Cigarette Smoke Extract Research Articles

Analysis of Influence of Cigarette Smoke on Signaling Pathways of Innate Immune System in Monocytes of Peripheral Blood

INTRODUCTION: Tobacco smoking is an important medical problem since it has a significant impact on the development and progression of chronic obstructive pulmonary disease (COPD). The components of tobacco smoke can initiate and support local and systemic inflammation with participation of monocytes and macrophages.
 AIM: To study molecular mechanisms associated with the impact of cigarette smoke on signaling pathways of the innate immune system in monocytes of peripheral blood.
 MATERIALS AND METHODS: The methods of in silico analysis was used to identify genes associated with the impact of tobacco smoke. On the basis of the data obtained, a cellular model of inflammation was created in vitro using tobacco smoke extract and monocytes of peripheral blood isolated by immunomagnetic separation. An enzyme-linked immunoassay (ELISA) kit was used to measure the concentration of tumor necrosis factor- (TNF-), interleukin-1 (IL-1) in cell supernatants, and of Toll-like receptor 4 (TLR4), ATP-binding cassette A1 (ABCA1) in homogenates of cell membranes of native monocytes and monocytes exposed to 4% tobacco smoke extract. These data were compared with the levels of TNF-, IL-1, TLR4 and ABCA1 in monocytes of peripheral blood of patients with COPD with frequent exacerbation phenotype and with obliterating atherosclerosis of lower limb arteries (OALLA). For statistical processing and visualization of the data, MedCalc 20.1.4 and R (version 4.2.2) software was used.
 RESULTS: Tobacco smoke influences TLR4, TNF- signaling pathways and lipid metabolism. Cigarette smoke extract enhanced the expression of proinflammatory cytokines TNF- and IL-1 in cell supernatants, increased the level of TLR4 and decreased that of ABCA1 in plasmolemma of monocytes of peripheral blood. In patients with COPD with frequent exacerbation phenotype and with OALLA, there were shown increase in the levels of proinflammatory TNF- and IL-1 cytokines in cell supernatants, increase in the level of TLR4 and reduction of the level of ABCA1 in plasmolemma of monocytes of peripheral blood compared to native monocytes of healthy individuals.
 CONCLUSION: Cigarette smoke enhances the production of proinflammatory TNF- and IL-1 cytokines, increases the levels of TLR4 protein and reduces the amount of ABCA1 transporter in membranes of monocytes of peripheral blood. This may partially explain the cause of the influence of cigarette smoke on development of the pulmonary and cardiovascular diseases. COPD with frequent exacerbation phenotype and OALLA are characterized by enhancement of inflammation with participation of monocytes.

MiR-216a reduces apoptosis of pulmonary microvascular endothelial cells in COPD by targeting DNMT1.

Abnormal apoptosis of pulmonary microvascular endothelial cells (PMVECs) participates in the pathogenesis of COPD. Studies have shown that microRNAs (miRNAs) contribute to the pathogenesis of pulmonary diseases by regulating cell apoptosis. The present study aimed to investigate the effects of miR-216a in cigarette smoke extract (CSE)-induced apoptosis of PMVECs in COPD and explore the potential mechanisms. The emphysema model mice were treated with CSE and CS exposure. The expression of miR-216a and DNA methyltransferase 1 (DNMT1) was assessed in emphysema mice and COPD patients. The miR-216a mimic and Lenti-DNMT1 were transfected into PMVECs to identify the underlying mechanisms. The expression levels of miR-216a and DNMT1 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) or Western blot. Moreover, cell apoptosis was examined by flow cytometry assays. The results show that the expression of miR-216a was decreased, whereas the expression of DNMT1 was increased in the lung tissue of emphysema mice and COPD patients. In addition, the expression of miR-216a was significantly reduced in CSE-treated PMVECs, and the overexpression of miR-216a attenuated CSE-induced PMVEC apoptosis. Furthermore, the expression of DNMT1 was increased in the CSE-induced PMVECs and then was reduced after the overexpression of miR-216a in the CSE-stimulated PMVECs. Luciferase reporter assays confirmed the target reaction between miR-216a and DNMT1. Also, the overexpression of DNMT1 was able to reverse the anti-apoptotic effect of miR-216a in CSE-induced PMVECs. The results indicate that miR-216a may play a crucial role in CSE-induced apoptosis by directly regulating its target gene DNMT1 in COPD. It provides insights into the function of MiR-216a/DNMT1 as a potential molecule in COPD.

