Experimental Lung Research最新文献

Background: Circular RNA (circRNA) is considered to be an important regulator of cancer malignant progression, including non-small cell lung cancer (NSCLC). Circ_0000735 has been found to be associated with NSCLC progression. Therefore, its role and molecular mechanism in NSCLC deserve further exploration.

Methods: Quantitative real-time PCR (qRT-PCR) was used to measure the expression of circ_0000735, microRNA (miR)-635 and family with sequence similarity 83 member F (FAM83F). Cell proliferation, migration, invasion and apoptosis were determined using cell counting kit 8 assay, colony formation assay, transwell assay and flow cytometry. Cell glycolysis were measured by detecting the glucose consumption and lactate production of cells. Western blot analysis was utilized to test the protein levels of glycolysis markers and FAM83F. The relationship between circ_0000735 and miR-635 or miR-635 and FAM83F was verified by dual-luciferase reporter assay. The effect of circ_0000735 on NSCLC tumor growth was evaluated by constructing xenograft models.

Results: Circ_0000735 was a highly expressed circRNA in NSCLC. Silenced circ_0000735 could inhibit NSCLC cell proliferation, migration, invasion, glycolysis, and increase apoptosis. MiR-635 could be sponged by circ_0000735, and its inhibitor could reverse the regulation of circ_0000735 silencing on NSCLC progression. Moreover, FAM83F was a target of miR-635, and circ_0000735 positively regulated FAM83F by sponging miR-635. The inhibitory effect of miR-635 on NSCLC progression could also be reversed by FAM83F overexpression. Additionally, circ_0000735 knockdown reduced NSCLC tumor growth through regulating miR-635/FAM83F axis.

Conclusion: Circ_0000735 promoted NSCLC progression by the miR-635/FAM83F axis, showing that circ_0000735 might be a promising biomarker for NSCLC. Highlights: Circ_0000735 knockdown represses NSCLC cell progression and tumor growth. Circ_0000735 functions as a miR-635 sponge. FAM83F is targeted by miR-635.

Background: Standard care in severe SARS-CoV-2 pneumonia complicated by severe dyspnea and respiratory failure, consists of symptom reduction, ultimately supported by mechanical ventilation. Patients with severe SARS-CoV-2, a prominent feature of COVID-19, show several similar symptoms to Critical Asthma Syndrome (CAS) patients, such as pulmonary edema, mucus plugging of distal airways, decreased tissue oxygenation, (emergent) exhaustion due to severe dyspnea and respiratory failure. Prior application of elective phosphodiesterase (PDE)3-inhibitors milrinone and enoximone in patients with CAS yielded rapid symptomatic relief and reverted the need for mechanical ventilation, due to their bronchodilator and anti-inflammatory properties. Based on these observations, we hypothesized that enoximone may be beneficial in the treatment of patients with severe SARS-CoV-2 pneumonia and prominent CAS-features.

Methods: In this case report enoximone was administered to four consecutive patients (1 M; 3 F; 46-70 y) with emergent respiratory failure due to SARS-CoV-2 pneumonia. Clinical outcome was compared with three controls who received standard care only.

Results: After an intravenous bolus of enoximone 20 mg followed by 10 mg/h via perfusor, a rapid symptomatic relief was observed: two out of four patients recovered within a few hours, the other two (with comorbid COPD GOLD II/III) responded within 24-36 h. Compared to the controls, in the enoximone-treated patients respiratory failure and further COVID-19-related deterioration was reverted and mechanical ventilation was prevented, leading to reduced hospital/ICU time.

Discussion: Our preliminary observations suggest that early intervention with the selective PDE3-inhibitor enoximone may help to revert respiratory failure as well as avert mechanical ventilation, and reduces ICU/hospital time in patients with severe SARS-CoV-2 pneumonia. Our findings warrant further research on the therapeutic potential of PDE3-inhibition, alone or in combination with other anti-COVID-19 strategies.

Objective: Glycogen phosphorylase B (PYGB), the rate-determining enzyme in glycogen degradation, plays a critical role in progression of various tumors. The present study focused on the potential molecular mechanism toward PYGB in non-small cell lung cancer (NSCLC) progression.

