Bronchial Epithelial Cells Research Articles

Integrated respiratory toxicity of municipal wastewater to human bronchial epithelial cells and 3D bronchospheres

Respiratory symptoms have been reported in wastewater treatment workers and residents living close to sewage treatment plant. However, toxicological research about the respiratory hazards of municipal wastewater is scarce. The present study aims to gain insight into the comprehensive respiratory hazards induced by the contaminant mixtures in municipal wastewater. The integrated respiratory hazards of effluents from four secondary wastewater treatment plants (SWTPs), a tertiary wastewater treatment plant (TTP), and a constructed wetland (CW) were evaluated using normal human bronchial epithelial cells (NHBE) bioassay, and toxicity reduction efficiency of various treatment techniques was analyzed. Effluents caused cytotoxicity, oxidative damage, inflammation response with the increased levels of IL-6 and CXCL8, and impaired barrier integrity with decreased expressions of ZO-1 and occludin in NHBE. Further, the effluents inhibited the development of 3D bronchospheres, increased irregular surface and cell debris, and suppressed the formation of luminal structures. TTP E effluent significantly increased the expression of MUC5AC in bronchospheres. The integrated biomarker response (IBR) of the influent was removed by 40.2% at SWTPs, 18.2% at TTP, and 36.6% at CW, respectively. The IBR of the final effluents from SWTPs, TTP, and CW were 7.2, 7.7, and 7.7, respectively. Significant correlation with toxicity biomarkers was frequently observed for stearyl alcohol, o-cresol, phenanthrene, butylated hydroxytoluene, and dimethyl phthalate. The present study provided human relevant evidence concerning the adverse respiratory effects associated with discharge. The necessity for deep water treatment, performance optimization, and the potential means were suggested for improving water quality and protecting respiratory health.

Progranulin derivative attenuates lung neutrophilic infiltration from diesel exhaust particle exposure.

Air pollutants, such as diesel exhaust particles (DEPs), induce respiratory disease exacerbation with neutrophilic infiltration. Progranulin (PGRN), an epithelial cell and macrophage-derived secretory protein, is associated with neutrophilic inflammation. PGRN is digested into various derivatives at inflammatory sites and is involved in several inflammatory processes. PGRN and its derivatives likely regulate responses to DEP exposure in allergic airway inflammation. To investigate the role of PGRN and its derivatives in the regulation of responses to DEP exposure in allergic airway inflammation. A murine model of allergic airway inflammation was generated in PGRN-deficient mice, and they were simultaneously exposed to DEP followed by intranasal administration of full-length recombinant PGRN (PGRN-FL) and a PGRN-derived fragment (FBAC). Inflammatory status was evaluated by bronchoalveolar lavage fluid and histopathologic analyses. Human bronchial epithelial cells were stimulated with DEPs and house dust mites (HDMs), and the effect of FBAC treatment was evaluated by assessing various intracellular signaling molecules, autophagy markers, inflammatory cytokines, and intracellular oxidative stress. DEP exposure exaggerated neutrophilic inflammation, enhanced IL-6 and CXCL15 secretions, and increased oxidative stress in the murine model; this effect was greater in PGRN-deficient mice than in wild-type mice. The DEP-exposed mice with PGRN-FL treatment revealed no change in neutrophil infiltration and higher oxidative stress status in the lungs. On the contrary, FBAC administration inhibited neutrophilic infiltration and reduced oxidative stress. In human bronchial epithelial cells, DEP and HDM exposure increased intracellular oxidative stress and IL-6 and IL-8 secretion. Decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and increased phosphor-p62 and LC3B expression were also observed. FBAC treatment attenuated oxidative stress from DEP and HDM exposure. FBAC reduced neutrophilic inflammation exaggerated by DEP exposure in a mouse model of allergic airway inflammation by reducing oxidative stress. PGRN and PGRN-derived proteins may be novel therapeutic agents in attenuating asthma exacerbation induced by air pollutant exposure.

Increased Lipocalin 2 detected by RNA sequencing regulates apoptosis and ferroptosis in COPD

