Experimental Lung Research最新文献

Purpose: The proteome during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice is unclear.

Materials and methods: In this study, eight-week-old male C57BL/6 mice were intraperitoneally injected with LPS and sacrificed 18 hours after LPS administration to identify protein expression levels in lung tissue using tandem mass tag (TMT) analysis for relative quantification. Hematoxylin-eosin (HE) staining was used to evaluate lung injury in mice. Immunohistochemical staining was used to calculate the production of myeloperoxidase (MPO) and TUNEL staining was performed to detect apoptosis. GO functional clustering and KEGG pathway enrichment analyses were performed to determine functions of differentially expressed proteins (DEPs) and transduction pathways. Domain annotation and subcellular localization analysis of the DEPs were also performed. Furthermore, parallel reaction monitoring (PRM) analysis was used to verify the top 30 DEPs.

Results: A total of 5188 proteins were found to be expressed in lung tissues from LPS- and saline-treated mice. Among these proteins, 293 were differentially expressed between the two groups; 255 proteins were upregulated in the LPS-treated ALI mice, while 38 were downregulated. GO analysis showed that the DEPs are mainly extracellular, and KEGG analysis suggested that the DEPs are mainly enriched in the NOD-like receptor signaling pathway, complement and coagulation cascades and natural killer cell-mediated cytotoxicity. Enrichment of the DEPs is mainly peptidase S1A, serine proteases, peptidase S1, and the serpin domain. 26.6% of the DEPs are in the nucleus, 24.6% are in the cytosol, 19.1% are in the extracellular space, and 18.8% are in the plasma membrane. PRM validation showed that the trend of 30 DEPs was same with TMT analysis. Among these, Cytochrome b-245 heavy chain (Cybb), Monocyte differentiation antigen CD14 (Cd14) and Neutrophil gelatinase-associated lipocalin (NGAL) were the most obvious change.

Conclusions: Our results may help to identify markers and therapeutic targets for LPS-induced ALI.

Purpose: Eosinophils are one of the main cells responsible to the inflammatory response in asthma by the release of inflammatory molecules such as cytokines, reactive oxygen species (ROS), cytotoxic granule, eosinophil extracellular trap (EET), and lipid mediators as cysteinyl leukotriene (cysLT). The interconnections between these molecules are not fully understood. Here, we attempted to investigate the cysLT participation in the mechanisms of EET formation in an asthma model of OVA challenge.

Materials and methods: Before intranasal challenge with OVA, BALB/cJ mice were treated with a 5-lipoxygenase-activating protein (FLAP) inhibitor (MK-886), or with a cysLT1 receptor antagonist (MK-571) and the lung and bronchoalveolar lavage fluid (BALF) were analyzed.

Results: We showed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in inflammatory cells, goblet cells hyperplasia, and eosinophil peroxidase (EPO) activity in the airway. However, only OVA-challenged mice treated with MK-571 had an improvement in lung function. Also, treatments with MK-886 or MK-571 decreased Th2 cytokines levels in the airway. Moreover, we observed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in EET formation in BALF. We also verified that EET release was not due to cell death because the cell viability remained the same among the groups.

Conclusion: We revealed that the decrease in cysLT production or cysLT1 receptor inhibition by MK-886 or/and MK-571 treatments, respectively reduced EET formation in BALF, showing that cysLT regulates the activation process of EET release in asthma.

Background: House dust mite has been well documented as a major source of allergen in asthma. Circular RNAs (circRNAs) vacuolar protein sorting 33A (circVPS33A, circ_0000455) is overexpressed in a murine asthma model. Herein, we sought to identify its critical action in Dermatophagoides pteronyssinus peptidase 1 (Der p1)-induced dysfunction of BEAS-2B cells.

Methods: The levels of circVPS33A, microRNA (miR)-192-5p, and high-mobility group box 1 (HMGB1) were assessed by quantitative real-time PCR (qRT-PCR) or western blot. Actinomycin D treatment and Ribonuclease R (RNase R) assay were used to characterize circVPS33A. Cell viability, proliferation, apoptosis, migration, and invasion were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to quantify interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6. Direct relationship between miR-192-5p and circVPS33A or HMGB1 was verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assay.

