Cigarette Smoke-induced Emphysema Research Articles

Blocking IL-23 Signaling Mitigates Cigarette Smoke-Induced Murine Emphysema.

Inflammatory cell infiltration is a characteristic feature of COPD and correlates directly with the severity of the disease. Interleukin-23 (IL-23) is a pro-inflammatory cytokine that regulates Th-17 inflammation, which mediates many pathophysiological events in COPD. The primary goal of this study was to determine the role of IL-23 as a mediator of key pathologic processes in cigarette smoke-induced COPD. In this study, we report an increase in IL23 gene expression in the lung biopsies of COPD patients compared to controls and identified a positive correlation between IL23 gene expression and disease severity. In a cigarette smoke-induced murine emphysema model, the suppression of IL-23 with a monoclonal blocking antibody reduced the severity of cigarette smoke-induced murine emphysema. Mechanistically, the suppression of IL-23 was associated with a reduction in immune cell infiltration, oxidative stress injury, and apoptosis, suggesting a role for IL-23 as an essential immune mediator of the inflammatory processes in the pathogenesis of CS-induced emphysema.

Ism1 deficiency in mice exacerbates bleomycin-induced pulmonary fibrosis with enhanced cellular senescence and delayed fibrosis resolution

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease with high morbidity and mortality but unclear etiology and incomplete pathophysiological understandings, making the discovery of effective therapeutics arduous. Currently, two drugs, nintedanib and pirfenidone, are available for IPF treatment which can slow down the fibrotic scarring in the lung but are unable to provide disease resolution. Hence, further revelation of the molecular mechanisms of IPF is critical for the development of novel therapeutics. Isthmin-1 (ISM1) is a secreted anti-inflammatory protein highly expressed in the mouse and human lung. Ism1−/− mice presented spontaneous and progressive lung emphysema, as well as heightened acute lung injury (ALI) upon lipopolysaccharide (LPS) treatment with an accompanied increase of post-LPS-ALI pulmonary fibrosis. ISM1 is important for lung homeostasis with airway-delivered recombinant ISM1 (rISM1) suppressing cigarette smoke-induced emphysema, LPS-ALI, and house-dust mites (HDM)-induced asthma-like symptoms in mice. However, the role of ISM1 in pulmonary fibrosis is yet to be clearly understood. In this work, we show that Ism1−/− mice presented heightened bleomycin-induced pulmonary fibrosis (BIPF), with enhanced immune cell infiltration, myofibroblast accumulation, and collagen deposition. ISM1 deficiency also led to increased cellular senescence in mouse lungs, isolated primary alveolar type II epithelial cells, and primary lung fibroblasts upon bleomycin treatment. Ism1−/− mice also showed delayed resolution of pulmonary fibrosis with reduced lipofibroblasts and downregulation of lipid synthesis-related genes. These results are in congruence with the RNA-seq data which demonstrated gene expression alterations in Ism1−/− lung that are linked to predisposition to pulmonary fibrosis and dysregulation of lipid metabolism pathways. Gene expression analyses showed that Ism1 is similarly dysregulated in the lungs of BIPF and human IPF patients. These findings reveal an anti-fibrotic role of ISM1 in mouse lungs and provide the foundation to further investigate possible therapeutic applications of ISM1 for pulmonary fibrosis in the future.

Early-life house dust mite aeroallergen exposure augments cigarette smoke-induced myeloid inflammation and emphysema in mice

