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.

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.

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.

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.

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.

Comparative study of the effects of cigarette smoke versus next-generation tobacco and nicotine product extracts on inflammatory biomarkers of human monocytes.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.

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.

Nicotine- and tar-removed cigarette smoke extract modulates the antigen presentation function of mouse bone marrow-derived dendritic cells.

Dendritic cells (DCs) take up antigens derived from pathogens such as bacteria and viruses, and from tumor cells and induce the activation of antigen-specific T cells through major histocompatibility complex (MHC)-mediated antigen presentation. Mainstream cigarette smoke extract (CSE) has various effects, and the effects of its major components, nicotine and tar, have been analyzed extensively. Recently, the physiological effects of nicotine- and tar-removed CSE (cCSE) have also been reported. However, the effects of cCSE on DC-mediated immune responses remain unknown. In this study, we found that cCSE enhanced lipopolysaccharide (LPS)-stimulated induction of the expression of MHC-I and MHC-II on the cell surface of mouse bone marrow-derived DCs (BMDCs). In contrast, cCSE suppressed the induction of CD86 induced by stimulation with curdlan and interferon-γ (IFN-γ). In addition, cCSE suppressed the production of IL-12, IL-23, and IL-10 by LPS and curdlan stimulation. In the presence of cCSE, LPS-stimulated BMDCs showed enhanced activation of CD4 and CD8 T cells and increased IL-2 production from T cells by antigen presentation in a mixed-leukocyte reaction assay. In contrast, cCSE did not affect the activation of T cells by curdlan- or IFN-γ-stimulated BMDCs, and curdlan-stimulated BMDCs suppressed IL-17 production from T cells and enhanced IFN-γ production. These results suggest that cCSE has different effects on the activation signals induced by LPS, curdlan, and IFN-γ in BMDCs and modulates the antigen presentation function of BMDCs.

Cigarette smoke extract-induced inflammatory response via inhibition of the TFEB-mediated autophagy in NR8383 cells.

Objective: Chronic pulmonary inflammation caused by long-term smoking is the core pathology of COPD. Alveolar macrophages (AMs) are involved in the pulmonary inflammation of COPD. The accumulation of damaged materials caused by impaired autophagy triggers inflammatory response in macrophages. As a key transcription regulator, transcription factor EB (TFEB) activates the transcription of target genes related autophagy and lysosome by binding to promoters, whereas it is unclarified for the relationship between inflammatory response induced by cigarette smoke extract (CSE) and TFEB-mediated autophagy. Thus, we investigated the role of TFEB-mediated autophagy in inflammatory response induced by CSE in NR8383 cells, and to explore its potential mechanism. Methods: Based on cell viability and autophagy, cells treated with 20% concentration of CSE for 24 h were selected for further studies. Cells were divided into control group, chloroquine (CQ, the autophagy inhibitor) group, CSE group, CSE + rapamycin (the autophagy inducer) group and CSE + fisetin (the TFEB inducer) group. The levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 in supernatant were detected by ELISA kits. The protein expressions were tested by western blot. The intensity of fluorescence of Lysosome-associated membrane protein 1 (LAMP1) and TFEB was detected by immunofluorescence. Lyso-Tracker Red staining was applied to detect the lysosome environment. Results: CSE inhibited the cell viability, increased the contents of TNF-α, IL-1ß, IL-6, the ratio of LC3II/I, and the level of P62 protein. Besides, CSE decreased the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining, as well as the ratio of nucleus/cytosol of TFEB protein. Activating autophagy with rapamycin alleviated CSE-induced inflammatory response. The activation of TFEB via fisetin alleviated CSE-induced autophagy impairment and lysosomal dysfunction, thus alleviated inflammatory response in NR8383 cells. Conclusion: CSE-induced inflammatory response in NR8383 cells, which may be related to the inhibition of TFEB-mediated autophagy.

Cyclophilin D Contributes to Airway Epithelial Mitochondrial Damage in Chronic Obstructive Pulmonary Disease.

