Bronchial Epithelial BEAS-2B Cells Research Articles

Palmitoleic acid inhibits Pseudomonas aeruginosa quorum sensing activation and protects lungs from infectious injury

BackgroundUnsaturated fatty acids targeting quorum sensing (QS) system have shown potential application in reducing bacterial virulence. We aim to investigate the effect of palmitoleic acid (PMA) on P. aeruginosa QS activation, and its impact on infection-induced lung injury.MethodsThe influence of PMA on QS signaling molecule (3OC12-HSL and C4-HSL) concentrations, pyocyanin production, and QS gene transcription levels were examined in wildtype PAO1 culture. The roles of PMA in reducing infection-induced injury were assessed in human bronchial epithelial BEAS-2B cells and mouse lung infection models, respectively. PMA levels and QS signaling molecule concentrations were tested in the bronchoalveolar lavage fluid (BALF) of bronchiectasis patients with first-time detection of P. aeruginosa infection.ResultsPMA administration dose-dependently suppressed the expression of QS signaling molecules, pyocyanin, and QS genes during the logarithmic stage of bacterial growth. In BEAS-2B cells, PMA-treated PAO1 filtrates significantly reduced cell apoptosis and expression of IL-8 and IL-6. In mouse lung infection models, prophylactically oral administration of PMA significantly downregulated the expression of P. aeruginosa QS signals and QS genes (lasR, rhlR, rhlI, lasB, rhlA, phzA1, phnA) in lungs, and relieved neutrophilic airway inflammation. Finally, PMA levels were negatively correlated with the concentrations of both 3OC12-HSL and C4-HSL in BALF of bronchiectasis patients, and positively correlated with their forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1.0).ConclusionOur findings show that PMA inhibits P. aeruginosa QS activation and protects lungs from injury caused by bacterial virulence. Hence, PMA may serve as a potential anti-QS agent against P. aeruginosa infection and would help to alleviate lung injury in bronchiectasis patients.

Ebeiedinone and peimisine inhibit cigarette smoke extract-induced oxidative stress injury and apoptosis in BEAS-2B cells.

Ebeiedinone and peimisine are the major active ingredients of Fritillariae Cirrhosae Bulbus. In this study, we looked at how these two forms of isosteroidal alkaloids protect human bronchial epithelial BEAS-2B cells from oxidative stress and apoptosis caused by cigarette smoke extract (CSE). First, the cytotoxicity was determined using the CCK8 assay, and an oxidative stress model was established. Then the antioxidative stress activity and mechanism were investigated by ELISA, flow cytometry, and Western blotting. By the CCK-8 assay, exposure to CSE (20%, 40%, and 100%) reduced the viability of BEAB-2S cells. The flow cytometry findings indicated that CSE-induced production of ROS (0.5% to maximum) and treatments with 10μM ebeiedinone and 20μM peimisine attenuated the production of ROS. The western blot assay results indicate that ebeiedinone and peimisine reduce CSE-induced oxidative stress, DNA damage, apoptosis, and autophagy dysregulation by inhibiting ROS, upregulating SOD and GSH/GSSG, and downregulating MDA, 4-HNE, and 8-OHdG through the NRF2/KEAP1 and JNK/MAPK-dependent pathways, thereby delaying the pathological progression of COPD caused by CS.Our data suggest that CSE causes oxidative stress, DNA damage, and apoptosis in BEAS-2B cells, as well as the progression of COPD. Ebeiedinone and peimisine fight CS-induced COPD by suppressing autophagy deregulation and apoptosis.

Metrnl/IL-41 Alleviates Lipopolysaccharide-Induced Acute Lung Injury by Suppressing Inflammatory Responses through the AMPK/SIRT1 Signaling Pathway