BackgroundLongitudinal studies have identified childhood asthma as a risk factor for obstructive pulmonary disease (COPD) and asthma-COPD overlap (ACO) where persistent airflow limitation can develop more aggressively. However, a causal link between childhood asthma and COPD/ACO remains to be established. Our study aimed to model the natural history of childhood asthma and COPD and to investigate the cellular/molecular mechanisms that drive disease progression.MethodsAllergic airways disease was established in three-week-old young C57BL/6 mice using house dust mite (HDM) extract. Mice were subsequently exposed to cigarette smoke (CS) and HDM for 8 weeks. Airspace enlargement (emphysema) was measured by the mean linear intercept method. Flow cytometry was utilised to phenotype lung immune cells. Bulk RNA-sequencing was performed on lung tissue. Volatile organic compounds (VOCs) in bronchoalveolar lavage-fluid were analysed to screen for disease-specific biomarkers.ResultsChronic CS exposure induced emphysema that was significantly augmented by HDM challenge. Increased emphysematous changes were associated with more abundant immune cell lung infiltration consisting of neutrophils, interstitial macrophages, eosinophils and lymphocytes. Transcriptomic analyses identified a gene signature where disease-specific changes induced by HDM or CS alone were conserved in the HDM-CS group, and further revealed an enrichment of Mmp12, Il33 and Il13, and gene expression consistent with greater expansion of alternatively activated macrophages. VOC analysis also identified four compounds increased by CS exposure that were paradoxically reduced in the HDM-CS group.ConclusionsEarly-life allergic airways disease worsened emphysematous lung pathology in CS-exposed mice and markedly alters the lung transcriptome.

Nebulized platelet-derived extracellular vesicles attenuate chronic cigarette smoke-induced murine emphysema

Chronic obstructive pulmonary disease (COPD) is a prevalent lung disease usually resulting from cigarette smoking (CS). Cigarette smoking induces oxidative stress, which causes inflammation and alveolar epithelial cell apoptosis and represents a compelling therapeutic target for COPD. Purified human platelet-derived exosome product (PEP) is endowed with antioxidant enzymes and immunomodulatory molecules that mediate tissue repair. In this study, a murine model of CS-induced emphysema was used to determine whether nebulized PEP can influence the development of CS-induced emphysema through the mitigation of oxidative stress and inflammation in the lung. Nebulization of PEP effectively delivered the PEP vesicles into the alveolar region, with evidence of their uptake by type I and type II alveolar epithelial cells and macrophages. Lung function testing and morphometric assessment showed a significant attenuation of CS-induced emphysema in mice treated with nebulized PEP thrice weekly for 4 weeks. Whole lung immuno-oncology RNA sequencing analysis revealed that PEP suppressed several CS-induced cell injuries and inflammatory pathways. Validation of inflammatory cytokines and apoptotic protein expression on the lung tissue revealed that mice treated with PEP had significantly lower levels of S100A8/A9 expressing macrophages, higher levels of CD4+/FOXP3+ Treg cells, and reduced NF-κB activation, inflammatory cytokine production, and apoptotic proteins expression. Further validation using in vitro cell culture showed that pretreatment of alveolar epithelial cells with PEP significantly attenuated CS extract-induced apoptotic cell death. These data show that nebulization of exosomes like PEP can effectively deliver exosome cargo into the lung, mitigate CS-induced emphysema in mice, and suppress oxidative lung injury, inflammation, and apoptotic alveolar epithelial cell death.

Apoptosis inhibitor of macrophage (AIM)/CD5L is involved in the pathogenesis of COPD

BackgroundAlveolar macrophages (AMs) and AM-produced matrix metalloprotease (MMP)-12 are known to play critical roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). The apoptosis inhibitor of the macrophages (AIM)/CD5 molecule-like (CD5L) is a multifunctional protein secreted by the macrophages that mainly exists in the blood in a combined form with the immunoglobulin (Ig)M pentamer. Although AIM has both facilitative and suppressive roles in various diseases, its role in COPD remains unclear.MethodsWe investigated the role of AIM in COPD pathogenesis using porcine pancreas elastase (PPE)-induced and cigarette smoke-induced emphysema mouse models and an in vitro model using AMs. We also analyzed the differences in the blood AIM/IgM ratio among nonsmokers, healthy smokers, and patients with COPD and investigated the association between the blood AIM/IgM ratio and COPD exacerbations and mortality in patients with COPD.ResultsEmphysema formation, inflammation, and cell death in the lungs were attenuated in AIM−/− mice compared with wild-type (WT) mice in both PPE- and cigarette smoke-induced emphysema models. The PPE-induced increase in MMP-12 was attenuated in AIM−/− mice at both the mRNA and protein levels. According to in vitro experiments using AMs stimulated with cigarette smoke extract, the MMP-12 level was decreased in AIM−/− mice compared with WT mice. This decrease was reversed by the addition of recombinant AIM. Furthermore, an analysis of clinical samples showed that patients with COPD had a higher blood AIM/IgM ratio than healthy smokers. Additionally, the blood AIM/IgM ratio was positively associated with disease severity in patients with COPD. A higher AIM/IgM ratio was also associated with a shorter time to the first COPD exacerbation and higher all-cause and respiratory mortality.ConclusionsAIM facilitates the development of COPD by upregulating MMP-12. Additionally, a higher blood AIM/IgM ratio was associated with poor prognosis in patients with COPD.Trial RegistrationThis clinical study, which included nonsmokers, healthy smokers, and smokers with COPD, was approved by the Ethics Committee of the Hokkaido University Hospital (012–0075, date of registration: September 5, 2012). The Hokkaido COPD cohort study was approved by the Ethics Committee of the Hokkaido University School of Medicine (med02-001, date of registration: December 25, 2002).