Methods: Expression of PYGB in NSCLC tissues and cell lines was evaluated via quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. Cell viability, proliferation and apoptosis were investigated using 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-bromo-2-deoxyuridine (BrdU) and flow cytometry, respectively. Cell migration and invasion ability were detected by wound healing and transwell invasion assays, respectively. The in vivo effect of PYGB on NSCLC tumor growth was determined via subcutaneous xenotransplanted tumor model.

Results: PYGB was upregulated in NSCLC tissues and cell lines, suggesting a poor prognosis in NSCLC patients. In vitro functional assays indicated that knockdown of PYGB suppressed cell viability, proliferation, migration and invasion, while promoted cell apoptosis in NSCLC. Mechanistically, we found that overexpression of PYGB could activate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, while these effects were effectively reversed by knockdown of PYGB. In vivo tumorigenesis and PI3K/AKT signaling pathway were also inhibited by PYGB knockdown.

Conclusions: Knockdown of PYGB suppressed NSCLC progression, suggesting PYGB as a novel biomarker and potential molecular therapeutic target for further investigation in NSCLC.

Purpose: This study aimed to explore the regulatory effects and mechanisms of long noncoding RNA H19 (H19) on pulmonary injury, inflammation, and fibrosis of acute respiratory distress syndrome (ARDS).

Materials and methods: A rat model of ARDS was established by intratracheal instillation of 2 mg/kg lipopolysaccharide (LPS). qRT-PCR was performed to detect the expression of H19, miR-423-5p, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF). Histology score was assessed by hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of proinflammatory cytokines and the content of VEGF in bronchoalveolar lavage fluid (BALF). The lung fibrosis was evaluated using western blot and Masson's trichrome staining. Dual-luciferase reporter gene assay was used for confirming the relationship between miR-423-5p and H19/FOXA1 in alveolar macrophage cells (MH-S) and alveolar epithelial cells (MLE-12). The regulatory effects of H19/miR-423-5p/FOXA1 axis on the inflammation and fibrosis were further analyzed in LPS-induced MH-S cells.

Results: The expression of H19 and FOXA1 was significantly up-regulated, while the expression of miR-423-5p was down-regulated in LPS-induced ARDS rats. Silencing of H19 decreased the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1, and VEGF, the contents of TNF-α, IL-1β, IL-6, and VEGF in BALF, and histology score in LPS-induced ARDS rats. H19 knockdown also reduced the fibrosis scores and the protein expression of vimentin and α-SMA, and elevated the protein expression of E-cadherin in LPS-induced ARDS rats. Furthermore, silencing of miR-423-5p and overexpression of FOXA1 reversed the inhibitory effects of si-H19 on the inflammation and fibrosis of LPS-induced MH-S cells.

Conclusions: Silencing of H19 relieved the pulmonary injury, inflammation and fibrosis of LPS-induced ARDS in rats. Silencing of H19 also alleviated the inflammation and fibrosis of LPS-induced MH-S cells through regulating the miR-423-5p/FOXA1 axis.

Purpose: Idiopathic pulmonary fibrosis (IPF) is a type of progressive lung fibrosis disease. The survival time of diagnosed IPF patients is often only 2 years. Currently much evidence showed that the epithelial-mesenchymal transition (EMT) process is the main cause of the occurrence and development of IPF. LncRNA cardiac hypertrophy related factor (CHRF) was reported to be related with IPF development. Here we explored the functions and regulatory mechanisms of CHRF on EMT in IPF.

Materials and methods: A549 cells were treated with transforming growth factor-β1 (TGF-β1) for 48 h to construct IPF cell model. CHRF and miR-146a expression were quantified using qPCR. The expression of L1 cell adhesion molecule (L1CAM) and EMT related indicators (E-cadherin, Vimentin, Slug and N-cadherin) were detected by qPCR and western blot. Dual luciferase reporter experiment was conducted to prove the molecular interaction of miR-146a and L1CAM, as well as CHRF and miR-146a.

Results: CHRF and L1CAM expression were significantly upregulated and promoted the EMT process in A549 after treatment of TGF-β1. MiR-146a was obviously down-regulated, and knockdown of CHRF inhibited the EMT process by up-regulating miR-146a, in A549 after treatment of TGF-β1. Meanwhile, overexpression of miR-146a inhibited EMT process via targeting L1CAM. In addition, L1CAM overexpression eliminated the inhibitory effect of sh-CHRF on the EMT process.