Introduction: Interleukin-41 (IL-41), also known as Metrnl, is a multifunctional adipokine recognized for its neurotrophic and anti-inflammatory properties that play a significant role in diseases such as sepsis and chronic obstructive pulmonary disease. Despite its crucial biological functions, the mechanisms by which IL-41 mitigates lipopolysaccharide (LPS)-induced acute lung injury (ALI) are not well understood. This study aimed to elucidate the protective effects of IL-41 against LPS-induced inflammation and apoptosis in a murine model and in vitro using bronchial epithelial cells (Beas-2b). Methods: We administered recombinant IL-41 to mice subjected to LPS-induced ALI and observed changes in lung histopathology, inflammation, and apoptosis. Concurrently, Beas-2b cells were transfected with IL-41 constructs, and the role of the AMPK/SIRT1 pathway was investigated using specific inhibitors and agonists. Results: Our results demonstrated that LPS-induced ALI is characterized by increased inflammatory cell chemotaxis in lung lavage fluid, enhanced phosphorylation of NFκB p65, and elevated Bax protein expression, coupled with a decrease in IL-41 protein levels. Treatment with recombinant IL-41 effectively mitigated these pathological changes by upregulating AMPK phosphorylation and SIRT1 expression and inhibiting IκB/NFκB p65 phosphorylation. In cellular assays, overexpression of IL-41 reversed LPS-induced oxidative stress, apoptosis, and the secretion of inflammatory cytokines, whereas suppression of IL-41 or inhibition of AMPK reversed these protective effects. Conclusions: In conclusion, IL-41 exerts significant protective effects against ALI by activating the AMPK/SIRT1 signaling pathway and reducing excessive inflammation and apoptosis. These findings suggest that IL-41 holds promise as a therapeutic target for ALI, potentially allowing for personalized treatments based on serum IL-41 levels.

Sennoside A represses the malignant phenotype and tumor immune microenvironment of non-small cell lung cancer cells by inhibiting the TRAF6/NF-κB pathway.

Non-small cell lung cancer (NSCLC) is a prominent cause of cancer death worldwide. Sennoside A (SA) is the primary anthraquinone active component from Rheum officinale Baill and exerts antitumor functions in multiple human tumors. This research aimed to elucidate the function and mechanism of SA in NSCLC. SA functions in NSCLC were determined using Cell Counting Kit-8 (CCK-8) assay, Terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis, Transwell assay, Enzyme-Linked Immunosorbent Assay (ELISA), and Western blot. Meanwhile, the SA mechanism in NSCLC was examined with Western blot, immunofluorescence assay, CCK-8 assay, Transwell analysis, and ELISA. Furthermore, SA functions in NSCLC growth in vivo were assessed by the establishment of a tumor xenograft model, hematoxylin-eosin staining, analysis of NSCLC tissue apoptosis, and immunohistochemistry assays. Functionally, less than 200µM SA had no significant effect on normal human bronchial epithelial cell BEAS-2B cell viability. Furthermore, H460 cell viability was decreased when the SA dose was greater than or equal to 25µM (IC50 = 53.34µM). A549 cell viability was reduced when the SA dose was greater than or equal to 12.5µM (IC50 = 48.21µM). Also, SA repressed NSCLC cell proliferation and boosted cell apoptosis. SA restrained NSCLC cell invasion and tumor microenvironment. Mechanically, SA weakened NSCLC cell proliferation, invasion, and tumor microenvironment, yet this impact was abolished after transfecting pcDNA3.1-TRAF6, which indicated that SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB. Moreover, SA reduced subcutaneous tumor volume and promoted NSCLC tissue apoptosis in vivo. SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB.

Dodecanoyl-L-tryptophan: A Novel Natural Antibiotic Isolated from Mesophotic Sponge-associated Salinicola sp. LHM and its Biological Function

Background: Sponge-associated microbiota plays a crucial role in maintaining host health by providing chemical defense through the synthesis of diverse secondary metabolites. However, research on these secondary metabolites is still in its early stages. Objective: The present study aimed to investigate new natural antibiotics from mesophotic sponge symbiotic microbiota and explore its in vitro antibacterial activity and preliminary biological function. Methods: Bacteria strain Salinicola sp. LHM was isolated from sponge L. birotulata L26 and identified based on the 16S rRNA gene analysis. Subsequently, the strain was fermented using a liquid M9 medium and screened for antibiotics with an antibacterial guiding assay. Extensive chromatographic methods were introduced to isolate the target compound, and its chemical structure was elucidated by spectroscopic analysis (LC-MS, NMR). The minimum inhibitory concentration (MIC) and cytotoxicity experiments evaluated the isolated compound's biological activity. Furthermore, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quartz crystal microbalance with dissipation (QCM-D) were used to study the bactericidal mechanism of DLT. Finally, the preliminary biological function was explored by performing the cell-feeding experiment. Results: We successfully identified one novel natural antibiotic, dodecanoyl-L-tryptophan (DLT), in Salinicola sp. LHM isolated from mesophotic sponge Lotrochota birotulata L26. DLT exhibited potent antibacterial activity against the Bacillus subtilis and Staphylococcus aureus, with MIC values of 32 μM and 16 μM, respectively. The bactericidal tests showed that DLT broke the cell membrane to cause cell death by leaking the cell's inner content. Furthermore, the cell-feeding experiment proved that DLT producer- Salinicola sp. LHM could feed on the inner content of death cells. In addition, DLT also exhibited cytotoxicity against bronchial epithelial cells BEAS-2B, with an EC50 value of 150 μM, indicating a favorable selectivity profile. result: We successfully identified one novel natural antibiotic, dodecanoyl-L-tryptophan (DLT), in Salinicola sp. LHM isolated from mesophotic sponge Lotrochota birotulata L26. DLT exhibited potent antibacterial activity against the Bacillus subtilis and Staphylococcus aureus, with MIC values of 32 μM and 16 μM, respectively. The bactericidal tests showed that DLT broke the cell membrane to cause cell death by leaking the cell's inner content. Furthermore, the cell-feeding experiment proved that DLT producer- Salinicola sp. LHM could feed on the inner content of death cells. In addition, DLT also exhibited cytotoxicity against bronchial epithelial cells BEAS-2B, with an EC50 value of 150 μM, indicating a favorable selectivity profile. Conclusion: This research identified one novel natural antibiotic DLT and provided initial insights into the chemical defense exerted by Salinicola sp. LHM with its secondary metabolite DLT.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host’s antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host’s innate antiviral immunity and thus provides insights into viral pathogenesis.

Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

BackgroundPyroptosis is an inflammatory form of regulated necrosis that has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the role of lipid peroxidation in pyroptosis and its underlying mechanisms in COPD remain unclear. MethodsIn vitro, human bronchial epithelial cells (Beas-2b cells) were exposed to cigarette smoke extract (CSE) for 24 h. In vivo, mice were exposed to cigarette smoke (CS) for 4 weeks. To investigate the role of xCT, we used siRNA and AAV6 to conditionally knock down xCT in vitro and in vivo, respectively. ResultsThe administration of ferrostatin-1 (Fer-1), a ferroptosis inhibitor that inhibits lipid peroxidation, significantly reduced the cytotoxicity of CSE to Beas-2b cells and mitigated inflammatory exudation, lung injury and mucus hypersecretion in mice with CS-induced COPD. Fer-1 suppressed gasdermin D (GSDMD)-mediated pyroptosis caused by CS in vitro and in vivo. However, in Beas-2b cells and the lung epithelial cells of mice, conditional knockdown of xCT (a negative regulatory factor of lipid peroxidation) inhibited the xCT/GPx4 axis, leading to more severe lipid peroxidation and GSDMD-mediated pyroptosis during cigarette smoke exposure. Moreover, we found that CS promoted the degradation of xCT through the ubiquitin proteasome system (UPS) and that treatment with MG132 significantly inhibited the degradation of xCT and downregulated the expression of pyroptosis-related proteins. ConclusionThe results of this study suggested that the ubiquitination-mediated degradation of xCT drives GSDMD-mediated pyroptosis in COPD and is a potential therapeutic target for COPD.

On the dose-response association of fine and ultrafine particles in an urban atmosphere: toxicological outcomes on bronchial cells at realistic doses of exposure at the Air Liquid Interface

Air pollution and particulate matter (PM) are the leading environmental cause of death worldwide. Exposure limits have lowered to increase the protection of human health; accordingly, it becomes increasingly important to understand the toxicological mechanisms on cellular models at low airborne PM concentrations which are relevant for actual human exposure. The use of air liquid interface (ALI) models, which mimic the interaction between airborne pollutants and lung epithelia, is also gaining importance in inhalation toxicological studies. This study reports the effects of ALI direct exposure of bronchial epithelial cells BEAS-2B to ambient PM1 (i.e. particles with aerodynamic diameter lower than 1 μm). Gene expression (HMOX, Cxcl-8, ATM, Gadd45-a and NQO1), interleukin (IL)-8 release, and DNA damage (Comet assay) were evaluated after 24 h of exposure. We report the dose-response curves of the selected toxicological outcomes, together with the concentration-response association and we show that the two curves differ for specific responses highlighting that concentration-response association may be not relevant for understanding toxicological outcomes. Noteworthy, we show that pro-oxidant effects may be driven by the deposition of freshly emitted particles, regardless of the airborne PM1 mass concentration. Furthermore, we show that reference airborne PM1 metrics, namely airborne mass concentration, may not always reflect the toxicological process triggered by the aerosol.These findings underscore the importance of considering different aerosol metrics to assess the toxicological potency of fine and ultrafine particles. To better protect human health additional metrics should be defined, than account for the properties of the entire aerosol mixture including specific as particle size (i.e. particles with aerodynamic diameter lower than 20 nm), the relevant aerosol sources (e.g., traffic combustion, secondary organic aerosol …) as well as their atmospheric processing (freshly emitted vs aged ones).