Secretoglobin 3A2 protects lung from developing cigarette smoke-induced pulmonary emphysema.

Secretoglobin (SCGB) 3A2 is a bioactive molecule exhibiting various functions such as improving allergic airway inflammation and pulmonary fibrosis and promoting bronchial branching and proliferation during lung development. To determine if and how SCGB3A2 is involved in chronic obstructive pulmonary disease (COPD), a multifactorial disease with both airway and emphysematous lesions, a COPD mouse model was created by exposing Scgb3a2-deficient (KO), Scgb3a2-lung-specific overexpressing (TG), and wild type (WT) mice to cigarette smoke (CS) for 6 months. The KO mice showed loss of lung structure under control condition, and CS exposure resulted in more expansion of airspace and destruction of alveolar wall than WT mouse lungs. In contrast, TG mouse lungs showed no significant changes after CS exposure. SCGB3A2 increased the expression and phosphorylation of signal transducers and activators of transcription (STAT)1 and STAT3, and the expression of α1-antitrypsin (A1AT) in mouse lung fibroblast-derived MLg cells and mouse lung epithelial-derived MLE-15 cells. In MLg cells, A1AT expression was decreased in Stat3-knockdown cells, and increased upon Stat3 overexpression. STAT3 formed a homodimer when cells were stimulated with SCGB3A2. Chromatin immunoprecipitation and reporter assays demonstrated that STAT3 binds to specific binding sites on the Serpina1a gene encoding A1AT and upregulates its transcription in lung tissues of mice. Furthermore, nuclear localization of phosphorylated STAT3 upon SCGB3A2 stimulation was detected by immunocytochemistry. These findings demonstrate that SCGB3A2 protects the lungs from the development of CS-induced emphysema by regulating A1AT expression through STAT3 signaling.

Probiotics supplementation reduces cigarette smoke-induced damage in the respiratory micro-architecture of mice

This study evaluated the protective efficacy of probiotics supplementation against cigarette smoke-induced lung emphysema, inflammation, and loss of cilia in mice. Probiotics are known to promote mucosal tolerance and mitigate respiratory injuries. Twenty-four adult mice were randomly divided into three groups: control (Ctr), cigarette smoke (CS), and cigarette smoke + probiotics (CS+P). Probiotics were given for 7 days before exposure to smoke in the CS+P group. Tissue samples of the trachea (goblet cell count and index, loss of cilia), lungs (airspace distention), and bronchoalveolar lavage fluid (BALF) were collected and processed. The results showed a significant increase in acidic and neutral goblet cells in the CS group compared to the Ctr and CS+P groups (P < 0.05). Overall, goblet cell number and index were lower in the CS+P group (41.71 ± 5.76, 0.67 ± 0.073) than CS group (56.28 ± 5.34, 1.31 ± 0.28). Inflammatory cells and loss of cilia significantly decreased in mice fed probiotics before exposure to cigarette smoke (P < 0.05). Lung emphysema was also significantly reduced in the CS+P group compared to the CS group (P < 0.05). In conclusion, dietary supplementation of probiotics reduced lung emphysema, inflammatory cells, goblet cell index, and loss of cilia under conditions of cigarette smoke exposure in mice.

HIV Increases the Risk of Cigarette Smoke-Induced Emphysema Through MMP-9.