Results: CircVPS33A was highly expressed in asthma plasma and Der p1-treated BEAS-2B cells. Knocking down circVPS33A suppressed Der p1-induced injury in BEAS-2B cells. CircVPS33A targeted miR-192-5p. MiR-192-5p directly targeted HMGB1, and miR-192-5p-mediated repression of HMGB1 alleviated Der p1-driven cell injury. Furthermore, circVPS33A modulated HMGB1 expression through miR-192-5p.

Conclusion: Our findings demonstrated that circVPS33A regulated house dust mite-induced injury in human bronchial epithelial cells at least partially depending on the modulation of the miR-192-5p/HMGB1 axis.

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature neonates. Classical BPD is caused by hyperoxia and high-pressure mechanical ventilation, whereas BPD in recent era is caused by impaired pulmonary angiogenesis and alveolarization in extreme prematurity. Although sildenafil was reported to be effective in a hyperoxia-induced rat BPD model, several clinical trials could not demonstrate any significant improvement in the respiratory statuses of BPD infants. Riociguat is a soluble guanylate cyclase stimulator that increases cyclic guanosine monophosphate activity in a nitric oxide independent manner. However, a beneficial effect in BPD has not been established yet.

Methods and results: We established BPD model in rats by injection of SU5416 on day 1 followed by maintenance under normoxia, which resulted in oversimplified alveoli, sparse pulmonary capillary vessels, severe pulmonary hypertension, and growth retardation, which mimicked the features observed in recent clinical management of BPD. We administered riociguat from day 10, when BPD rats exhibited growth retardation. Histological analyses demonstrated that riociguat treatment significantly but partially ameliorated lung alveolarization, vascularization, and pulmonary hypertension. However, the survival rate was not significantly improved by riociguat treatment.

Conclusions: Riociguat could ameliorate pulmonary alveolarization, vascularization, and hypertension in the SU5416 induced BPD rat model, but could not improve the overall survival.

Aim of the study: The current gold standard to assess respiratory mechanics in mice is oscillometry, a technique from which several readouts of the respiratory system can be deduced, such as resistance and elastance. However, these readouts are often not altered in mouse models of asthma. This is in stark contrast with humans, where asthma is generally associated with alterations when assessed by either oscillometry or other techniques. In the present study, we have used double-chamber plethysmography (DCP) to evaluate the breathing pattern and the degree of airflow obstruction in a mouse model of asthma.

Materials and methods: Female C57BL/6 and BALB/c mice were studied at day 1 using DCP, as well as at day 11 using both DCP and oscillometry following a once-daily exposure to either house-dust mite (HDM) or saline for 10 consecutive days.

Results: All DCP readouts used to describe either the breathing pattern (e.g., tidal volume and breathing frequency) or the degree of airflow obstruction (e.g., specific airway resistance) were different between mouse strains at day 1. Most of these strain differences persisted at day 11. Most oscillometric readouts (e.g., respiratory system resistance and elastance) were also different between strains. Changes caused by HDM were obvious with DCP, including decreases in tidal volume, minute ventilation, inspiratory time and mid-tidal expiratory flow and an increase in specific airway resistance. HDM also caused some strain specific alterations in breathing pattern, including increases in expiratory time and end inspiratory pause, which were only observed in C57BL/6 mice. Oscillometry also detected a small but significant increase in tissue elastance in HDM versus saline-exposed mice.

Conclusions: DCP successfully identified differences between C57BL/6 and BALB/c mice, as well as alterations in mice from both strains exposed to HDM. We conclude that, depending on the study purpose, DCP may sometimes outweigh oscillometry.

