Soluble epoxide hydrolase deficiency attenuates airway inflammation in COPD via IRE1α/JNK/AP-1 signaling pathway.

BACKGROUND: Soluble Epoxide Hydrolase (sEH) metabolizes anti-inflammatory epoxyeicosatrienoic acids and critically affects airway inflammation in chronic obstructive pulmonary disease (COPD). Considering the excessive endoplasmic reticulum stress is associated with the earlier onset of COPD. The role of sEH and endoplasmic reticulum stress in the pathogenesis of COPD remains unknown. METHOD: 16 weeks of cigarette-exposed mice were used to detect the relationship between sEH and endoplasmic reticulum stress in COPD. Human epithelial cells were used in vitro to determine the regulation mechanism of sEH in endoplasmic reticulum stress induced by cigarette smoke. RESULTS: sEH deficiency helps reduce emphysema formation after smoke exposure by alleviating endoplasmic reticulum stress response. sEH deficiency effectively reverses the upregulation of phosphorylation IRE1α and JNK and the nuclear expression of AP-1, alleviating the secretion of inflammatory factors induced by cigarette smoke extract. Furthermore, the treatment with endoplasmic reticulum stress and IRE1α inhibitor downregulated cigarette smoke extract-induced sEH expression and the secretion of inflammatory factors. CONCLUSION: sEH probably alleviates airway inflammatory response and endoplasmic reticulum stress via the IRE1α/JNK/AP-1 pathway, which might attenuate lung injury caused by long-term smoking and provide a new pharmacological target for preventing and treating COPD.

Endoplasmic Reticulum Stress in Chronic Obstructive Pulmonary Disease: Mechanisms and Future Perspectives.

The endoplasmic reticulum (ER) is an integral organelle for maintaining protein homeostasis. Multiple factors can disrupt protein folding in the lumen of the ER, triggering ER stress and activating the unfolded protein response (UPR), which interrelates with various damage mechanisms, such as inflammation, apoptosis, and autophagy. Numerous studies have linked ER stress and UPR to the progression of chronic obstructive pulmonary disease (COPD). This review focuses on the mechanisms of other cellular processes triggered by UPR and summarizes drug intervention strategies targeting the UPR pathway in COPD to explore new therapeutic approaches and preventive measures for COPD.

Quantitative evaluation of diaphragmatic motion during forced breathing in chronic obstructive pulmonary disease patients using dynamic chest radiography.

Objective: To quantitatively evaluate the bilateral diaphragmatic motion difference during forced breathing between chronic obstructive pulmonary disease (COPD) patients and healthy individuals using dynamic chest radiography technique. Methods: This prospective study included the COPD patients (n: 96, f/m: 17/79, age: 66 ± 8 years old) and healthy individuals (n: 50, f/m: 42/8, age: 53 ± 5 years old) that underwent dynamic chest radiography with a flat panel X-ray detector system during forced breathing in a standing position. After analyzing the excursions, duration and velocity of diaphragmatic motion were automatically calculated using the postprocessing software. The parameters of diaphragmatic motion including excursion, duration, velocity, inhalation/exhalation times were assessed in all subjects for both diaphragms. The correlation between lung function parameters and diaphragmatic motion excursions were further evaluated. Results: The excursions of diaphragmatic motion in COPD patients were significantly decreased in COPD patients compared with healthy individuals during forced breathing (P < 0.05). The excursion in COPD patients was 35.93 ± 13.07 mm vs. 41.49 ± 12.07 mm in healthy individuals in the left diaphragm, and 32.05 ± 12.29 mm in COPD patients vs. 36.88 ± 10.96 mm in healthy individuals in the right diaphragm. The duration of diaphragmatic motion significantly decreased in COPD patients, compared with the healthy individuals (P < 0.05). The inhalation time in COPD patients was 2.03 ± 1.19 s vs. 2.53 ± 0.83 s in healthy individuals in the left diaphragm and 1.94 ± 1.32 s in COPD patients vs. 2.23 ± 1.21 s in healthy individuals in the right diaphragm. The exhalation time was 4.77 ± 1.32 s in COPD patients vs. 6.40 ± 2.73 s in healthy individuals in the left diaphragm and 4.94 ± 3.30 s in COPD patients vs. 6.72 ± 2.58 s in healthy individuals in the right diaphragm. The peak velocity of diaphragmatic motion showed no significant difference between COPD and healthy groups. The excursions of bilateral diaphragmatic motion showed moderate correlation with FEV1/FVC (r = 0.44, P < 0.001). Multi-linear regression analysis showed that the excursions of bilateral diaphragm are significantly associated with COPD occurrence (P < 0.05). Conclusion: The excursions and duration of diaphragmatic motion during forced breathing are significantly decreased in COPD patients, compared with healthy individuals. Our study showed that precise bilateral diaphragmatic motion activity can be evaluated by dynamic chest radiography.