HIV is associated with an increased risk for emphysema. Matrix metalloproteinase 9 (MMP-9) is a lung tissue remodeling enzyme associated with emphysema. We previously found MMP-9 activity increases with increases in oxidative stress and that HIV increases alveolar oxidative stress. We hypothesized that HIV proteins would increase the risk of cigarette smoke-induced emphysema due to MMP-9. HIV-1 transgenic rats and wild-type littermates were exposed to cigarette smoke or sham for 8 weeks. Lung compliance and histology were assessed. Bronchoalveolar lavage (BAL), primary alveolar macrophages (AM), and serum samples were obtained. A rat alveolar macrophage cell line was exposed to the HIV protein Tat, and MMP-9 levels were assessed by Western immunoblotting. MMP-9 protein expression and activity were assessed in AM from the HIV rat model by ELISA and cytoimmunofluoresence, respectively. Serum from human subjects with and without HIV and tobacco dependence was assessed for MMP-9 levels. MMP-9 expression was significantly increased in rat alveolar macrophages after Tat exposure. HIV-1 transgenic rats developed emphysema while wild-type littermates did not. MMP-9 expression was also increased in the serum, BAL, and AM of HIV-1 transgenic rats after exposure to cigarette smoke compared with wild-type rats. In parallel, serum samples from HIV+ smokers had higher levels of MMP-9 than subjects without HIV and those who did not smoke. The combination of HIV and cigarette smoke increases MMP-9 expression in experimental rat HIV models and human subjects. HIV and cigarette smoke both induce alveolar oxidative stress and thereby increase MMP-9 activity.

Glycyl-L-histidyl-L-lysine-Cu2+ attenuates cigarette smoke-induced pulmonary emphysema and inflammation by reducing oxidative stress pathway.

Background: Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder manifested as chronic airway inflammation and persistent airflow limitation with the essential mechanism as inflammatory response and oxidative stress induced by toxic exposures such as cigarette smoke (CS). Glycyl-L-histidyl-L-lysine (GHK) is a nontoxic tripeptide involved in the process of healing and regeneration as a natural product. With the combination of Cu(II), glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) improves antioxidative and anti-inflammatory bioavailability, and they might offer potential therapeutic properties for COPD. Thus, the present study aimed to identify the potential effects of GHK-Cu on emphysema induced by cigarette smoke. Methods: In the in vivo experiment, C57BL/6J mice were exposed to CS for 12 weeks to induce pulmonary emphysema. GHK-Cu was injected intraperitoneally at doses of 0.2, 2 and 20 μg/g/day in 100 µl of saline on alternative days from the 1st day after CS exposure. The effects of GHK-Cu on the morphology of CS-induced emphysema, the inflammatory response and oxidative stress were evaluated. The antioxidative effect of GHK-Cu on human alveolar epithelial A549 cells was assessed in vitro. Results: GHK-Cu treatment attenuated the CS-induced emphysematous changes and partially reversed the matrix metalloprotein -9 (MMP-9)/tissue inhibitor of metalloproteinases-1 (TIMP-1) imbalance in the lung tissue. GHK-Cu reduced the inflammation and oxidation by decreasing the expression of inflammatory cytokines (IL-1β and TNF-α) in the bronchoalveolar lavage and the enzymatic activity of MPO and MDA in the lung homogenate while restoring the T-AOC and GSH content. Furthermore, administration of GHK-Cu reversed the increase in NF-κB expression induced by CS and increased the Nrf2 level, as an antioxidant defense component, in mice with chronic CS exposure. In CSE-exposed human alveolar epithelial A549 cells, GHK-Cu also inhibited oxidative stress by suppressing MDA levels and restoring T-AOC and GSH levels, which were modulated by upregulating Nrf2 expression. Conclusion: GHK-Cu treatment attenuated CS-induced emphysema by anti-inflammation by downregulating NF-κB and antioxidation via upregulation of the Nrf2/Keap1 in lung tissues.

Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice.

Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01-1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.