INTRODUCTION: Airway epithelial mitochondrial injury is an important pathogenesis of chronic obstructive pulmonary disease (COPD). Cyclophilin D (CypD) is a component of mitochondrial permeability transition pore and related to mitochondrial damage. However, the role of CypD in airway epithelial mitochondrial injury and COPD pathogenesis remains unclear. METHODS: CypD expression in human airway epithelium was determined by immunohistochemistry, and mitochondrial structure of airway epithelial cell was observed under the transmission electron microscopy. The expression of CypD signaling pathway in cigarette smoke extract (CSE)-treated airway epithelial cells was measured by real-time PCR and Western-blot. CSE-induced damage of airway epithelial cell and mitochondria was further studied. RESULTS: Immunohistochemistry and transmission electron microscopy analysis revealed that CypD expression in airway epithelium was significantly increased associated with notable airway epithelial mitochondrial structure damage in the patients with COPD. The mRNA and protein expression of CypD was significantly increased in concentration- and time-dependent manners when airway epithelial cells were treated with CSE. CypD siRNA pretreatment significantly suppressed the increases of CypD and Bax expression, and reduced the decline of Bcl-2 expression in 7.5% CSE-treated airway epithelial cells. Furthermore, CypD silencing significantly attenuated mitochondrial damage and cell apoptosis, and increased cell viability when airway epithelial cells were stimulated with 7.5% CSE. CONCLUSION: These data suggest that CypD signaling pathway is involved in the pathogenesis of COPD and provide a potential therapeutic target for COPD.

Osthole, an ingredient from Cnidium monnieri, reduces the pyroptosis and apoptosis in bronchial epithelial cells.

Osthole is the prominent active ingredient isolated from Cnidium. The role of osthole in chronic obstructive pulmonary disease (COPD) was investigated herein. Bronchial epithelial 16HBE cells were exposed to cigarette smoke extract (CSE) to generate injury models. The concentration of CSE had an inverse correlation with cell viability. Osthole suppressed inflammation, oxidative stress, apoptosis, and pyroptosis in 16HBE cells, along with a decrease in RIPK2 level. RIPK2 overexpression reversed the effects of osthole on the abovementioned aspects. This study found that the osthole could reduce RIPK2 level, inhibit pyroptosis, and alleviate the damage in 16HBE cells under CSE stimulation.

Aurintricarboxylic acid mitigates cigarette smoke extract induced oxidative stress and pulmonary inflammation via inhibition of NF-Ò¡B/p65 signaling.

Cigarette smoke (CS) induced emphysema and chronic pulmonary inflammation are major comorbidities of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. CS exposure exacerbates pulmonary inflammation and compromises immunity to various infections. Aurintricarboxylic acid (ATA) is a polyanionic aromatic compound especially recognized for its anti-inflammatory, nucleic acid, and protein interaction inhibition properties. The study was designed to investigate the anti-inflammatory role of ATA against cigarette smoke extract (CSE) induced pulmonary inflammation. Nicotine concentration was quantified in CSE by UPLC/MS technique. In vitro, fluorescence microscopy, and flow cytometry was performed in CSE stimulated alveolar epithelial cells to determine the effect of ATA on oxidative stress-mediated cellular apoptosis. In vivo, pulmonary inflammation was induced in male Wistar rats via a modified non-invasive intratracheal instillation of cigarette smoke extract (100 µl/animal) twice a week for 8 weeks and post-treated with ATA (10 mg/kg) intraperitoneally for 15 days. Lung homogenates were assessed for MDA and GSH. Lung tissues were subjected to western blotting and histopathological analysis. As result, ATA reduced CSE-induced chromatin condensation, fragmentation, cellular apoptosis in alveolar epithelial cells, and apoptotic biomarkers expression including BAX and Caspase-3 in the lungs. ATA reduced inflammation by normalizing redox balance reflected by MDA/GSH levels. ATA obviated airspace enlargement, fiber deposition, and immune cell infiltration. Reduced inflammation was accompanied by inhibition of inflammatory biomarkers TNF-α, TNFR1, TWEAK, and NF-Ò¡B/p65 activation and nuclear translocation. ATA efficaciously diminished the oxidative stress and pulmonary inflammation associated with lung pathogenesis through TNF-α/TNFR1/NF-Ò¡B/p65 signaling pathway. HIGHLIGHTSATA treatment attenuates CSE-stimulated chromatin condensation, fragmentation, and cellular apoptosis in alveolar epithelial cells.ATA treatment inhibits CSE stimulated activation and nuclear translocation of NF-Ò¡B/p65.ATA treatment diminishes CSE-induced oxidant injury, apoptosis, and emphysema-like phenotypic changes in the lungs.ATA inhibits lung inflammation via suppression of the NF-Ò¡B/p65 signaling pathway.