Role of specialized pro-resolving lipid mediators in pulmonary inflammation diseases: mechanisms and development.

Inflammation is an essential mechanism of various diseases. The development and resolution of inflammation are complex immune-modulation processes which induce the involvement of various types of immune cells. Specialized pro-resolving lipid mediators (SPMs) have been demonstrated to be signaling molecules in inflammation. SPMs are involved in the pathophysiology of different diseases, especially respiratory diseases, including asthma, pneumonia, and chronic obstructive pulmonary disease. All of these diseases are related to the inflammatory response and its persistence. Therefore, a deeper understanding of the mechanisms and development of inflammation in respiratory disease, and the roles of the SPM family in the resolution process, might be useful in the quest for novel therapies and preventive measures for pulmonary diseases.

Role of ß2-adrenergic receptors in chronic obstructive pulmonary disease.

Beta-2 adrenergic receptors (ß2-ARs) have important roles in the pathogenesis and treatment of chronic obstructive pulmonary disease (COPD). In recent years, progress has been made in the study of ß2-ARs. Here, we introduce the basic concepts of ß2-ARs, related pathways, as well as application of blockers/agonists of ß2-ARs, and ß2-AR autoantibodies in COPD. Drugs targeting the ß2-AR are being developed rapidly, and we expect them to improve the symptoms and prognosis of COPD patients in the future.

Slow-pull capillary technique versus suction technique in endobronchial ultrasound-guided transbronchial needle aspiration for diagnosing diseases involving hilar and mediastinal lymph node enlargement.

BACKGROUND: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a widely used, safe, and accurate technique for obtaining pathological specimens to be used in the diagnosis of diseases involving lung hilar and mediastinal lymph node (LN) enlargement. However, application of the suction technique during EBUS-TBNA remains controversial. In addition, the effectiveness of the slow-pull capillary technique for the diagnosis of pancreatic masses was recently reported. The aim of this study was to compare the diagnostic accuracy of EBUS-TBNA using these two techniques. METHODS: The accuracy, sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and availability of tissue cores of the suction and slow-pull capillary techniques were studied retrospectively in patients who underwent EBUS-TBNA for the diagnosis of diseases involving lung hilar and mediastinal LN enlargement. RESULTS: A total of 97 patients with hilar and mediastinal LN enlargement underwent EBUS-TBNA; 30 patients underwent the suction technique, 56 patients underwent the slow-pull capillary technique, 5 patients underwent both techniques, and 6 patients had failed operations. The accuracy, sensitivity, specificity, NPV, PPV, and the number of tissue cores obtained with the suction and slow-pull capillary techniques were 66.67% versus 85.71% (p = 0.039), 43.75% versus 85.42% (p < 0.001), 92.86% versus 87.5% (p > 0.05), 59.09% versus 50% (p > 0.05), 87.5% versus 97.62% (p > 0.05), and 19 versus 50 (p = 0.004), respectively. In both univariate and multivariate analyses, the acquisition of tissue core was significantly associated with the diagnostic accuracy of EBUS-TBNA. Moreover, the slow-pull capillary technique was significantly associated with the acquisition of tissue core in EBUS-TBNA. There were no significant differences between the two groups in the blood contamination of samples. CONCLUSIONS: Use of the slow-pull capillary technique in EBUS-TBNA can significantly increase the accuracy related to the diagnosis of diseases involving hilar and mediastinal LN enlargement by improving the acquisition of tissue core. The reviews of this paper are available via the supplemental material section.