Diallyl disulfide prevents cigarette smoke-induced emphysema in mice

IntroductionCigarette smoke (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and pulmonary emphysema. The use of antioxidants has emerged as a potential therapeutic strategy to treat airway inflammation and lung diseases. In the current study, we investigated the potential therapeutic impact of diallyl disulfide (Dads) treatment in a murine model of CS-induced emphysema. MethodsC57BL/6 mice were exposed to CS for 60 consecutive days and treated with vehicle or Dads (30, 60 or 90 mg/kg) by oral gavage for the last 30 days, three times/week. The control group was sham-smoked and received vehicle treatment. All mice were euthanized 24 h after day 60; bronchoalveolar lavage (BAL) was performed and lungs were processed for further experimentation. Histological (HE stained sections, assessment of mean linear intercept (Lm)), biochemical (nitrite, superoxide dismutase (SOD), glutathione transferase (GST), and malondialdehyde (MDA) equivalents), and molecular biology (metalloproteinase (MMP) 12, SOD2, carbonyl reductase 1 (CBR1), nitrotyrosine (PNK), 4-hydroxynonenal (4-HNE), and CYP2E1) analyses were performed. ResultsTreatment with Dads dose-dependently reduced CS-induced leukocyte infiltration into the airways (based on BAL fluid counts) and improved lung histology (indicated by a reduction of Lm). Furthermore, CS exposure dramatically reduced the activity of the antioxidant enzymes SOD and GST in lung tissue and increased nitrite and MDA levels in BAL; these effects were all effectively counteracted by Dads treatment. Western blot analysis further confirmed the antioxidant potential of Dads, showing that treatment prevented the CS-induced decrease in SOD2 expression and increase in lung damage markers, such as CBR1, PNK, and 4-HNE. Furthermore, increased MMP12 (an important hallmark of CS-induced emphysema) and CYP2E1 lung protein levels were significantly reduced in mice receiving Dads treatment. ConclusionOur findings demonstrate that treatment with Dads is effective in preventing multiple pathological features of CS-induced emphysema in an in vivo mouse model. In addition, we have identified several proteins/enzymes, including 4-HNE, CBR1, and CYP2E1, that are modifiable by Dads and could represent specific therapeutic targets for the treatment of COPD and emphysema.

Astragalin Inhibits Cigarette Smoke-Induced Pulmonary Thrombosis and Alveolar Inflammation and Disrupts PAR Activation and Oxidative Stress-Responsive MAPK-Signaling.

Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10–20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1–20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress–MAPK signaling–inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.

The effect of long-term doxycycline treatment in a mouse model of cigarette smoke-induced emphysema and pulmonary hypertension.

Chronic obstructive pulmonary disease (COPD) is a major cause of death and a still incurable disease, comprising emphysema and chronic bronchitis. In addition to airflow limitation, patients with COPD can suffer from pulmonary hypertension (PH). Doxycycline, an antibiotic from the tetracycline family, in addition to its pronounced antimicrobial activity, acts as a matrix metalloproteinase (MMP) inhibitor and has anti-inflammatory properties. Furthermore, doxycycline treatment exhibited a beneficial effect in several preclinical cardiovascular disease models. In preclinical research, doxycycline is frequently employed for gene expression modulation in Tet-On/Tet-Off transgenic animal models. Therefore, it is crucial to know whether doxycycline treatment in Tet-On/Tet-Off systems has effects independent of gene expression modulation by such systems. Against this background, we assessed the possible curative effects of long-term doxycycline administration in a mouse model of chronic CS exposure. Animals were exposed to cigarette smoke (CS) for 8 mo and then subsequently treated with doxycycline for additional 3 mo in room air conditions. Doxycycline decreased the expression of MMPs and general pro-inflammatory markers in the lungs from CS-exposed mice. This downregulation was, however, insufficient to ameliorate CS-induced emphysema or PH. Tet-On/Tet-Off induction by doxycycline in such models is a feasible genetic approach to study curative effects at least in established CS-induced emphysema and PH. However, we report several parameters that are influenced by doxycycline and use of a Tet-On/Tet-Off system when evaluating those parameters should be interpreted with caution.