Hsa_circ_0001776 targeting miR-1265 regulates the development of lung squamous cell carcinoma and clinical significance

Objective: To further explore the role and mechanism of hsa_circ_0001776 and mir-1265 in lung squamous carcinoma by verifying the expression level of hsa_circ_0001776 in plasma, tissues, and cells of lung squamous carcinoma. Methods: Plasma was collected from patients with lung squamous carcinoma treated at Tangshan People's Hospital and healthy individuals from 2020 to 2022. Lung squamous carcinoma tissue microarrays purchased from Shanghai Xinchao Biotechnology Company in 2022. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of hsa_circ_0001776 in lung squamous carcinoma plasma, tissues, and cells, and fluorescence in situ hybridization was used to verify the expression of hsa_circ_0001776 in lung squamous carcinoma. The localization of hsa_circ_0001776 in NCI-H1703 was verified by fluorescence in situ hybridization. The lung squamous carcinoma cells NCI-H1703 and NCI-H226 were cultured in vitro and divided into the circ-negative control (NC) group, hsa_circ_0001776 overexpression group, miR-NC group, miR-1265 mimic group, hsa_circ_0001776+miR-NC group, and hsa_circ_0001776+miR-1265 mimic group.The cell proliferation, motility and apoptosis were detected by the cell counting kit-8 (CCK-8) method, clone formation, Transwell invasion and migration, and scratch assay, and flow cytometry, respectively. The downstream of hsa_circ_0001776 was predicted by circular RNA interactome website, and the interaction between hsa_circ_0001776, miR-1265 was further determined by dual luciferase reporter gene assay, and nude mice subcutaneous tumorigenesis assay detected the growth of transplanted tumors. Results: Fluorescence in situ hybridization results showed that the fluorescence intensity of hsa_circ_0001776 in lung squamous carcinoma tissues was lower than that in paracancerous tissues, and the fluorescence intensity of miR-1265 in lung squamous carcinoma tissues was higher than that in paracancerous tissues (both P＜0.05). The expression level of hsa_circ_0001776 in the plasma of lung squamous carcinoma patients was lower than that in the plasma of healthy people, and the expression level of miR-1265 was higher than that in the plasma of healthy people (both P＜0.05). The expression levels of hsa_circ_0001776 in lung squamous carcinoma cells NCI-H1703, NCI-H226 and SK-MES-1 were lower than that in bronchial epithelial cells BEAS-2B (all P＜0.05), and the relative expression levels of miR-1265 in NCI-H1703 and NCI-H226 were higher than that in human bronchial epithelial cells BEAS -2B (all P＜0.05). The expression of hsa_circ_0001776 was correlated with age, lymph node metastasis, clinical stage, and tumor stage in patients with lung squamous carcinoma (all P＜0.05). Fluorescence in situ hybridization results showed that hsa_circ_0001776 was mainly expressed in the cytoplasm. The results of dual-luciferase reporter assay showed complementary binding of miR-1265 to hsa_circ_0001776. The absorbance values of the hsa_circ_0001776 overexpression group in NCI-H1703 and NCI-H226 cells were lower than that of the circ-NC group (P＜0.05). The number of cell clones in the hsa_circ_0001776 overexpressed group was (52±3) and (53±4), the number of migrating cells was (476±17) and (113±7), the number of invading cells was (100±2) and (184±2), and the cell migration rate was (25.00±4.36)% and (36.02±5.55)%, which were lower than those of the circ-NC group [(104±4) and (106±2), (783±29) and (517±16), (657±45) and (473±9), (48.95±8.69)% and (48.70±1.57)%, all P＜0.05]. The apoptosis rates in the overexpression hsa_circ_0001776 group were (24.77±2.303)% and (19.67±1.16)%, respectively, both higher than those in the circ-NC group [(11.83±1.15)% and (9.50±0.66)%, respectively, both P＜0.05]. MiR-1265 mimic group had a higher apoptotic rate in the NCI-H1703 and NCI-H226 than those of the miR-NC groups (P＜0.05). miR-1265 mimic group had (56±13) and (51±8) cell clones, (556±13) and (405±6) migrating cells, (486±6) and (359±7) invading cells, cell migration rates of (68.56±5.51)%, (81.74±8.04)%, were higher than those of miR-NC group [(31±4) and (21±8), (154±19) and (186±5), (227±6) and (176±7), (25.83±4.26)% and (53.12±4.14) %, all P＜0.05]. The apoptotic rates in the miR-1265 mimic group were (11.83±2.55)% and (17.50±1.05)%, respectively, which were lower than those in the miR-NC group [(32.67±4.44)% and (39.90±2.88)%, respectively, both P<0.05]. The absorbance values of NCI-H1703 and NCI-H226 in the overexpression of hsa_circ_0001776+miR-1265 mimic group were higher than those of the overexpression of hsa_circ_0001776+miR-NC group (P<0.05). The overexpression of hsa_circ_0001776+miR-1265 mimic group had (128±15) and (133±8) cell clones, (623±10) and (310±7) migrating cells, (643±16) and (420±7) invading cells, (66.39±4.46)% cell migration rate and (68.60±3.53)%, were higher than those of the hsa_circ_0001776+miR-NC group [(86±7) and (80±16), (380±11) and (115±5), (152±7) and (94±4), respectively, (31.41±5.91)% and (30.94±0.67)%, all P＜0.05]. The apoptotic rates in the overexpression of hsa_circ_0001776+miR-1265 mimic group were (19.27±0.15)% and (11.53±0.75)%, respectively, both lower than those in the overexpression of hsa_circ_0001776+miR-NC group [(27.77±1.29)% and (18.43±0.71)%, both P＜0.05]. The results of the subcutaneous tumorigenesis assay in nude mice showed that the volume of tumors in the overexpression of hsa_circ_0001776 group was lower than that in the circ-NC group (P＜0.05). Conclusion: hsa_circ_0001776 is downregulated in lung squamous cell carcinoma, and hsa_circ_0001776 can inhibit the development of lung squamous cell carcinoma by targeting miR-1265.