Silencing USP19 alleviates cigarette smoke extract-induced mitochondrial dysfunction in BEAS-2B cells by targeting FUNDC1.

Chronic obstructive pulmonary disease (COPD) is commonly caused by smoking. FUN14 domain-containing protein 1 (FUNDC1) plays a fundamental role in mitochondrial autophagy and apoptosis in cigarette smoke extract (CSE)-treated BEAS-2B cells. The present study investigated the mechanism of action of FUNDC1 in mitochondrial dysfunction and apoptosis in CSE-treated BEAS-2B cells. The interaction between ubiquitin-specific peptidase 19 (USP19) and FUNDC1 was analyzed using co-immunoprecipitation. Effects of USP19 knockdown and/or FUNDC1 overexpression on the survival, apoptosis, mitochondrial membrane potential, and oxygen consumption rate (OCR) of BEAS-2B cells treated with 15% CSE were determined. In BEAS-2B cells, CSE inhibited cell survival, promoted apoptosis, increased the expression of USP19 and FUNDC1, increased the ratio of LC3 II to LC3 I (LC3 II/I), and decreased mitochondrial membrane potential and TOM20 levels. In CSE-treated BEAS-2B cells, USP19 knockdown reduced FUNDC1 and LC3 II/I, increased the levels of TOM20, improved cell survival, mitochondrial membrane potential, and OCR, and inhibited apoptosis. USP19 deubiquitinates FUNDC1. FUNDC1 overexpression inhibited the effect of USP19 knockdown in CSE-treated BEAS-2B cells. Overall, decreasing USP19 expression alleviates CSE-induced mitochondrial dysfunction in BEAS-2B cells by downregulating FUNDC1, providing novel insights into the molecular mechanism of FUNDC1 regulation in COPD.

Honokiol ameliorates cigarette smoke-induced damage of airway epithelial cells via the SIRT3/SOD2 signalling pathway.

Cigarette smoking can cause damage of airway epithelial cells and contribute to chronic obstructive pulmonary disease (COPD). Honokiol is originally isolated from Magnolia obovata with multiple biological activities. Here, we investigated the protective effects of honokiol on cigarette smoke extract (CSE)-induced injury of BEAS-2B cells. BEAS-2B cells were treated with 300 mg/L CSE to construct an in vitro cell injury model, and cells were further treated with 2, 5 and 10 μM honokiol, then cell viability and LDH leakage were analysed by CCK-8 and LDH assay kits, respectively. Apoptosis was detected by flow cytometry analysis. ELISA was used to measure the levels of tumour necrosis factor (TNF)-ɑ, IL-1β, IL-6, IL-8 and MCP-1. The results showed that honokiol (0.5-20 μM) showed non-toxic effects on BEAS-2B cells. Treatment with honokiol (2, 5 and 10 μM) reduced CSE (300 mg/L)-induced decrease in cell viability and apoptosis in BEAS-2B cells. Honokiol also decreased CSE-induced inflammation through inhibiting expression and secretion of inflammatory cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-8 and MCP-1. Moreover, honokiol repressed CSE-induced reactive oxygen species (ROS) production, decrease of ATP content and mitochondrial biogenesis, as well as mitochondrial membrane potential. Mechanistically, honokiol promoted the expression of SIRT3 and its downstream target genes, which are critical regulators of mitochondrial function and oxidative stress. Silencing of SIRT3 reversed the protective effects of honokiol on CSE-induced damage and mitochondrial dysfunction in BEAS-2B cells. These results indicated that honokiol attenuated CSE-induced damage of airway epithelial cells through regulating SIRT3/SOD2 signalling pathway.