ß-Arrestin2 Inhibits Expression of Inflammatory Cytokines in BEAS-2B Lung Epithelial Cells Treated with Cigarette Smoke Condensate via Inhibition of Autophagy.

BACKGROUND/AIMS: ß-arrestin2 has been shown to have a role in human inflammatory disease. However, the role of ß-arrestin2 in cigarette smoke-induced inflammation in the lung remains unknown. The aims of this study were to investigate the effects of ß-arrestin2 on cigarette smoke condensate (CSC)-induced expression of inflammatory cytokines in the BEAS-2B human bronchial epithelial cell line in vitro, and the mechanisms involved. METHODS: The MTT assay determined cell viability of cultured BEAS-2B cells. Autophagy was assessed by western blot, adenoviral mRFP-GFP-LC3 transfection, and immunofluorescence. The effects of ß-arrestin2 shRNA knockdown were studied by western blot and real-time reverse transcription-polymerase chain reaction (RT-PCR). Western blot evaluated the AMPK/mTOR signaling pathway. Levels of inflammatory cytokines, interleukin (IL)-6, IL-8, and MCP-1 were measured in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). RESULTS: CSC suppressed expression of ß-arrestin2 in BEAS-2B cells, activated the AMPK/mTOR signaling pathway, increased cell autophagy and the expression of IL-6, IL-8, and MCP-1,pretreatment with the ß-arrestin2 biased ligands, propranolol, and ICI118551 reversed these changes. Inhibition of autophagy reduced the expression of inflammatory cytokines following CSC. CONCLUSION: In the human bronchial epithelial cell line, BEAS-2B, ß-arrestin2 reduced the expression of CSC-induced inflammatory cytokines by inhibiting autophagy, most likely via the AMPK/mTOR signaling pathway.

Role of p62/SQSTM1 in lipopolysaccharide (LPS)-induced mucus hypersecretion in bronchial epithelial cells.

AIMS: The aims of this study were to evaluate the effects of p62/SQSTM1 expression levels on lipopolysaccharide (LPS)-induced mucus secretion in BEAS-2B bronchial epithelial cells by measuring expression levels of the MUC5AC gene and the Mucin-5AC (MUC5AC) protein. MATERIALS AND METHODS: Bronchial epithelial cells, BEAS-2B, were treated with LPS at different time points. Rapamycin, an autophagy agonist, was added to the BEAS-2B cells 30 min before LPS treatment. Lentivirus transfection was then used to knock down the expression of p62/SQSTM1 (Sequestosome 1) to investigate changes in the downstream signaling pathway. Western blotting and immunofluorescence were used to study the expression levels of MUC5AC, and reverse transcription-polymerase chain reaction (RT-PCR) was used to study the expression of MUC5AC mRNA. KEY FINDINGS: LPS treatment of BEAS-2B cells inhibited autophagy, activated the nuclear factor kappa B (NF-κB) signaling pathway and increased the expression of MUC5AC. The autophagy agonist, rapamycin, activated autophagy, inhibited the NF-κB signaling pathway and decreased LPS-induced expression of MUC5AC. Knockdown of p62/SQSTM1 expression reduced activation of the NF-κB signaling pathway and reduced LPS-induced mucus secretion by BEAS-2B cells in vitro. SIGNIFICANCE: In this in vitro study, which utilized BEAS-2B bronchial epithelial cells, p62/SQSTM1 was shown to have a role in LPS-induced mucus hypersecretion by activating the NF-κB signaling pathway.

Correlation between autophagy levels in peripheral blood mononuclear cells and clinical parameters in patients with chronic obstructive pulmonary disease.