Calcitriol/vitamin D receptor system alleviates PM2.5-induced human bronchial epithelial damage through upregulating mitochondrial bioenergetics in association with regulation of HIF-1α/PGC-1α signaling

PM2.5 exposure causes lung injury by triggering oxidative stress, mitochondrial dysfunction, and modulating HIF-1α signaling. Calcitriol activates VDR, which regulates cellular homeostasis. This study evaluated the protective role of the calcitriol/VDR system in PM2.5-induced damage to BEAS-2B bronchial epithelial cells by reducing oxidative stress, upregulating mitochondrial bioenergetics, and downregulating HIF-1α. We found that the calcitriol/VDR system decreased ROS formation and restored mitochondrial bioenergetics in PM2.5-treated cells. This improvement correlated with reduced HIF-1α nuclear translocation and increased PGC-1α protein and mitochondrial gene expressions. This study is the first to suggest that targeting the calcitriol/VDR system could be a promising pharmacological strategy for mitigating PM2.5-induced lung epithelial damage by promoting mitochondrial bioenergetics and regulating PGC-1α and HIF-1α signaling.

Exosome-transmitted circ_0004664 suppresses the migration and invasion of cadmium-transformed human bronchial epithelial cells by regulating PTEN expression via miR-942-5p

Exosomes play a crucial role in regulating extracellular communication between normal and cancer cells within the tumor microenvironment, thereby affecting tumor progression through their cargo molecules. However, the specific impact of exosomal circular RNAs (circRNAs) on the development of cadmium-induced carcinogenesis remains unclear. To address this, we investigated whether exosomes derived from normal human bronchial epithelial BEAS-2B (N–B2B) cells could transmit circRNA to cadmium-transformed BEAS-2B (Cd–B2B) cells and the potential effects on Cd–B2B cells. Our findings demonstrated a significant downregulation of circ_0004664 in Cd–B2B cells compared to N–B2B cells (P < 0.01). Overexpression of circ_0004664 in Cd–B2B cells led to a significant inhibition of cell migration and invasion (P < 0.01 or P < 0.05). Furthermore, N–B2B cells could transfer circ_0004664 into recipient Cd–B2B cells via exosomes, subsequently inhibiting cell migration and invasion (P < 0.05 or P < 0.01). Mechanistic investigations revealed that exosomal circ_0004664 functioned as a competitive endogenous RNA for miR-942-5p, resulting in an upregulation of PTEN (P < 0.05). Our study highlights the involvement of exosomal circ_0004664 in cell-cell communication during cadmium carcinogenesis, providing a novel insight into the role of exosomal circRNA in this process.