BackgroundChronic obstructive pulmonary disease (COPD) is a complex respiratory condition influenced by environmental and genetic factors. Using next-generation sequencing, we aimed to identify dysregulated genes and potential therapeutic targets for COPD.MethodsPeripheral blood leukocyte RNA profiles from COPD patients and healthy controls were analyzed using next-generation sequencing. Key genes involved in COPD pathogenesis were identified through protein–protein interaction network analysis. In vitro, bronchial epithelial cells treated with cigarette smoke extract (CSE) were used to study the effects on gene expression, cell viability, apoptosis, and ferroptosis. Additionally, Lipocalin 2 (LCN2) inhibition experiments were conducted to elucidate its role in COPD-related cellular processes.ResultsAnalysis of RNA profiles revealed consistent downregulation of 17 genes and upregulation of 21 genes across all COPD groups. Among these, Cathelicidin Antimicrobial Peptide(CAMP), Defensin Alpha 4(DEFA4), Neutrophil Elastase(ELANE), LCN2 and Lactotransferrin(LTF) were identified as potentially important players in COPD pathogenesis. Particularly, LCN2 exhibited a close association with COPD and was found to be involved in cellular processes. In vitro experiments demonstrated that CSE treatment significantly increased LCN2 expression in bronchial epithelial cells in a concentration-dependent manner. Moreover, CSE-induced apoptosis and ferroptosis were observed, along with alterations in cell viability, Glutathione content, Fe2 + accumulation, ROS: Reactive Oxygen Species and Malondialdehyde levels, Lactate Dehydrogenase(LDH) release and Glutathione Peroxidase 4(GPX4) expression. Inhibition of LCN2 expression partially reversed these effects, indicating the pivotal role of LCN2 in COPD-related cellular processes.ConclusionOur study identified six candidate genes: CAMP, DEFA4, ELANE, LCN2, and LTF were upregulated, HSPA1B was downregulated. Notably, LCN2 emerges as a significant biomarker in COPD pathogenesis, exerting its effects by promoting apoptosis and ferroptosis in bronchial epithelial cells.

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.

Synergistic Action of Thymol-citral is Associated with Cell Cycle Arrest and Intracellular ROS Generation in A549 Cells

Objective: NSCLC is the predominant form of lung cancer, often exhibiting resistance to chemotherapy. Thymol and citral have shown promise as anticancer agents in different cancer cell lines but have not been evaluated in combination against NSCLC. Hence, we planned to investigate the anticancer effect of thymol-citral combination and explore its mechanisms of action against A549 cells. Methods: A549 cells were exposed to varying concentrations of thymol and citral, alone and in combination. Cell proliferation, plasma membrane integrity, apoptotic markers, reactive oxygen species (ROS) levels, cell cycle distribution, senescence induction, and migration potential were assessed. Additionally, in vitro safety was evaluated in human bronchial epithelial cells (HBECs) and human red blood cells (RBCs). Results: Thymol and citral showed synergistic action against A549 cells, with a CI value of 0.75. After 24 h, they induced apoptosis, caused G0/G1 phase arrest, and increased ROS levels, suggesting oxidative stress as the mechanism. This combination also induced cell senescence, significantly inhibited A549 cell migration, and was non-toxic to human RBCs and HBECs. Conclusion: Overall, the thymol-citral synergistic combination was found to be a safe and effective therapy option for non-small cell lung cancer.

TRIM7 knockdown protects against LPS-induced autophagy, ferroptosis, and inflammatory responses in human bronchial epithelial cells.

Asthma is one of the most common respiratory diseases in pediatric department. Several asthma-associated events including inflammatory responses, autophagy, and ferroptosis have been identified as typical pathological processes. TRIM7 is a member of TRIM proteins family associated with several types of diseases. Nevertheless, its role in asthma is still elusive. The current research showed that TRIM7 was involved in the pathogenesis of asthma mainly by regulating the Akt signaling pathway. In detail, we found that TRIM7 was highly expressed in patients with asthma and in an in vitro model of asthma. The following analysis indicated that TRIM7 knockdown attenuated the expression and secretion of inflammatory cytokines including TNF-α, IL-1β and IL-6 in lipopolysaccharide (LPS)-exposed human bronchial epithelial cells (HBECs). Meanwhile, knockdown of TRIM7 exerted inhibitory effects on LPS-induced autophagy and ferroptosis. Further mechanistic studies showed that TRIM7 knockdown inhibited LPS-induced activation of Akt pathway, while overexpression of Akt attenuated the inhibitory effects of TRIM7 knockdown on LPS-exposed HBECs. Collectively, we reported here that TRIM7 knockdown inhibited LPS-induced autophagy, ferroptosis, and inflammatory cytokine secretion in HBECs via regulating the Akt pathway, providing a new insight into the strategies for improving asthma treatments.

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK’s versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK’s performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK’s ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells.

