines, total ROS (reactive oxygen species), and lipid ROS were measured using the corresponding assay kits. The acetylation level of GNPAT was determined through immunoprecipitation. We assessed the expression levels of molecules involved in plasmalogen biosynthesis (FAR1, AGPS, and GNPAT), GPX4, and SIRT4 using quantitative real-time PCR, western blot analysis, and immunofluorescence staining. RESULTS: CSE-induced lung tissue damage was initially observed, accompanied by oxidative stress, ferroptosis, and increased plasmalogen biosynthesis molecules (FAR1, AGPS, and GNPAT). CSE also induced ferroptosis in A549 cells, resulting in reduced cell viability, GSH, and GPX4 levels, along with increased LDH, ROS, MDA (malondialdehyde) levels, oxidized lipids, and elevated FAR1, AGPS, and GNPAT expression. Knockdown of GNPAT mitigated CSE-induced ferroptosis. Furthermore, we found that CSE regulated the acetylation and protein levels of GNPAT by modulating SIRT4 expression. Importantly, the overexpression of GNPAT countered the inhibitory effects of SIRT4 on ferroptosis. CONCLUSIONS: Our study revealed GNPAT could be deacetylated by SIRT4, providing novel insights into the mechanisms underlying the relationship between CSE-induced ferroptosis and COPD.

Lung Fibroblasts from Chronic Obstructive Pulmonary Disease Subjects Have a Deficient Gene Expression Response to Cigarette Smoke Extract Compared to Healthy.

Background and aim: Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (COPD) but more mechanistic studies are needed. Cigarette smoke extract (CSE) can elicit a strong response in many COPD-related cell types, but no studies have been performed in lung fibroblasts. Therefore, we aimed to investigate the effect of CSE on gene expression in lung fibroblasts from healthy and COPD subjects. Patients and methods: Primary lung fibroblasts, derived from six healthy and six COPD subjects (all current or ex-smokers), were either unstimulated (baseline) or stimulated with 30% CSE for 4 h prior to RNA isolation. The mRNA expression levels were measured using the NanoString nCounter Human Fibrosis V2 panel (760 genes). Pathway enrichment was assessed for unique gene ontology terms of healthy and COPD. Results: At baseline, a difference in the expression of 17 genes was found in healthy and COPD subjects. Differential expression of genes after CSE stimulation resulted in significantly less changes in COPD lung fibroblasts (70 genes) than in healthy (207 genes), with 51 genes changed in both. COPD maintained low NOTCH signaling throughout and upregulated JUN >80%, indicating an increase in apoptosis. Healthy downregulated the Mitogen-activated protein kinase (MAPK) signaling cascade, including a ≥50% reduction in FGF2, CRK, TGFBR1 and MEF2A. Healthy also downregulated KAT6A and genes related to cell proliferation, all together indicating possible cell senescence signaling. Conclusion: Overall, COPD lung fibroblasts responded to CSE stimulation with a very different and deficient expression profile compared to healthy. Highlighting that stimulated healthy cells are not an appropriate substitute for COPD cells which is important when investigating the mechanisms of COPD.

The effect of cigarette smoke extract exposure on the size and sexual behaviour of Drosophila melanogaster.

Drosophila melanogaster is a widely used animal model in human diseases and to date it has not been applied to the study of the impact of tobacco use on human sexual function. Hence, this report examines the effects of different concentrations of cigarette smoke extract (CSE) exposure on the size and sexual behavior of D. melanogaster. Wild-type flies were held in vials containing CSE-infused culture media at concentrations of 10%, 25%, and 50% for three days, and their offspring were reared under the same conditions before measuring their body size and mating behavior. CSE exposure during development reduced the tibia length and body mass of emerging adult flies and prolonged the time required for successful courtship copulation success, while courtship behaviors (wing extension, tapping, abdomen bending, attempted copulation) remained largely unchanged. Our findings indicate that CSE exposure negatively affects the development of flies and their subsequent reproductive success. Future experiments should investigate the CSE effect on male female fertility.