Sea buckthorn extract mitigates chronic obstructive pulmonary disease by suppression of ferroptosis via scavenging ROS and blocking p53/MAPK pathways

Ethnopharmacological relevanceSea buckthorn (Hippophae rhamnoides), a traditional Tibetan medicinal herb, exhibits protective effects against cardiovascular and respiratory diseases. Although Sea buckthorn extract (SBE) has been confirmed to alleviate airway inflammation in mice, its therapeutic effect and underlying mechanism on chronic obstructive pulmonary disease (COPD) requires further clarification. Aim of the studyTo elucidate the alleviative effect and molecular mechanism of SBE on lipopolysaccharides (LPS)/porcine pancreatic elastase (PPE)-induced COPD by blocking ferroptosis. MethodsThe anti-ferroptotic effects of SBE were evaluated in human BEAS-2B bronchial epithelial cells using CCK8, RT-qPCR, western blotting, and transmission electron microscopy. Transwell was employed to detect chemotaxis of neutrophils. COPD model was induced by intranasally administration of LPS/PPE in mice and measured by alterations of histopathology, inflammation, and ferroptosis. RNA-sequencing, western blotting, antioxidant examination, flow cytometry, DARTS, CETSA, and molecular docking were then used to investigate its anti-ferroptotic mechanisms. ResultsIn vitro, SBE not only suppressed erastin- or RSL3-induced ferroptosis by suppressing lipid peroxides (LPOs) production and glutathione (GSH) depletion, but also suppressed ferroptosis-induced chemotactic migration of neutrophils via reducing mRNA expression of chemokines. In vivo, SBE ameliorated LPS/PPE-induced COPD phenotypes, and inhibited the generation of LPOs, cytokines, and chemokines. RNA-sequencing showed that p53 pathway and mitogen-activated protein kinases (MAPK) pathway were implicated in SBE-mediated anti-ferroptotic action. SBE repressed erastin- or LPS/PPE-induced overactivation of p53 and MAPK pathway, thereby decreasing expression of diamine acetyltransferase 1 (SAT1) and arachidonate 15-lipoxygenase (ALOX15), and increasing expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Mechanistically, erastin-induced elevation of reactive oxygen species (ROS) was reduced by SBE through directly scavenging free radicals, thereby contributing to its inhibition of p53 and MAPK pathways. CETSA, DARTS, and molecular docking further showed that ROS-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) may be the target of SBE. Overexpression of NOX4 partially impaired the anti-ferroptotic activity of SBE. ConclusionOur results demonstrated that SBE mitigated COPD by suppressing p53 and MAPK pro-ferroptosis pathways via directly scavenging ROS and blocking NOX4. These findings also supported the clinical application of Sea buckthorn in COPD therapy.

ETS1 and RBPJ transcriptionally regulate METTL14 to suppress TGF-β1-induced epithelial-mesenchymal transition in human bronchial epithelial cells

Asthma is a chronic respiratory disease characterized by airway inflammation and remodeling. Epithelial-mesenchymal transition (EMT) of bronchial epithelial cells is considered to be a crucial player in asthma. Methyltransferase-like 14 (METTL14), an RNA methyltransferase, is implicated in multiple pathological processes, including EMT, cell proliferation and migration. However, the role of METTL14 in asthma remains uncertain. This research aimed to explore the biological functions of METTL14 in asthma and its underlying upstream mechanisms. METTL14 expression was down-regulated in asthmatic from three GEO datasets (GSE104468, GSE165934, and GSE74986). Consistent with this trend, METTL14 was decreased in the lung tissues of OVA-induced asthmatic mice and transforming growth factor-β1 (TGF-β1)-stimulated human bronchial epithelial cells (Beas-2B) in this study. Overexpression of METTL14 caused reduction in mesenchymal markers (FN1, N-cad, Col-1 and α-SMA) in TGF-β1-treated cells, but caused increase in epithelial markers (E-cad), thus inhibiting EMT. Also, METTL14 suppressed the proliferation and migration ability of TGF-β1-treated Beas-2B cells. Two transcription factors, ETS1 and RBPJ, could both bind to the promoter region of METTL14 and drive its expression. Elevating METTL14 expression could reversed EMT, cell proliferation and migration promoted by ETS1 or RBPJ deficiency. These results indicate that the ETS1/METTL14 and RBPJ/METTL14 transcription axes exhibit anti-EMT, anti-proliferation and anti-migration functions in TGF-β1-induced bronchial epithelial cells, implying that METTL14 may be considered an alternative candidate target for the treatment of asthma.