Conclusions: These results provided evidence that CHRF promoted EMT process in A549 after treatment of TGF-β1, which proposed a new insight for depth understanding the pathological mechanisms of IPF.

Aim of the study: Ketogenic diet (KD) has been identified as an effective strategy in treating multiple diseases. KD is capable of inducing autophagy which is an important therapeutic target for pulmonary fibrosis (PF). This study aimed to investigate the effect of KD treatment on PF progression. Materials and Methods: Intratracheal instillation of bleomycin (BLM, 5 mg/kg) to establish PF model in male Kunming mice fed either KD or standard diet. The survival of mice was recorded every day for 3 weeks. The pulmonary tissues were weighed on day 21 and the pulmonary index was calculated. The histopathological changes of pulmonary tissues were analyzed by hematoxylin and eosin staining and Masson staining, and the collagen deposition by hydroxyproline assay. Then the content of proinflammatory factors in pulmonary tissues was measured using enzyme-linked immunosorbent assay, and the expression of profibrogenic cytokines, autophagy markers and PI3K/AKT/mTOR pathway-related proteins in pulmonary tissues using western blotting or immunohistochemistry. Results: KD treatment significantly restored the BLM-induced increase of pulmonary index and had a tendency to increase the survival rate of PF mice. Furthermore, KD treatment restored the BLM-induced damage of alveolar structure, infiltration of inflammatory cells and collagen deposition and decreased hydroxyproline content. In addition, the BLM-induced secretion of tumor necrosis factor-alpha, interleukin-6 and interleukin-1β and expression of transforming growth factor β1, phospho-Smad2/3, connective tissue growth factor, α-smooth muscle actin and collagen type III alpha 1 chain were inhibited by KD. KD treatment also up-regulated the expression of light chain 3 II/I and Beclin1 and down-regulated the expression of p62, phospho-AKT, phospho-mTOR and phospho-p70S6K, suggesting that KD induced autophagy and suppressed the BLM-induced activation of PI3K/AKT/mTOR signaling pathway. Conclusions: These findings indicate that KD can alleviate PF in vivo by regulating autophagy and PI3K/AKT/mTOR signaling pathway, which provides a novel therapeutic strategy for PF.

Methods: We conducted a cross-sectional study of adults between 18 and 55 years old. Inclusion criteria were: exclusive e-cigarette use or cigarette smoking for ≥ 1 year or no history of tobacco use. Participants with a history of pulmonary illness, atopy, medications (except birth control pills), marijuana, and illegal substance use were excluded. Custom Multiplex ELISA was used to measure YKL-40 and other biomarker levels in the serum and induced sputum of the participants. Multivariable linear regression was used to compare the levels of YLK-40 in healthy participants, e-cigarette, and cigarette users after adjusting for age, sex, and BMI.

Results: We recruited 20 healthy controls, 23 cigarette smokers, and 22 exclusive e-cigarette users. Serum YKL-40 (ng/ml) was significantly higher in e-cigarette users (Median 21.2 [IQR 12.1-24.0] ng/ml) when compared to controls (12.2 [IQR 8.7-18.1] ng/ml, p = 0.016) but comparable to cigarette smokers (21.6 [IQR 11.62-51.7] ng/ml, p = 0.31). No significant differences were found in the serum or sputum of the other biomarkers tested.

Conclusion: The inflammatory biomarker, YKL-40 is elevated in the serum but not the sputum of e-cigarette users with no reported pulmonary disease. Further research is necessary to characterize this association.