Oxidative stress mediates the apoptosis and epigenetic modification of the Bcl-2 promoter via DNMT1 in a cigarette smoke-induced emphysema model

BackgroundEmphysema is a crucial pathological characteristic of chronic obstructive pulmonary disease (COPD). Oxidative stress, apoptosis and epigenetic mechanisms contribute to the pathogenesis of emphysema. However, an attempt to accurately identify whether these mechanisms interact with each other and how they are triggered has never been conducted.MethodThe total reactive oxygen species (ROS) level, pulmonary apoptosis and B-cell lymphoma/leukemia-2 (Bcl-2) expression, an apoptosis regulator, were detected in samples from COPD patients. Bisulfite sequencing PCR (BSP) was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), a vital DNA methyltransferase enzyme, in the lungs of patients was confirmed through western blotting. To find out interactions between oxidative stress and DNA methylation in emphysema, mouse models were built with antioxidant treatment and DNMT1 silencing, and were examined with the pulmonary apoptosis, Bcl-2 and DNMT1 levels, and epigenetic alterations of Bcl-2.ResultsHigher ROS levels and pulmonary apoptosis were observed in COPD patients than in healthy controls. Downregulated Bcl-2 expression with increased promoter methylation and DNMT1 protein expression was found in COPD patients. Antioxidant treatment reduced the level of ROS, DNMT1 protein and emphysematous progression in the smoking models. Following DNMT1 blockade, smoking models showed improved lung function, pulmonary apoptosis, emphysematous progression, and increased Bcl-2 protein level with less promoter methylation than emphysema mice.ConclusionCigarette-induced oxidative stress mediates pulmonary apoptosis and hypermethylation of the Bcl-2 promoter in emphysema models through DNMT1.

Thymoquinone (Tq) protects necroptosis induced by autophagy/mitophagy-dependent oxidative stress in human bronchial epithelial cells exposed to cigarette smoke extract (CSE).

Chronic obstructive pulmonary disease (COPD) is characterized by cigarette smoke-induced emphysema. Herein, we demonstrate protective effects of Thymoquinone (Tq), an active constituent from Nigella sativa, against cigarette smoke extract (CSE)-induced abnormalities in bronchial epithelial cells. Dose-dependent reduction in cell viability was observed in BEAS-2B cells when exposed to different CSE concentrations, which was significantly reversed by Tq evident by LDH release. Levels of SOD, CAT, GR , GSH, and mitochondrial membrane ATPases were significantly reduced upon CSE exposure, an event, again, antagonized in presence of Tq. Similarly, Tq treatment significantly blocked CSE-induced 4HNE elevations. Further, Tq-improved mitochondrial dysfunction caused by CSE and significantly decreased autophagy/mitophagy markers like LC3II and p-Drp. Tq also reduced necroptosis markers such as p-MLKL, RIP-1, and RIP-3, by stabilizing PINK-1 levels. In summary, Tq possesses protective properties against human bronchial epithelial cell autophagy/mitophagy-dependent necroptosis caused by CSE, which warrants considerable attention for further preclinical evaluations. PRACTICAL APPLICATIONS: This study demonstrates Thymoquinone (Tq), a natural plant extract to possess protective properties against human bronchial epithelial cell autophagy/mitophagy-dependent necroptosis caused by cigarette smoke extract. The demonstrated efficacy of Tq will throw light for further preclinical evaluation of this molecule in CSE-mediated complications. A detailed in vivo research is recommended.

Astaxanthin Suppresses Cigarette Smoke-Induced Emphysema through Nrf2 Activation in Mice.

Oxidative stress plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key cellular defense mechanism against oxidative stress. Recent studies have shown that astaxanthin protects against oxidative stress via Nrf2. In this study, we investigated the emphysema suppression effect of astaxanthin via Nrf2 in mice. Mice were divided into four groups: control, smoking, astaxanthin, and astaxanthin + smoking. The mice in the smoking and astaxanthin + smoking groups were exposed to cigarette smoke for 12 weeks, and the mice in the astaxanthin and astaxanthin + smoking groups were fed a diet containing astaxanthin. Significantly increased expression levels of Nrf2 and its target gene, heme oxygenase-1 (HO-1), were found in the lung homogenates of astaxanthin-fed mice. The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was significantly decreased, and emphysema was significantly suppressed. In conclusion, astaxanthin protects against oxidative stress via Nrf2 and ameliorates cigarette smoke-induced emphysema. Therapy with astaxanthin directed toward activating the Nrf2 pathway has the potential to be a novel preventive and therapeutic strategy for COPD.

Salidroside mitigates skeletal muscle atrophy in rats with cigarette smoke-induced COPD by up-regulating myogenin and down-regulating myostatin expression.