Tobacco toxins induce osteoporosis through ferroptosis

Clinical epidemiological studies have confirmed that tobacco smoking disrupts bone homeostasis and is an independent risk factor for the development of osteoporosis. The low viability and inferior osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) are important etiologies of osteoporosis. However, few basic studies have elucidated the specific mechanisms that tobacco toxins devastated BMSCs and consequently induced or exacerbated osteoporosis. Herein, our clinical data showed the bone mineral density (BMD) values of femoral neck in smokers were significantly lower than non-smokers, meanwhile cigarette smoke extract (CSE) exposure led to a significant decrease of BMD in rats and dysfunction of rat BMSCs (rBMSCs). Transcriptomic analysis and phenotype experiments suggested that the ferroptosis pathway was significantly activated in CSE-treated rBMSCs. Accumulated intracellular reactive oxygen species activated AMPK signaling, furtherly promoted NCOA4-mediated ferritin-selective autophagic processes, increased labial iron pool and lipid peroxidation deposition, and ultimately led to ferroptosis in rBMSCs. Importantly, in vivo utilization of ferroptosis and ferritinophagy inhibitors significantly alleviated BMD loss in CSE-exposed rats. Our study innovatively reveals the key mechanism of smoking-related osteoporosis, and provides a possible route targeting on the perspective of BMSC ferroptosis for future prevention and treatment of smoking-related bone homeostasis imbalance.

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by β-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by β-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

Vardenafil alleviates cigarette smoke-induced chronic obstructive pulmonary disease by activating autophagy via the AMPK/mTOR signalling pathway: an in vitro and in vivo study.

Chronic obstructive pulmonary disease (COPD) has always attracted global attention with its high prevalence, incidence rate, and mortality. Exposure to cigarette smoke is one of main causes of COPD. Therefore, it is still necessary to study its pathogenesis and find new therapeutic strategies for early COPD prevention and treatment. Vardenafil, a type 5 phosphodiesterase (PDE5) inhibitor, is known to have an efficient therapy in some cardiovascular, pulmonary, and vascular diseases, which is an important mechanism for COPD. However, it still loss relevant research on whether vardenafil is effective in COPD and its mechanism. In this study, the cigarette smoke inhalation was performed to establish cigarette smoke-induced COPD model using C57BL/6 mice and 16HBE cells were treated with cigarette smoke extract (CSE). Mice were treated with vardenafil for 30 d. Then condition of lung injury was evaluated using histological analysis. The content of cytokines and the number of inflammatory cells in lung tissues or bronchoalveolar lavage fluid were measured. Additionally, western blot analysis was employed to evaluate the activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR)-mediated autophagy in vitro. The results showed that vardenafil abolished CSE's effect by activating autophagy via the AMPK/mTOR signalling pathway in vitro. Vardenafil attenuated cigarette smoke-induced lung injury and inflammation response by activating autophagy via the AMPK/mTOR signalling pathway in vivo. These results provide valuable insights into the molecular mechanisms underlying vardenafil's beneficial effects in cigarette smoke-induced COPD treatment. In conclusion, vardenafil alleviates cigarette smoke-induced experimental COPD by activating autophagy via the AMPK/mTOR signalling pathway.

Mechanism of KLF9 in airway inflammation in chronic obstructive pulmonary.