Autophagy serves a role in the pathogenesis of chronic inflammatory diseases. The aim of the present study was to compare the autophagy levels in the peripheral blood mononuclear cells (PBMCs) of patients with chronic obstructive pulmonary disease (COPD) and healthy individuals and to assess the association between autophagy and the clinical parameters of COPD. Samples of peripheral blood from 20 patients with stable COPD and 20 healthy controls were collected. PBMCs were harvested using Ficoll density gradient centrifugation. Levels of the autophagy­associated proteins ubiquitin­binding protein p62 (p62), microtubule­associated proteins 1A/1B light chain 3A (LC3I/II) and beclin­1 in PBMCs were detected by western blotting. Enzyme­linked immunosorbent assay kits were used to detect the serum concentrations of interleukin (IL)­6, IL­8 and tumor necrosis factor (TNF)­α. Associations between the levels of autophagy and forced expiratory volume in 1 sec % predicted (FEV1%) and pro­inflammatory factors were assessed. Western blotting demonstrated that the protein expression of p62 was decreased, but LC3II/I and beclin­1 levels increased in patients with COPD compared with healthy controls. Serum levels of IL­6, IL­8 and TNF­α were increased in patients with COPD. The extent of PBMC autophagy was negatively correlated with FEV1% predicted, but positively correlated with levels of pro­inflammatory cytokines. The levels of autophagy in PBMCs in patients with COPD were increased and were negatively correlated with FEV1% predicted and positively correlated with circulating levels of pro­inflammatory cytokines. Autophagy may serve a role as a biomarker of the severity of COPD or as a therapeutic target for treatment of COPD.

Cigarette Smoke-Induced Pulmonary Inflammation and Autophagy Are Attenuated in Ephx2-Deficient Mice.

Cigarette smoke (CS) increases the risk of chronic obstructive pulmonary disease (COPD) by causing inflammation, emphysema, and reduced lung function. Additionally, CS can induce autophagy which contributes to COPD. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) have promising anti-inflammatory properties that may protect the heart and liver by regulating autophagy. For this reason, the effect of decreased soluble epoxide hydrolase (sEH, Ephx2)-mediated EET hydrolysis on inflammation, emphysema, lung function, and autophagy was here studied in CS-induced COPD in vivo. Adult male wild-type (WT) C57BL/6J and Ephx2-/- mice were exposed to air or CS for 12 weeks, and lung inflammatory responses, air space enlargement (emphysema), lung function, and autophagy were assessed. Lungs of Ephx2-/- mice had a less pronounced inflammatory response and less autophagy with mild distal airspace enlargement accompanied by restored lung function and steady weight gain. These findings support the idea that Ephx2 may hold promise as a therapeutic target for COPD induced by CS, and it may be protective property by inhibiting autophagy.

14,15-Epoxyeicosatrienoic acid suppresses cigarette smoke condensate-induced inflammation in lung epithelial cells by inhibiting autophagy.

Epoxyeicosatrienoic acids (EETs) are metabolic products of free arachidonic acid, which are produced through cytochrome P-450 (CYP) epoxygenases. EETs have anti-inflammatory, antiapoptotic, and antioxidative activities. However, the effect of EETs on cigarette smoke-induced lung inflammation is not clear. Autophagy is believed to be involved in the pathogenesis of chronic obstructive pulmonary disease. In addition, nuclear erythroid-related factor 2 (Nrf2), a transcription factor that regulates many antioxidant genes, is thought to regulate antioxidant defenses in several lung diseases. In addition, interaction between EETs, autophagy, and Nrf2 has been reported. The aim of this study was to explore the effect of 14,15-EET on cigarette smoke condensate (CSC)-induced inflammation in a human bronchial epithelial cell line (Beas-2B), and to determine whether the underlying mechanisms involved in the regulation of Nrf2 through inhibition of autophagy. Autophagy and expression of autophagy signaling pathway proteins (LC3B, p62, PI3K, Akt, p-Akt, and p-mTOR) and anti-inflammatory proteins (Nrf2 and HO-1) were assessed via Western blot analysis. Autophagosomes and autolysosomes were detected by adenoviral mRFP-GFP-LC3 transfection. Inflammatory factors (IL-6, IL-8, and MCP-1) were detected by ELISA. Lentiviral vectors carrying p62 short hairpin RNA were used to interfere with p62 expression to evaluate the effect of p62 on Nrf2 expression. Nrf2 expression was determined through immunocytochemistry. 14,15-EET treatment resulted in a significant reduction in IL-6, IL-8, and MCP-1 secretion, and increased accumulation of Nrf2 and expression of HO-1. In addition, 14,15-EET inhibited CSC-induced autophagy in Beas-2B cells. The mechanism of the anti-inflammatory effect of 14,15-EET involved inhibition of autophagy and an increase in p62 levels, followed by translocation of Nrf2 into the nucleus, which then upregulated expression of the antioxidant enzyme HO-1. 14,15-EET protects against CSC-induced lung inflammation by promoting accumulation of Nrf2 via inhibition of autophagy.