Complement activation and lung injury in Japanese patients with COVID-19: a prospective observational study

Background The production of inflammatory cytokines is reportedly increased in patients with coronavirus disease 2019 (COVID-19), causing the migration of neutrophils and monocytes to lung tissues. This disrupts the air–blood barrier by damaging the bronchial epithelial and vascular endothelial cells. As multiorgan dysfunction in sepsis is considered to be partly caused by complement activation, which can cause lung injury in patients with COVID-19. There are limited studies examining the link between complement activation in patients with COVID-19. This study aimed to AQinvestigate the association of complement activation with the pathophysiology of COVID-19. Twenty-seven patients with COVID-19 were enrolled in this study and classified into two groups depending on the indication for mechanical ventilation. Plasma complement factors (C3a, C5a, Ba, and sC5b-9), complement regulators (sCD59 and factor H), interleukin-6 (IL-6), and syndecan-1 levels were measured using Enzyme-linked immunosorbent assay (ELISA). Results: All complement factors and regulators, IL-6, and syndecan-1 levels were significantly elevated in patients with COVID-19 compared with those in healthy controls. C5a and sC5b-9 levels were decreased significantly in the invasive mechanical ventilation (IMV) group compared with those in the non-IMV group. Syndecan-1 levels were significantly increased in the IMV group compared with those in the non-IMV group. Conclusions: Complement activation is an exacerbating factor for lung injury in patients with COVID-19. Complement factors are nonessential predictors of mechanical ventilation; however, syndecan-1 could be a biomarker of COVID-19 severity in patients.

Interferon Epsilon-Mediated Antiviral Activity Against Human Metapneumovirus and Respiratory Syncytial Virus

Background: Interferon epsilon (IFN-ε) is a type I IFN that plays a critical role in the host immune response against pathogens. Despite having demonstrated antiviral activity in macrophages and mucosal tissues such as the female reproductive tract and the constitutive expression in mucosal tissues such as the lung, the relevance of IFN-ε against respiratory viral infections remains elusive. Results: We present, for the first time, the expression of IFN-ε in alveolar epithelial cells and primary human bronchial epithelial cells grown in an air–liquid interface (ALI) in response to human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) infection. The molecular characterization of the IFN-ε induction by the viruses indicates that the expression of RIG-I is necessary for an optimal IFN-ε expression. Furthermore, treatment of the airway epithelial cells with rhIFN-ε induced the expression of IFN-stimulated genes (ISGs) and significantly restricted the viral replication of HMPV and RSV. Conclusions: These findings underscore the relevance of IFN-ε against viral infections in the respiratory tract.

Interaction between CD244 and SHP2 regulates inflammation in chronic obstructive pulmonary disease via targeting the MAPK/NF-κB signaling pathway.

This study delved into the interplay between CD244 and Src Homology 2 Domain Containing Phosphatase-2 (SHP2) in chronic obstructive pulmonary disease (COPD) pathogenesis, focusing on apoptosis and inflammation in cigarette smoke extract (CSE)-treated human bronchial epithelial (HBE) cells. Analysis of the GSE100153 dataset identified 290 up-regulated and 344 down-regulated differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) highlighted the turquoise module had the highest correlation with COPD samples. Functional enrichment analysis linked these DEGs to critical COPD processes and pathways like neutrophil degranulation, protein kinase B activity, and diabetic cardiomyopathy. Observations on CD244 expression revealed its upregulation with increasing CSE concentrations, suggesting a dose-dependent relationship with inflammatory cytokines (IL-6, IL-8, TNF-α). CD244 knockdown mitigated CSE-induced apoptosis and inflammation, while overexpression exacerbated these responses. Co-immunoprecipitation (Co-IP) confirmed the physical interaction between CD244 and SHP2, emphasizing their regulatory connection. Analysis of Concurrently, the Nuclear Factor-kappa B (NF-κB) and Mitogen-activated protein kinase (MAPK) signaling pathways showed that modulating CD244 expression impacted key pathway components (p-JNK, p-IKKβ, p-ERK, p-P38, p-lkBα, p-P65), an effect reversed upon SHP2 knockdown. These findings underscore the pivotal role of the CD244/SHP2 axis in regulating inflammatory and apoptotic responses in CSE-exposed HBE cells, suggesting its potential as a therapeutic target in COPD treatment strategies.

Nrf-2/HO-1 activation protects against oxidative stress and inflammation induced by metal welding fume UFPs in 16HBE cells

As one of the main occupational hazards, welding fumes can cause oxidative damage and induce series of diseases, such as COPD or asthma. To clarify the effects of the metal fume ultrafine particulates (MF-UFPs) of welding fumes on oxidative damage, UFPs were collected by melt inert gas (MIG) and manual metal arc (MMA) welding, and the composition was confirmed. Human bronchial epithelial 16HBE cells were treated with 0–1000 µg/cm2 MF-UFPs to analyse the cytotoxicity, oxidative stress and cytokines. The protein and mRNA expression of Keap1-Nrf-2/antioxidant response elements (AREs) signalling pathway components were also analysed. After 4 h of treatment, the cell viability decreased 25% after 33.85 and 32.81 µg/cm2 MIG/MMA-UFPs treated. The intracellular ATP concentrations were also decreased significantly, while LDH leakage was increased. The decreased mitochondrial membrane potential and increased ROS suggested the occurrence of oxidative damage, and the results of proteome profiling arrays also showed a significant increase in IL-6 and IL-8. The expression of AREs which related to antioxidant and anti-inflammatory were also increased. These results indicate that the MF-UFPs can cause oxidative stress in 16HBE cells and activate the Nrf-2/ARE signalling pathway to against oxidative damage.