Objectives: The present study aimed at investigating the therapeutic effect of Salidroside on skeletal muscle atrophy in a rat model of cigarette smoking-induced chronic obstructive pulmonary disease (COPD) and its potential mechanisms.Methods: Male Wistar rats were randomized, and treated intraperitoneally (IP) with vehicle (injectable water) or a low, medium or high dose of Salidroside, followed by exposure to cigarette smoking daily for 16 weeks. A healthy control received vehicle injection and air exposure. Their lung function, body weights and gastrocnemius (GN) weights, grip strength and cross-section area (CSA) of individual muscular fibers in the GN were measured. The levels of TNF-α, IL-6, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) in serum and GN tissues as well as myostatin and myogenin expression in GN tissues were measured.Results: In comparison with that in the healthy control, long-term cigarette smoking induced emphysema, significantly impaired lung function, reduced body and GN weights and CSA values in rats, accompanied by significantly increased levels of TNF-α, IL-6 and MDA, but decreased levels of SOD and GSH in serum and GN tissues. Furthermore, cigarette smoking significantly up-regulated myostatin expression, but down-regulated myogenin expression in GN tissues. Salidroside treatment decreased emphysema, significantly ameliorated lung function, increased antioxidant, but reduced MDA, IL-6 and TNF-α levels in serum and GN tissues of rats, accompanied by decreased myostain, but increased myogenin expression in GN tissues.Conclusion: Salidroside mitigates the long-term cigarette smoking-induced emphysema and skeletal muscle atrophy in rats by inhibiting oxidative stress and inflammatory responses and regulating muscle-specific transcription factor expression.

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung's structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Emphysema assessment for personalized immunotherapy response prediction in lung cancer patients.

After editorial request, we considered the expert opinion on the link between emphysema, immune suppression and lung cancer, expressed in two invited editorials on our published article entitled “Cigarette Smoke-Induced Emphysema Exhausts Early Cytotoxic CD8+ T Cell Responses against Nascent Lung Cancer Cells” (1).

Rapamycin attenuates Tc1 and Tc17 cell responses in cigarette smoke-induced emphysema in mice.

Chronic exposure to cigarette smoke promotes airway inflammation and emphysema accompanied by enhanced CD8+ interferon (IFN)-γ+ T(Tc1) and CD8+ interleukin (IL)-17+ T(Tc17) cell responses. The mammalian target of rapamycin (mTOR) has been involved in the pathogenesis of emphysema. Inhibiting mTOR by rapamycin has been reported to alleviate emphysema, but the mechanism is not fully understood. We aimed to explore the effect of rapamycin on Tc1 and Tc17 cell responses induced by cigarette smoke exposure. Male C57BL/6 mice were exposed to cigarette smoke or room air for 24weeks. Half of the smoke-exposed mice received rapamycin in the last 12weeks. The severity of emphysema in those mice was evaluated by mean linear intercept (MLI), mean alveolar airspace area (MAA) and destructive index (DI). Bronchoalveolar lavage was collected and analyzed. Phosphorylated (p-) mTOR in CD8+ T cells, Tc1 and Tc17 cells were detected by flow cytometry. The relative expression of p-mTOR in lungs was determined by western blot analysis. IFN-γ and IL-17A levels were detected by enzyme-linked immunosorbent assays. IFN-γ, mTOR and RAR-related orphan receptor (ROR)γt mRNA levels were evaluated by the real-time polymerase chain reaction. Elevated p-mTOR expression in CD8+ T cells and lung tissue was accompanied by the enhanced Tc1 and Tc17 cell responses in lungs of mice exposed to cigarette smoke. Rapamycinreduced inflammatory cells in BALF and decreased MLI, DI and MAA in lungs. Rapamycin decreased p-mTOR expression, and down-regulation of mTOR and RORγt mRNA levels along with the attenuation of Tc1 and Tc17 cell responses in mice with emphysema. The mTOR was activated in CD8+ T cells accompanied by the enhanced Tc1 and Tc17 cell responses in cigarette smoke-related pulmonary inflammation. Rapamycin ameliorated emphysema and attenuated Tc1 and Tc17 cell responses probably caused by inhibiting mTOR in cigarette smoke-exposed mice.