Decreased Histone Deacetylase 2 (HDAC2) in Peripheral Blood Monocytes (PBMCs) of COPD Patients.

BACKGROUND: Histone deacetylase 2 (HDAC2) is a class I histone deacetylase family member that plays a critical role in suppressing inflammatory gene expression in the airways, lung parenchyma, and alveolar macrophages in patients with chronic obstructive pulmonary disease (COPD). However, the expression of HDAC2 in peripheral blood monocytes (PBMCs), nuclear factor kappa B (NF-κB) p65, and serum inflammatory cytokine levels in COPD patients, smokers, and non-smokers remains unclear. METHODS: PBMCs were obtained from COPD patients, healthy smokers, and healthy nonsmokers. The HDAC2 and NF-κB p65 expression were quantified by Western Blot. HDAC activity was assessed by an HDAC fluorometric immunoprecipitation activity assay kit. Serum tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) levels were measured by ELISA. RESULTS: HDAC2 expression and HDAC activity were decreased in PBMCs in COPD patients compared with smokers and non-smokers. Increased NF-κB p65 expression, serum TNF-α and IL-8 levels were observed in COPD patients compared with nonsmokers. The FEV1%pred was positively correlated with HDAC2 expression and HDAC activity in COPD patients. Smokers had decreased HDAC activity, increased NF-κB p65 expression and serum TNF-α compared with nonsmokers. CONCLUSIONS: HDAC2 expression was decreased in PBMCs of COPD patients and was correlated with disease severity. The reduction of HDAC2 expression not only directly enhances the expression of inflammatory genes, but may account for the activation of NF-κB mediated inflammation. Decreased HDAC2 may serve as a potential biomarker of COPD and predict the decline of lung function.

14,15-epoxyeicosatrienoic Acid suppresses cigarette smoke extract-induced apoptosis in lung epithelial cells by inhibiting endoplasmic reticulum stress.

BACKGROUND/AIMS: Epoxyeicosatrienoic acids (EETs), a type of lipid mediators produced by cytochrome P450 epoxygenases, exert anti-inflammatory, angiogenic, anti-oxidative and anti-apoptotic effects. However, the role of EETs in cigarette smoke-induced lung injury and the underlying mechanisms are not fully known. The aim of this study was to explore the effects of CYP2J2-EETs on cigarette smoke extracts (CSE)-induced apoptosis in human bronchial epithelial cell line (Beas-2B) and the possible mechanisms involved. METHODS: Cytochrome P450 epoxygenase 2J2 (CYP2J2) and its metabolites EETs were assessed by western blotting or LC-MS-MS. Cell viability and apoptosis were determined by MTT assay and AnnexinV-PI staining. Reactive oxygen species (ROS) were assessed by measuring H2DCFDA. Caspase-3, HO-1, MAPK and endoplasmic reticulum (ER) stress-related markers GRP78, p-elF2a, and CHOP were evaluated by western blotting. RESULTS: CSE suppressed expression of both CYP2J2 and EET by Beas-2B cells. CSE also induced apoptosis, the generation of ROS and the ER stress in Beas-2B cells. These changes were abolished by pretreatment with exogenous 14,15-EET while pretreatment with 14,15-EEZE, a selective EET antagonist, abolished the protective effects of 14,15-EET. In addition, EETs increased the expression of antioxidant enzyme HO-1. Furthermore, 14,15-EET reduced CSE-induced activation of p38 and JNK. CONCLUSION: The data suggest that CYP2J2-derived EETs protect against CSE-induced lung injury possibly through attenuating ER stress.

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat.

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.