Purpose/aim: TIMAP (TGF-β-inhibited membrane-associated protein) is a regulatory subunit of protein phosphatase 1 (PP1). The N-terminal region contains a binding motif for the catalytic subunit of PP1 (PP1c) and a nuclear localization signal (NLS). Phosphorylation of TIMAP on Ser331, Ser333 and Ser337 side chains was shown to regulate the activity of the TIMAP-PP1c complex. Several studies, however, reported an additional side chain of TIMAP. Ser69 is located near to the PP1c binding motif and NLS, therefore, we hypothesized that the phosphorylation of this side chain perhaps may regulate the interaction between TIMAP and PP1c, or may affect the nuclear transport of TIMAP. Materials and Methods: To study the significance of Ser69 phosphorylation, GST-tagged or c-myc-tagged wild type, phosphomimic S69D and phosphonull S69A recombinant TIMAP proteins were expressed in bacteria or endothelial cells, respectively. Protein-protein interactions of the wild type or mutant forms of TIMAP were studied by pull-down and Western blot. Localization of TIMAP S69 mutants in pulmonary artery endothelial cells was detected by immunofluorescent staining and expression and localization of the recombinants were investigated by subcellular fractionation and Western blot. Results: Modifications of Ser69 of TIMAP had no effect on binding of PP1c, ERM or RACK1. However, S69D TIMAP showed enhanced membrane localization and an increased number of membrane protrusions were observed in the cells overexpressing this phosphomimic mutant. Furthermore, significantly faster wound healing and migration rate of the S69D mutant overexpressing cells were detected by endothelial barrier resistance measurements (ECIS). Specific interaction was shown between TIMAP and polo-like kinase 4 (PLK4), a potential kinase to phosphorylate Ser69. Conclusions: Altogether, our results indicate that Ser69 phosphorylation by PLK4 may evoke an enrichment of TIMAP in the plasma membrane region and may play an important role in endothelial cell migration without affecting the PP1c binding ability of TIMAP.

Purpose. Behind armor blunt trauma (BABT) is a non-penetrating injury caused by the rapid deformation of body armor, by a projectile, which may in extreme circumstances cause death. The understanding of the mechanisms is still low, in relation to what is needed for safety threshold levels. High velocity projectile BABT causes immediate and severe hypoxia by increased venous admixture (Q's/Q't), but it is not known whether the level of hypoxia correlates to the kinetic energy (E_k) of the projectile.Materials and Methods. We constructed a 65 mm BABT-simulator to measure the E_k absorbed by the thorax. The simulator was validated to 7.62 mm high velocity BABT (swine with removed organs) for 7.62 mm (n = 7) and 65 mm (n = 12). Physiological measurements during 60 minutes were performed in 40 anesthetized swine in groups control (n = 9), 7.62 mm (n = 7), 65 mm weight variation (n = 24), 65 mm speed variation (n = 12, included in the weight variation group). New calculations were done for a previously studied group of 7.62 mm with backing (n = 9).Results. 65 mm BABT simulation and 7.62 mm BABT had similar back-face signatures (24 mm), and maximum thoracic impression speed (24-34 m/s). Back-face signatures correlated linearly to E_k (R²=0.20). Rib fractures had a 50% likelihood at back-face signature 23.0 mm (95% CI 18.5 to 29.0 mm, area under ROC curve 0.93). E_k correlated linearly to pO₂ (R²=0.34, p = 0.0026) and venous admixture (R²=0.37, p = 0.0046). The extrapolated E_k at 5 minutes for pO₂=0 kPa was 587 J and for venous admixture = 100% 574 J.Conclusions. Hypoxia and venous admixture correlated linearly to E_k, allowing for a calculated predicted lethal E_k to ≥574 J, which should be verified in survival studies. Lethality predictions from lung physiology is an alternative to clay impressions and may facilitate the development of ballistic safety equipment and new BABT safety criteria.Supplemental data for this article is available online at https://doi.org/10.1080/01902148.2021.1950869 .

Background: Circular RNAs (circRNAs) have been implicated in the molecular etiology of pediatric pneumonia. Here, we investigated the precise action of circRNA tropomodulin 3 (circTMOD3, hsa_circ_0035292) in cell injury and inflammation induced by lipopolysaccharide (LPS). Methods: Cell viability was gauged by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis and cycle distribution were assessed by flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to measure interleukin-6 (IL-6), IL-1β and tumor necrosis factor alpha (TNF-α) production. The levels of circTMOD3, microRNA (miR)-146b-3p, and C-X-C motif chemokine receptor 1 (CXCR1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease (RNase) R, Actinomycin D and subcellular localization assays were done to characterize circTMOD3. The direct relationship between miR-146b-3p and circTMOD3 or CXCR1 was confirmed by dual-luciferase reporter assays. Results: Our data showed that LPS induced the expression of circTMOD3 in WI-38 cells. CircTMOD3 was resistant to RNase R and was mainly present in the cytoplasm. Silencing endogenous circTMOD3 alleviated WI-38 cell injury and inflammation triggered by LPS. Mechanistically, circTMOD3 directly targeted miR-146b-3p, and CXCR1 was a direct and functional target of miR-146b-3p. CircTMOD3 regulated LPS-induced cell inflammation and injury by targeting miR-146b-3p, and miR-146b-3p-mediated suppression of CXCR1 impacted LPS-evoked cytotoxicity and inflammation. Furthermore, circTMOD3 functioned as a competing endogenous RNA (ceRNA) for miR-146b-3p to induce CXCR1 expression. Conclusion: Our findings demonstrated the regulation of circTMOD3 in LPS-induced cell injury and inflammation at least partially via miR-146b-3p-independent modulation of CXCR1.