Purpose of the study: Macrolide therapy is effective in reducing chronic obstructive pulmonary disease (COPD) exacerbations. Our recent study has shown the effectiveness of taking azithromycin in COPD patients, not only ex-smokers but also current smokers. Beyond their anti-microbial effects, macrolides have anti-inflammatory and immunomodulatory properties. The aim of this study was to determine if pretreatment with azithromycin modulates cigarette smoke-induced inflammation in airway epithelial cells. We hypothesized that pretreatment with azithromycin decreases exacerbation frequency by modulating inflammation in human airway epithelial cells exposed to cigarette smoke. Materials and methods: BEAS-2B bronchial epithelial cells were incubated with 5% cigarette smoke extract (CSE) for 3 h, 6 h, and 24 h. Then, airway epithelial cells were pretreated with azithromycin and exposed to 5% CSE. In each stage, the expression and release of IL-6 and IL-8 mRNA were analyzed by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results: There was a significant increase of IL-6 and IL-8 mRNA, as well as an increase in extracellular IL-8 protein following exposure to 5% CSE. When cells were pretreated with azithromycin and exposed to 5% CSE for 3 h, there was a significant dose-dependent decrease in the expression of IL-6 mRNA. A final concentration of 9 µg/mL of azithromycin was sufficient to decrease IL-6, IL-8 mRNA, and extracellular IL-8 levels. Conclusion: Pretreatment with azithromycin decreased the expression of IL-6 and IL-8 mRNA and the release of IL-8 in bronchial epithelial cells exposed to cigarette smoke. These results demonstrate the direct effect of azithromycin on inflammatory mediators in bronchial epithelial cells exposed to cigarette smoke.

Background: Smoking is a cause behind many diseases, including tuberculosis, and it is a risk factor for tuberculosis infection and mortality. Moreover, smoking is associated with a poor tuberculosis treatment outcome.

Objectives: In this study, we focus on the effects of cigarette smoke on an infected cell culture treated with anti-tuberculosis drugs.

Materials and methods: Cytotoxicity on THP-1, J774A.1 and MH-S cell lines and growth of Mycobacterium tuberculosis exposed to a reference or a commercial cigarette was evaluated. THP-1 cell line was exposed to cigarette smoke, infected with Mycobacterium tuberculosis and treated with anti-tuberculosis drugs. Apoptosis and death cell were also tested on M. bovis BCG infected cells. Minimal inhibitory concentrations of anti-tuberculosis drugs were analyzed.

Results: All cells lines showed viability values higher than 80% when exposed to cigarette smoke extract. However, THP-1 cell line infected with M. bovis BCG and exposed to Marlboro cigarette smoke showed up to a 54% reduction of apoptotic cells than cells unexposed to smoke. M. tuberculosis exposed to Marlboro cigarette smoke for 11 days had an optical density 16% lower than unexposed bacteria. When cells were infected with M. tuberculosis, the intracellular recovery of CFUs showed up to a 0.66 log reduction in cells exposed to cigarette smoke extract because of a potential impairment in the phagocytosis. Macrophages treated with drugs showed up to a 2.55 log reduction in the intracellular load burden compared with non-treated ones. Despite poor treatment outcome on TB smoker patients, minimal inhibitory concentration of rifampicin increased only 2-fold in M. tuberculosis exposed to cigarette smoke.

Conclusion: Smoking interferes with tuberculosis treatment impairing the immunity of the host.

Purpose: The regulation effect and mechanism of respiratory syncytial virus (RSV) infection on the expression of tachykinin substance P (SP) in airway epithelial cells was investigated.

Methods: The regulation of SP expression by RSV was investigated in the BEAS-2B airway epithelial cell line. RT-qPCR, immunofluorescence, and ELISA assay were used to examine the expression of the SP encoding gene TAC1, the intracellular SP protein expression, and the extracellular SP secretion.

Results: The mRNA expression of TAC1 and the intracellular SP protein level in BEAS-2B cells were significantly enhanced by RSV infection with multiplicity of infection (MOI) values of both 1 and 0.1 at 48 hours post infection. Heat-inactivated and UV-inactivated RSV, but not live RSV, significantly induced SP secretion in both control BEAS-2B cells and CX3CR1 receptor knockout cells without affecting the TAC1 gene expression or cell viability. RSV G protein (2-10 μg/ml) and fractalkine (10-50 ng/ml), both CX3CR1 receptor ligands, did not affect SP secretion in BEAS-2B cells. Inhibition of STAT1 phosphorylation by fludarabine (1 μM) markedly reduced the RSV-induced TAC1 gene expression and antagonized the inhibition of RSV replication by interferon-α in BEAS-2B cells.

Conclusions: STAT1 participates in RSV infection-induced SP expression in airway epithelial cells.