Crosstalk between gut microbiota and lung inflammation in murine toxicity models of respiratory exposure or co-exposure to carbon nanotube particles and cigarette smoke extract.

Carbon nanotubes (CNTs) are emerging environmental and occupational toxicants known to induce lung immunotoxicity. While the underlying mechanisms are evolving, it is yet unknown whether inhaled CNTs would cause abnormalities in gut microbiota (dysbiosis), and if such microbiota alteration plays a role in the modulation of CNT-induced lung immunotoxicity. It is also unknown whether co-exposure to tobacco smoke will modulate CNT effects. We compared the effects of lung exposure to multi-wall CNT, cigarette smoke extract (CSE), and their combination (CNT + CSE) in a 4-week chronic toxicity mouse model. The exposures induced differential perturbations in gut microbiome as evidenced by altered microbial α- and ß- diversity, indicating a lung-to-gut communication. The gut dysbiosis due to CNTs, unlike CSE, was characterized by an increase in Firmicutes/Bacteroidetes ratio typically associated with proinflammatory condition. Notably, while all three exposures reduced Proteobacteria, the CNT exposure and co-exposure induced appearance of Tenericutes and Cyanobacteria, respectively, implicating them as potential biomarkers of exposure. CNTs differentially induced certain lung proinflammatory mediators (TNF-α, IL-1ß, CCL2, CXCL5) whereas CNTs and CSE commonly induced other mediators (CXCL1 and TGF-ß). The co-exposure showed either a component-dominant effect or a summative effect for both dysbiosis and lung inflammation. Depletion of gut microbiota attenuated both the differentially-induced and commonly-induced (TGF-ß) lung inflammatory mediators as well as granulomas indicating gut-to-lung communication and a modulatory role of gut dysbiosis. Taken together, the results demonstrated gut dysbiosis as a systemic effect of inhaled CNTs and provided the first evidence of a bidirectional gut-lung crosstalk modulating CNT lung immunotoxicity.

CircRNA circ_0006892 regulates miR-24/PHLPP2 axis to mitigate cigarette smoke extract-induced bronchial epithelial cell injury.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disorder mainly resulted from cigarette smoke exposure. The dysregulated circular RNAs (circRNAs) are relevant to the pathogenesis of COPD. This study aims to explore the function and mechanism of circRNA hsa_circ_0006892 (circ_0006892) in cigarette smoke extract (CSE)-induced bronchial epithelial injury. The lung tissues were collected from 17 nonsmokers and 23 smokers with COPD. The bronchial epithelial cells (BEAS-2B and 16HBE) were stimulated via CSE. Circ_0006892, microRNA-24 (miR-24), and PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) abundances were examined via a quantitative reverse transcription polymerase chain reaction or Western blot. Cell viability, apoptosis, and inflammatory response were assessed via cell counting kit-8 (CCK-8), flow cytometry, and enzyme-linked immunosorbent assay (ELISA). The target relationship of miR-24 and circ_0006892 or PHLPP2 was tested via dual-luciferase reporter analysis, RNA immunoprecipitation, and RNA pull-down. Circ_0006892 expression was reduced in lung tissues of smokers with COPD and CSE-stimulated bronchial epithelial cells. Circ_0006892 overexpression alleviated CSE-induced viability reduction and promotion of apoptosis and inflammatory response. MiR-24 was bound via circ_0006892, and miR-24 overexpression reversed the effect of circ_0006892 on CSE-induced injury. PHLPP2 was targeted via miR-24, and miR-24 knockdown mitigated CSE-induced viability reduction and promotion of apoptosis and inflammatory response via regulating PHLPP2. Circ_0006892 could promote PHLPP2 expression via regulating miR-24. Circ_0006892 attenuated CSE-induced bronchial epithelial cell apoptosis and inflammatory response via regulating miR-24/PHLPP2 axis.