Benzo(a)pyrene promotes the malignant progression of malignant-transformed BEAS-2B cells by regulating YTH N6-methyladenosine RNA binding protein 1 to inhibit ferroptosis

Benzo[a]pyrene (BaP) is associated with the development of lung cancer, but the underlying mechanism has not been completely clarified. Here, we used 10 μM BaP to induce malignant transformation of human bronchial epithelial BEAS-2B cells, named BEAS-2B-T. Results indicated that BaP (6.25, 12.5 and 25 μM) treatment significantly promoted the migration and invasion of BEAS-2B-T cells. Meanwhile, BaP exposure inhibited ferroptosis in BEAS-2B-T, ferroptosis-related indexes Fe2+, malondialdehyde (MDA), lipid peroxidation (LPO) and reactive oxygen species (ROS) decreased significantly. The protein level of ferroptosis-related molecule transferrin receptor (TFRC) decreased significantly, while solute carrier family 7 membrane 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) increased significantly. The intervention of ferroptosis dramatically effected the migration and invasion of BEAS-2B-T induced by BaP. Furthermore, the expression of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) was markedly increased after BaP exposure. YTHDF1 knockdown inhibited BEAS-2B-T migration and invasion by promoting ferroptosis. In the meantime, the contents of Fe2+, MDA, LPO and ROS increased significantly, TFRC was markedly increased, and SLC7A11, FTH1, and GPX4 were markedly decreased. Moreover, overexpression of YTHDF1 promoted BEAS-2B-T migration and invasion by inhibiting ferroptosis. Importantly, knockdown of YTHDF1 promoted ferroptosis and reduced BEAS-2B-T migration and invasion during BaP exposure, and overexpression of YTHDF1 increased migration and invasion of BEAS-2B-T by inhibiting ferroptosis during BaP exposure. RNA immunoprecipitation assays indicated that the binding of YTHDF1 to SLC7A11 and FTH1 markedly increased after YTHDF1 overexpression. Therefore, we concluded that BaP promotes the malignant progression of BEAS-2B-T cells through YTHDF1 upregulating SLC7A11 and FTH1 to inhibit ferroptosis. This study reveals new epigenetic and ferroptosis markers for preventing and treating lung cancer induced by environmental carcinogens.

Acute exposure to dihydroxyacetone promotes genotoxicity and chromosomal instability in lung, cardiac, and liver cell models.

Inhalation exposures to dihydroxyacetone (DHA) occur through spray tanning and e-cigarette aerosols. Several studies in skin models have demonstrated that millimolar doses of DHA are cytotoxic, yet the genotoxicity was unclear. We examined the genotoxicity of DHA in cell models relevant to inhalation exposures. Human bronchial epithelial cells BEAS-2B, lung carcinoma cells A549, cardiomyocyte Ac16, and hepatocellular carcinoma HepG3 were exposed to DHA, and low millimolar doses of DHA were cytotoxic. IC90 DHA doses induced cell cycle arrest in all cells except the Ac16. We examined DHA's genotoxicity using strand break markers, DNA adduct detection by Repair Assisted Damage Detection (RADD), metaphase spreads, and a forward mutation assay for mutagenesis. Similar to results for skin, DHA did not induce significant levels of strand breaks. However, RADD revealed DNA adducts were induced 24 h after DHA exposure, with BEAS-2B and Ac16 showing oxidative lesions and A549 and HepG3 showing crosslink-type lesions. Yet, only low levels of reactive oxygen species or advanced glycation end products were detected after DHA exposure. Metaphase spreads revealed significant increases in chromosomal aberrations in the BEAS-2B and HepG3 with corresponding changes in ploidy. Finally, we confirmed the mutagenesis observed using the supF reporter plasmid. DHA increased the mutation frequency, consistent with methylmethane sulfonate, a mutagen and clastogen. These data demonstrate DHA is a clastogen, inducing cell-specific genotoxicity and chromosomal instability. The specific genotoxicity measured in the BEAS-2B in this study suggests that inhalation exposures pose health risks to vapers, requiring further investigation.

Hazard assessment of hexagonal boron nitride and hexagonal boron nitride reinforced thermoplastic polyurethane composites using human skin and lung cells

Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.

Ezrin Contributes to the Damage of Airway Epithelial Barrier Related to Diabetes Mellitus.