Chronic obstructive pulmonary disease (COPD) is an airway-associated lung disorder, resulting in airway inflammation. This article aimed to explore the role of the krüppel-like factor 9 (KLF9)/microRNA (miR)-494-3p/phosphatase and tensin homolog (PTEN) axis in airway inflammation and pave a theoretical foundation for the treatment of COPD. The COPD mouse model was established by exposure to cigarette smoke, followed by measurements of total cells, neutrophils, macrophages, and hematoxylin and eosin staining. The COPD cell model was established on human lung epithelial cells BEAS-2B using cigarette smoke extract. Cell viability was assessed by cell counting kit-8 assay. miR-494-3p, KLF9, PTEN, and NLR family, pyrin domain containing 3 (NLRP3) levels in tissues and cells were measured by quantitative real-time polymerase chain reaction or Western blot assay. Inflammatory factors (TNF-α/IL-6/IL-8/IFN-γ) were measured by enzyme-linked immunosorbent assay. Interactions among KLF9, miR-494-3p, and PTEN 3'UTR were verified by chromatin immunoprecipitation and dual-luciferase assays. KLF9 was upregulated in lung tissues of COPD mice. Inhibition of KLF9 alleviated airway inflammation, reduced intrapulmonary inflammatory cell infiltration, and repressed NLRP3 expression. KLF9 bound to the miR-494-3p promoter and increased miR-494-3p expression, and miR-494-3p negatively regulated PTEN expression. miR-494-3p overexpression or Nigericin treatment reversed KLF9 knockdown-driven repression of NLRP3 inflammasome and inflammation. KLF9 bound to the miR-494-3p promoter and repressed PTEN expression, thereby facilitating NLRP3 inflammasome-mediated inflammation.

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

Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/β-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/β-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/β-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15min with 5AU/mL CS extract (CSE), and subsequent 6h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100nMTg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/β-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/β-catenin signalling pathway.

CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation

Ovarian tumor family deubiquitinase 4 (OTUD4), a member of the OTU deubiquitinating enzyme, is implicated to decrease in cancer to regulate cell apoptosis. However, the role of OTUD4 in cigarette smoke induced epithelial cell apoptosis and its mechanism have not been elucidated. In this study, we showed that OTUD4 protein reduced in CSE treated mice and airway epithelial cells. OTUD4 silence aggravated cell apoptosis and emphysematous change in the lung tissue of cigarette smoke extract (CSE) treated mice. Additionally, restoration of OTUD4 in the lung of mice alleviated CSE induced apoptosis and emphysematous morphology change. The effect of OTUD4 on cell apoptosis was also confirmed in vitro. Through protein profile screening, we identified that OTUD4 may interact with plasminogen activator inhibitor 1(PAI-1). We further confirmed that OTUD4 interacted with PAI-1 for de-ubiquitination and inhibiting CSE induced PAI-1 degradation. Furthermore, the protective role of OTUD4 in airway epithelial cells apoptosis was blocked by PAI-1 deactivation. Taken together, our data suggest that OTUD4 regulates cigarette smoke (CS)-triggered airway epithelial cell apoptosis via modulating PAI-1 degradation. Targeting OUTD4/PAI-1 signaling might potentially provide a therapeutic target against the lung cell apoptosis in cigarette smoke (CS)-induced emphysema.

Astaxanthin attenuated cigarette smoke extract-induced apoptosis via decreasing oxidative DNA damage in airway epithelium

Chronic obstructive pulmonary disease (COPD) is a lung inflammatory disease that is associated with environmental allergic component exposure. Cigarette smoke is an environmental toxicant that induces lung malfunction leading to various pulmonary diseases. Astaxanthin (AST) is a carotenoid that shows antioxidant and anti-inflammatory activities which might be a promising candidate for COPD therapy. In this study, we released that AST could attenuate cigarette smoke-induced DNA damage and apoptosis in vivo and in vitro. AST administration ameliorated cigarette smoke extract (CSE)-induced activation of Caspase-3 and apoptosis. Pretreated mice with AST significantly decrease CSE-induced DNA damage which shows lower nuclear γ-H2AX level. AST treatment also dramatically reduces the production of intracellular reactive oxygen species (ROS) by suppressing the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 (NOX4) and dual oxidase 1 (DUOX1). Taken together, this study suggested that AST can decrease CSE-induced DNA damage and apoptosis by inhibiting NOX4/DUOX1 expression that promotes ROS generation. AST may be a potential protective agent against CSE-associated lung disease that is worth in-depth investigation.