Purpose of the study: The involvement of the IL-23/IL23R pathway is well known in the disease pathogenesis of sarcoidosis and other inflammatory diseases. To date, the pathogenic mechanism of IL-23 is most notably described on CD4⁺ Th17 lymphocytes. However, the function of the IL23R on myeloid cells in sarcoidosis is poorly understood. Thus, the aim of the study is to investigate the role of the IL23R on myeloid cell in pulmonary granuloma formation. Methods: We generated IL23RLysMCre mice lacking the IL23R gene in myeloid cells. The importance of IL23R in myeloid cells for the development of sarcoidosis was studied in a mouse model of inflammatory lung granuloma formation through embolization of PPD from Mycobacterium bovis-coated Sepharose beads into previously PPD-immunized mice. In addition the function of IL23R on myeloid cells was studied in LPS or IFNγ stimulated BMDMs and BMDCs. The mRNA and protein expression levels of relevant cytokines were analyzed by RT-PCR (TaqMan) and ELISA. The composition of immune cells in BALF was quantified by flow cytometry and alteration in granuloma sizes were observed by H&E stained lung sections. Results: Mycobacterium Ag-elicted pulmonary granulomas tend to be smaller in IL23RLysMCre mice and NF-κB dependent Th1 cytokines in the murine lungs are reduced compared to wildtype mice. In line, we observed that IL23R-deficient bone marrow-derived macrophages show a reduced production of Th1 cytokines after LPS stimulation. Conclusion: We here for the first time demonstrate a role for IL23R on myeloid cells in pulmonary inflammation and granuloma formation. Our findings provide essential insights in the pathogenesis of inflammatory lung diseases like sarcoidosis, which might be useful for the development of novel therapeutics targeting distinct immunological pathways like IL-23/IL23R.

Objective: Chronic obstructive pulmonary disease (COPD) is a respiratory disease with high morbidity and mortality worldwide, so far there is no ideal treatment method. Previous studies have shown that hydrogen (H₂) is involved in the treatment of COPD as an antioxidant. In this study, the effect of H₂ on M1/M2 polarization of alveolar macrophages in COPD rats was observed, and its anti-inflammatory mechanism was further elucidated. Methods: Twenty-four Sprague-Dawley rats were randomly divided into three groups including the control, COPD and H₂ group. A rat model of COPD was established by cigarette exposure combined with lipopolysaccharide (LPS) induction. H₂ therapy was administered 2 hours per day for 14 days. Lung function and pathology were assessed. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1 and IL-10 in bronchoalveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA, protein expression and immunoreactivity of inducible nitric oxide synthase (iNOS) and arginase (Arg)-1 in lung were observed by quantitative real-time PCR, western blot and immunohistochemistry. Results: Compared with the control rats, there were a significant decline in lung function, a marked inflammatory infiltration and pulmonary parenchymal remodeling and the increases of IL-6, TNF-α and TGF-β1 levels in BALF and lung tissue, but a lower expression of IL-10 in COPD rats. The iNOS mRNA and protein expression, as well as its optical density (OD), were increased significantly in lung tissue, while those of Arg-1 decreased significantly. H₂ treatment improved the lung function and the parenchymal inflammation, reversed the increased levels of IL-6, TNF-α and TGF-β1, and the lower IL-10. Meanwhile, H₂ also down-regulated the expression of iNOS, but up-regulated expression of Arg-1 in lung tissue. Conclusion: H₂ reduces inflammation in the lung of COPD, which may be related to its inhibition of M1 type polarization and activation of M2 type polarization of alveolar macrophage.