Cigarette smoke extract combined with LPS reduces ABCA3 expression in chronic pulmonary inflammation may be related to PPARγ/ P38 MAPK signaling pathway.

ABCA3 (ATP-binding cassette class A3) is a transmembrane transporter that plays a positive role in chronic pulmonary inflammation by regulating lipid metabolism. However, it is not completely clear whether ABCA3 and its signaling factors are involved in chronic pulmonary inflammation induced by the combination of CSE (cigarette smoke extract) and LPS (lipopolysaccharide). In this study, we used the method of combining CSE and LPS which was widely used to study lung inflammation-related diseases and has been proven effective in our group's studies to create in vivo and in vitro pulmonary inflammation models. The result showed that, after CSE in combination with LPS treatment, ABCA3 expression was downregulated in rat lung in vivo and in a human alveolar cell line in vitro. ABCA3 expression was upregulated, and related inflammatory factors were downregulated in the state of overexpression of PPARγ or inhibition of the p38 MAPK pathway, while PPARγ deletion or MAPK14 overexpression showed the opposite results. The level of PPARγ remained unchanged, and the expression of ABCA3 was upregulated in the state of the p38 MAPK pathway was inhibited under overexpression of PPARγ. These results indicate that CSE combined with LPS can result in downregulation of ABCA3 under conditions of inflammation, and that the p38 MAPK signaling pathway mediated by PPARγ can regulate the expression changes of ABCA3, thus providing new targets for treating chronic pulmonary inflammation.

Angiopoietin-like 4 knockdown attenuates cigarette smoke extract-induced oxidative stress and apoptosis in lung bronchial epithelial cells by inhibiting NADPH oxidase.

It has been found that angiopoietin-like 4 (ANGPTL4) expression is increased in the serum of patients with chronic obstructive pulmonary disease (COPD). Herein, cigarette smoke extract (CSE) was used to stimulate oxidative stress in bronchial epithelial cells BEAS-2B, and the role and potential mechanism of ANGPTL4 in smoking-induced lung dysfunction were explored. The roles of different concentrations of CSE (0, 1, 2.5, 5, or 10%) in cell viability and ANGPTL4 levels were evaluated. Following ANGPTL4 being knocked down, the effects of ANGPTL4 knockdown on oxidative stress and apoptosis were determined. Moreover, the level of NADPH oxidase 2 (NOX2) was upregulated to assess the mediated role of NOX in the regulation of ANGPTL4, along with JNK/p38 MAPK signaling. CSE treatment elevated the level of ANGPTL4, and ANGPTL4 knockdown reduced CSE-induced oxidative stress, apoptosis, and NOX level in BEAS-2B cells. The greatest degree of alteration was found in NOX2, and additional NOX2 overexpression broke the inhibitory influences of ANGPTL4 knockdown on oxidative stress and apoptosis. Otherwise, ANGPTL4 knockdown hindered the activation of JNK/p38 MAPK signaling, whereas NOX2 overexpression activated this signaling pathway. Together, ANGPTL4 knockdown attenuated CSE-induced oxidative stress, apoptosis, and activation of JNK/MAPK signaling by inhibiting NOX.

Effect of erythromycin on the ultrastructure of human macrophages exposed to cigarette smoke extract in vitro.