Chronic exposure to cigarette smoke extract increases nicotine withdrawal symptoms in adult and adolescent male rats.

The aim of the current study was to determine whether non-nicotine constituents of cigarette smoke contribute to nicotine dependence in adolescent and adult male Sprague Dawley rats. For 10days animals were given three times daily intravenous injections of nicotine (1.5mg/kg/day) or cigarette smoke extract (CSE) containing an equivalent dose of nicotine. Both spontaneous and mecamylamine-precipitated withdrawal were then measured. Chronic treatment with CSE induced significantly greater somatic and affective withdrawal signs than nicotine in both adolescents and adults. Mecamylamine-precipitated somatic signs were similar at both ages. In contrast, animals spontaneously withdrawn from chronic drug treatment exhibited significant age differences: whereas adolescents chronically treated with nicotine did not show somatic signs, those treated with CSE showed similar physical withdrawal to those of adults. Mecamylamine did not precipitate anxiety-like behavior at either age. However, both adolescents and adults showed significant anxiety in a light-dark box test 18h after spontaneous withdrawal. Anxiety-like behavior was still evident in an open field test 1month after termination of drug treatment, with adolescents showing significantly greater affective symptoms than adults. Our findings indicate that non-nicotine constituents of cigarette smoke do contribute to dependence in both adolescents and adults and emphasize the importance of including smoke constituents with nicotine in animal models of tobacco dependence.

Naringenin attenuated airway cilia structural and functional injury induced by cigarette smoke extract via IL-17 and cAMP pathways

BackgroundCigarette smoke impairs mucociliary clearance via mechanisms such as inflammatory response and oxidative injury, which in turn induces various respiratory diseases. Naringenin, a naturally occurring flavonoid in grapes and grapefruit, has exhibited pharmacological properties such as anti-inflammatory, expectorant, and antioxidant properties. However, it is still unclear whether naringenin protects airway cilia from injury caused by cigarette smoke. PurposeThis study aimed to investigate the effect of naringenin on cigarette smoke extract (CSE)-induced structural and functional abnormalities in airway cilia and highlight the potential regulatory mechanism. MethodsInitially, network pharmacology was used to predict the mechanism of action of naringenin in ciliary disease. Next, HE staining, immunofluorescence, TEM, qRT-PCR, western blot, and ELISA were performed to assess the effects of naringenin on airway cilia in tracheal rings and air-liquid interface (ALI) cultures of Sprague Dawley rats after co-exposure to CSE (10% or 20%) and naringenin (0, 25, 50, 100 μM) for 24 h. Finally, transcriptomics and molecular biotechnology methods were conducted to elucidate the mechanism by which naringenin protected cilia from CSE-induced damage in ALI cultures. ResultsThe targets of ciliary diseases regulated by naringenin were significantly enriched in inflammation and oxidative stress pathways. Also, the CSE decreased the number of cilia in the tracheal rings and ALI cultures and reduced the ciliary beat frequency (CBF). However, naringenin prevented CSE-induced cilia damage via mechanisms such as the downregulation of cilia-related genes (e.g., RFX3, DNAI1, DNAH5, IFT88) and ciliary marker proteins such as DNAI2, FOXJ1, and β-tubulin IV, the upregulation of inflammatory factors (e.g., IL-6, IL-8, IL-13), ROS and MDA. IL-17 signaling pathway might be involved in the protective effect of naringenin on airway cilia. Additionally, the cAMP signaling pathway might also be related to the enhancement of CBF by naringenin. ConclusionIn this study, we first found that naringenin reduces CSE-induced structural disruption of airway cilia in part via modulation of the IL-17 signaling pathway. Furthermore, we also found that naringenin enhances CBF by activating the cAMP signaling pathway. This is the first report to reveal the beneficial effects of naringenin on airway cilia and the potential underlying mechanisms.