Diabetes mellitus predisposes individuals to respiratory infections. The airway epithelial barrier provides defense against inhaled antigens and pathogens. Ezrin, is a component of the membrane-cytoskeleton that maintains the cellular morphology, intercellular adhesion, and barrier function of epithelial cells. This study aimed to explore the role of ezrin in airway epithelial barrier damage and correlate its expression and activation with diabetes mellitus. This study was performed in a murine model of diabetes mellitus and with human bronchial epithelial BEAS-2B cells using real-time PCR, Western blotting, immunohistochemical and immunofluorescence staining. Ezrin was knocked down in BEAS-2B cells using siRNA. Ezrin phosphorylation levels were measured to determine activation status. The integrity of the airway epithelial barrier was assessed in vivo by characterizing morphological structure, and in vitro in BEAS-2B cells by measuring tight junction protein expression, transepithelial electrical resistance (TER) and permeability. We demonstrated that ezrin expression levels were lower in the lung tissue and airway epithelium of diabetic mice than those in control mice. The morphological structure of the airway epithelium was altered in diabetic mice. High glucose levels downregulated the expression and distribution of ezrin and connexin 43, reduced the expression of tight junction proteins, and altered the epithelial barrier characteristics of BEAS-2B cells. Ezrin knockdown had effects similar to those of high glucose levels. Moreover, a specific inhibitor of ezrin Thr567 phosphorylation (NSC305787) inhibited epithelial barrier formation. These results demonstrate that ezrin expression and activation are associated with airway epithelial damage in diabetes mellitus. These findings provide new insights into the molecular pathogenesis of pulmonary infections in diabetes mellitus and may lead to novel therapeutic interventions for airway epithelial barrier damage.

TMEM2 suppresses TLR3-mediated IFN-β/ISG56/CXCL10 expression in BEAS-2B bronchial epithelial cells.

Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-β and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-β. The expression of TMEM2, IFN-β, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-β, CXCL10, and ISG56, while IFN-β-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. TMEM2 knockdown enhanced TLR3-mediated IFN-β, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.

Metabolic alterations and mitochondrial dysfunction in human airway BEAS-2B cells exposed to vanadium pentoxide

Vanadium pentoxide (V+5) is a hazardous material that has drawn considerable attention due to its wide use in industrial sectors and increased release into environment from human activities. It poses potential adverse effects on animals and human health, with pronounced impact on lung physiology and functions. In this study, we investigated the metabolic response of human bronchial epithelial BEAS-2B cells to low-level V+5 exposure (0.01, 0.1, and 1 ppm) using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Exposure to V+5 caused extensive changes to cellular metabolism in BEAS-2B cells, including TCA cycle, glycolysis, fatty acids, amino acids, amino sugars, nucleotide sugar, sialic acid, vitamin D3, and drug metabolism, without causing cell death. Altered mitochondrial structure and function were observed with as low as 0.01 ppm (0.2 μM) V+5 exposure. In addition, decreased level of E-cadherin, the prototypical epithelial marker of epithelial-mesenchymal transition (EMT), was observed following V+5 treatment, supporting potential toxicity of V+5 at low levels. Taken together, the present study shows that V+5 has adverse effects on mitochondria and the metabolome which may result in EMT activation in the absence of cell death. Furthermore, results suggest that high-resolution metabolomics could serve as a powerful tool to investigate metal toxicity at levels which do not cause cell death.

Toxicity of particles derived from combustion of Ethiopian traditional biomass fuels in human bronchial and macrophage-like cells

The combustion of traditional fuels in low-income countries, including those in sub-Saharan Africa, leads to extensive indoor particle exposure. Yet, the related health consequences in this context are understudied. This study aimed to evaluate the in vitro toxicity of combustion-derived particles relevant for Sub-Saharan household environments. Particles (< 2.5 µm) were collected using a high-volume sampler during combustion of traditional Ethiopian biomass fuels: cow dung, eucalyptus wood and eucalyptus charcoal. Diesel exhaust particles (DEP, NIST 2975) served as reference particles. The highest levels of particle-bound polycyclic aromatic hydrocarbons (PAHs) were found in wood (3219 ng/mg), followed by dung (618 ng/mg), charcoal (136 ng/mg) and DEP (118 ng/mg) (GC–MS). BEAS-2B bronchial epithelial cells and THP-1 derived macrophages were exposed to particle suspensions (1–150 µg/mL) for 24 h. All particles induced concentration-dependent genotoxicity (comet assay) but no pro-inflammatory cytokine release in epithelial cells, whereas dung and wood particles also induced concentration-dependent cytotoxicity (Alamar Blue). Only wood particles induced concentration-dependent cytotoxicity and genotoxicity in macrophage-like cells, while dung particles were unique at increasing secretion of pro-inflammatory cytokines (IL-6, IL-8, TNF-α). In summary, particles derived from combustion of less energy dense fuels like dung and wood had a higher PAH content and were more cytotoxic in epithelial cells. In addition, the least energy dense and cheapest fuel, dung, also induced pro-inflammatory effects in macrophage-like cells. These findings highlight the influence of fuel type on the toxic profile of the emitted particles and warrant further research to understand and mitigate health effects of indoor air pollution.