Macrophages serve an active role in the pathophysiology of chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been verified as an effective treatment for COPD. However, there are few studies on the effect of EM on the ultrastructure of macrophages exposed to cigarette smoke extract (CSE). In the present study, human macrophages were randomly divided into three groups: The control, CSE and the CSE+EM group, using electron microscopy, the effect of EM was evaluated by comparing the ultrastructural changes between these groups. The macrophages were additionally divided into a further four groups: The control, CSE, CSE+EM 24 h and CSE+EM 48 h groups. The generation of reactive oxygen species (ROS) in each group was evaluated by detecting fluorescence intensity. It was observed that the cellular ultrastructure of the CSE group exhibited abnormal changes, though this effect was reversed back to the level of the control in the CSE+EM group. Compared with the control group, the ROS expression level was significantly increased in the CSE group (P < .05); however, compared with the CSE group, the ROS concentration was decreased in the CSE+EM 24 h (P < .05) and CSE+EM 48 h groups (P < .05), though this was more apparent in the EM 48 h group. It was concluded that EM protects human macrophages against CSE. Moreover, it was hypothesized that EM may reduce the symptoms of patients with COPD by protecting the macrophage ultrastructure from the effects of CSE, resulting in the decreased generation of ROS, inhibiting autophagy and reducing endoplasmic reticulum stress.

Different Effects of Cigarette Smoke, Heated Tobacco Product and E-Cigarette Vapour on Orbital Fibroblasts in Graves' Orbitopathy; a Study by Real Time Cell Electronic Sensing.

Thyroid autoimmunity in Graves' disease (GD) is accompanied by Graves' orbitopathy (GO) in 40% of the cases. Orbital fibroblasts (OF) play a key role in the pathogenesis and cigarette smoking is a known deteriorating factor. Alongside conventional cigarettes (CC) new alternatives became available for smokers, including heated tobacco products (HTP) and E-cigarettes (ECIG). We aimed to study the cellular effects of smoke extracts (SE) in orbital fibroblasts. Primary OF cultures from GO and NON-GO orbits were exposed to different concentrations of SE (1%, 50%) and the changes were followed using Real Time Cell Electronic Sensing (RT-CES). Untreated GO and NON-GO cells had different maximum cell index (CI) values of 3.3 and 2.79 respectively (p < 0.0001). CC, HTP and ECIG treated NON-GO fibroblasts exhibited peak CIs of 2.62, 3.32 and 3.41 while treated GO cells' CIs were higher, 5.38, 6.25 and 6.33, respectively (p < 0.0001). The metabolic activity (MTT) decreased (p < 0.001) and hyaluronan production doubled (p < 0.02) after 50% of CC SE treatment in all cell cultures. GO fibroblasts were more sensitive to low concentration SE then NON-GO fibroblasts (p < 0.0001). The studied SEs exerted different effects. RT-CES is a sensitive technique to detect the effects of very low concentration of SE on fibroblasts.

Ginsenoside Rb3 Alleviates CSE-induced TROP2 Upregulation through p38 MAPK and NF-κB Pathways in Basal Cells.

Smoking-mediated reprogramming of the phenotype and function of airway basal cells (BCs) disrupts airway homeostasis and is an early event in chronic obstructive pulmonary disease (COPD)-associated airway remodeling. Here, we examined the expression and regulation of the transmembrane glycoprotein TROP2 (trophoblast antigen 2), a putative stem cell marker in airway BCs, in lung tissue samples from healthy smokers and healthy nonsmokers and in models in culture to identify therapeutic targets. TROP2 expression was upregulated in the airway epithelia of smokers and positively correlated with the smoking index. In vitro, cigarette smoke extract (CSE) induced TROP2 expression in airway BCs in a time- and dose-dependent manner. The p38 MAPK and NF-κB pathways were also activated by CSE, and their specific antagonists inhibited CSE-induced TROP2 expression. A therapeutic component derived from traditional Chinese medicine, ginsenoside Rb3, inhibited CSE-induced TROP2 expression as well as activation of the p38 MAPK and NF-κB pathways in BCs in monolayer culture. Furthermore, ginsenoside Rb3 prevented the increase in TROP2 expression and antagonized CSE-induced BC hyperplasia and expression of inflammatory factors and epithelial-mesenchymal transition changes in an air-liquid culture model. Thus, CSE-induced TROP2 is a possible biomarker for early changes in the epithelium of smokers, and ginsenoside Rb3 may serve as a therapeutic molecule, preventing the disruption of epithelial homeostasis in COPD.