Comparing Effects of Short- and Long-Term Exposure of Cigarette Smoke Extract on Human Vocal Fold Fibroblasts

To explore the effects of short- and long-term cigarette smoke extract (CSE) stimulation on the expression of extracellular matrix (ECM) components and inflammatory cytokines in an in vitro model for studying Reinke's edema using human vocal fold fibroblasts (hVFF). Experimental pilot study using intervention with CSE in vitro. Immortalized hVFF were pretreated with 5% CSE or control medium over a period of 2 or 8weeks, followed by a final 3-day incubation time. We evaluated cell proliferation and examined gene and protein expression of control- and CSE-treated cells using quantitative polymerase chain reaction, Western Blot and enzyme linked immunosorbent assay. Cell numbers of CSE-treated hVFF strongly decreased after 8weeks and limited the overall duration of the experiment. We observed significant upregulations in gene expression and protein levels of inflammatory markers (cyclooxygenase COX1, COX2) and ECM components (decorin, matrix metalloproteinase 1, transglutaminase 2, gremlin 2) induced by CSE after 2 and 8weeks. Interleukin 1 receptor 1, prostaglandin I2 synthase, collagen- and hyaluronan-related gene expression showed minor upregulations. The majority of the observed genes were similarly regulated at both time points. However, the CSE-induced mRNA level of COX1 was ablated after 8weeks. Long-term treatment did not yield results significantly different from the short-term protocol. Therefore, we propose that prolonged CSE exposure is not superior to short-term settings, which save both time and materials.

Baicalin Relieves Airway Inflammation in COPD by Inhibiting miR-125a.

To investigate the effects of Baicalin on the apoptosis of human bronchial epithelial cells (16HBE) induced by cigarette smoke extract (CSE) and the release of inflammatory factors, and to clarify its possible mechanism. CSE was used to treat 16HBE cells and construct COPD cell model. The activity of 16HBE cells was detected by CCK-8 and BrdU. Real-time fluorescence quantitative PCR (RT-QPCR) was used to detect the expression level of miR-125a in each group of 16HBE cells. At the same time, the levels of 16HBE inflammatory cytokines IL-1β, IL-8, IL-6, and TNF-α were detected. The apoptosis rate of 16HBE cells in each group was detected by TUNEL. Compared with the control group, the proliferation of 16HBE cells in CSE group was decreased. Baicalin reversed the effect of 2% CSE on the proliferation of 16HBE cells. Baicalin also reversed the effect of 2% CSE on apoptosis and inflammatory factors in 16HBE cells. miR-125a is highly expressed in COPD, and Baicalin can inhibit the expression of miR-125a. Silencing miR-125a reduces apoptosis and inflammatory response of 16HBE cells in COPD. miR-125a reversed the effects of Baicalin on apoptosis and inflammation of 16HBE cells. Baicalin can reduce CSE-induced apoptosis of human bronchial epithelial cells and release of inflammatory factors, and its mechanism may be related to the inhibition of miR-125a.

Cigarette Smoking Contributes to Th1/Th2 Cell Dysfunction via the Cytokine Milieu in Chronic Obstructive Pulmonary Disease.

Dysregulation and pyroptosis of T-helper (Th) cells and inflammatory cytokines have been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immune response mechanisms as a consequence of tobacco smoke exposure are not fully understood. We hypothesized that cigarette smoke-induced inflammation could be modulated through the cytokine milieu and T-cell nicotinic acetylcholine receptors (nAChRs). The proportions of peripheral blood Th1 and Th2 cells from patients with COPD, smokers without airway obstruction and healthy nonsmokers were analyzed using flow cytometry. The levels of plasma proinflammatory cytokines and their potential association with pulmonary function were also measured. The influence of cigarette smoke extract (CSE) on the conditioned differentiation of T helper cell subsets was further examined in vitro. Significantly higher Th1 cell and plasma IFN-γ and IL-18 levels but lower levels of Th2 cells were found in the peripheral blood from patients with COPD. The increased plasma levels of IFN-γ and IL-18 were negatively correlated with pulmonary function (FEV1% predicted value). Pyroptosis participates in COPD development probably through the activation of the NLRP3 inflammasome upon exposure to CSE. CSE does not directly induce the differentiation of T helper cells; however, under conditioned medium, CSE promotes Th1 development through α7 nAChR modification, while it does not substantially interfere with Th2 differentiation. The differences in the cytokine milieu play a key role in the effects of CSE on the immune response in patients with COPD.

P14-24: Identifying the lung adverse effect of Heat-Not-Burn whole cigarette smoke extract on repeated intratracheal instillation

P14-24: Identifying the lung adverse effect of Heat-Not-Burn whole cigarette smoke extract on repeated intratracheal instillation

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

Smoking induces WEE1 expression to promote docetaxel resistance in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) patients have poor clinical outcomes, with an overall 5-year survival rate of 20%. Smoking is a significant risk factor for EAC. The role of WEE1, a nuclear kinase that negatively regulates the cell cycle in normal conditions, in EAC tumorigenesis and drug resistance is not fully understood. Immunohistochemistry staining shows significant WEE1 overexpression in human EAC tissues. Nicotine, nicotine-derived nitrosamine ketone, or 2% cigarette smoke extract treatment induces WEE1 protein expression in EAC, detected by western blot and immunofluorescence staining. qRT-PCR and reporter assay indicates that smoking induces WEE1 expression through miR-195-5p downregulation in EAC. ATP-Glo cell viability and clonogenic assay confirmed that WEE1 inhibition sensitizes EAC cells to docetaxel treatment invitro. A TE-10 smoking machine with EAC patient-derived xenograft mouse model demonstrated that smoking induces WEE1 protein expression and resistance to docetaxel invivo. MK-1775 and docetaxel combined treatment improves EAC patient-derived xenograft mouse survival invivo. Our findings demonstrate, for the first time, that smoking-induced WEE1 overexpression through miRNA dysregulation in EAC plays an essential role in EAC drug resistance. WEE1 inhibition is a promising therapeutic method to overcome drug resistance and target treatment refractory cancer cells.

Cigarette Smoke Extract Contributes to the Inception and Aggravation of Asthmatic Inflammation by Stimulating Innate Immunity

Purpose: Smoking is a risk factor for the development of asthma and worsens the long-term prognosis of asthma. This study investigated the effect of cigarette smoke extract (CSE) on innate immune cells such as innate lymphoid cells (ILCs) and macrophages in a murine model of induced asthma. Methods: Six-week-old female BALB/C mice were exposed to ovalbumin (OVA) via an intranasal route with or without CSE for 8 weeks to establish a chronic murine asthma model. Airway hyperresponsiveness (AHR), airway inflammatory cells from bronchoalveolar lavage fluid, and the population of CD4+ T cells, ILCs, and macrophages in the lungs were studied to evaluate the effect of chronic CSE exposure on asthma. Results: Mice intranasally exposed to CSE along with OVA treatment (CSE/OVA) had significantly enhanced AHR, eosinophilic inflammation, increased IL- 13 and IL-17 producing CD4+ T cells compared to mice intranasally exposed to OVA only. On the contrary, the frequency of Foxp3+ in CD4+ T cells was reduced in the CSE/ OVA group. CSE enhanced the dendritic cell (DC) population, especially MHCII+ DC with antigen-presenting capacity. Among ILCs, the CSE/OVA group showed a significant increase of IL-13-producing type 2 ILCs, but not interferon-γ+ ILC1s and IL-17+ ILC3s. Among macrophages, alveolar macrophage and Ym-1 and FIZZ1 positive M2 macrophage populations were significantly induced by CSE exposure alone and when combined with OVA treatment. Conclusion: In this study, we showed that long-term exposure to cigarette smoke contributes to the inception and aggravation of asthmatic inflammation by enhancing DCs, ILC2, and M2 alveolar macrophage populations in the mouse model.