Air Fixation and AFM: A Comparative Study of Nanoparticle-Induced Topographical Changes in Lung Cells

Gold nanoparticles (AuNPs) have emerged as promising agents in biomedical applications due to their unique physicochemical properties. This study investigates the cellular interactions of AuNPs with A549 (non-small cell lung adenocarcinoma) and BEAS-2B (normal bronchial epithelial) cell lines. AuNPs were synthesized via the citrate reduction method, resulting in 20, 50, and 70 nm particles. Cells were incubated with AuNPs for increasing durations (30 minutes, 4 hours, and 24 hours). Post-incubation, cells were washed with PBS, air-fixed, and subsequently analyzed using Atomic Force Microscopy (AFM) to obtain detailed topographical maps. AFM imaging revealed distinct interactions between AuNPs and the two cell lines. A549 cells displayed darker regions on the cell surface, indicative of topographical depressions likely resulting from nanoparticle-induced membrane collapse. In contrast, BEAS-2B cells did not exhibit such depressions, which is consistent with the literature that suggests cancer cells are mechanically softer than normal cells. The surface roughness analysis results indicated that the preservation of surface integrity post-fixation validates the air-fixation methodology for obtaining reliable mechanical data from AFM analyses.

Benzo(a)pyrene-loaded gypsum dust-induced apoptosis of human bronchial epithelial cells through the Ahr signaling pathway

Benzo(a)pyrene-loaded gypsum dust-induced apoptosis of human bronchial epithelial cells through the Ahr signaling pathway

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.

Acetamiprid elicits oxidative stress, pro-inflammatory response, and cellular proliferation in human bronchial epithelial cells in vitro and in silico: alleviative implications of the mixture of heat-killed Lactobacillus strains

BackgroundAcetamiprid (ACE), a neonicotinoid insecticide, has been extensively used to control pests in agricultural and industrial environments. It has been reported that ACE is detrimental to the lungs. Nevertheless, the extent to which the activation of oxidative stress, inflammation, and cellular proliferation contributes to the pulmonary toxicity induced by ACE exposure remains insufficiently understood. This study explored the mechanism of toxicological consequences after ACE exposure in bronchial epithelial cells (BEAS-2B cells). The research also examined the potential ameliorative effects of the mixture of heat-killed Lactobacillus delbrueckii and Lactobacillus fermentum (HKL) on the toxicities of ACE.ResultsFollowing 14 days of exposure to ACE at 0.5 and 1 μM, oxidative stress was induced, as evidenced by the decreased levels of reduced glutathione, catalase, glutathione peroxidase, and superoxide dismutase, along with increased levels of malondialdehyde. Also, ACE exposure results in overexpression and raised protein levels of the IL-25, NF-κB1, NF-κB2, IL-33, TSLP, and NF-κB target genes, which induce inflammatory responses. In addition, ACE boosted Ki-67-positive BEAS-2B cells. The molecular docking of ACE with target genes and their proteins demonstrated a potent binding affinity, further supported by the presence of hydrophobic contacts, electrostatic interactions, and hydrogen bonds. The post-treatment of HKL following the ACE (1 μM) exhibited its antioxidant, anti-inflammatory, and antiproliferative activities in suppressing ACE-induced toxicity.ConclusionsOur research revealed that ACE toxicity in BEAS-2B cells is caused by driving oxidative stress, pro-inflammatory response, and cellular proliferation. This study would give us a strategy to alleviate ACE-induced lung impairment by heat-killed probiotic supplements. As a result, dietary supplements that contain these microorganisms may potentially be beneficial in countries with high levels of pesticide contamination.

Chrysene contribution to bronchial asthma: Activation of TRPA1 disrupts bronchial epithelial barrier via ERK pathway

BackgroundElevated polycyclic aromatic hydrocarbon (PAH) levels are associated with exacerbation of asthma. Chrysene is one of the most prevalent unsubstituted PAHs in the environment. Transient receptor potential ankyrin 1 (TRPA1) can be used as a chemoreceptor to detect inhaled stimuli and plays an important role in the occurrence and deterioration of asthma. Whether exposure to a high concentration of chrysene in the environment can activate TRPA1 and contribute to the development of asthma, potentially through the dysfunction of the bronchial epithelial barrier, remains unclear. MethodsA cell-based assay was performed to verify the downregulation of the expression of E-cadherin and tight junction (TJ) proteins by chrysene in bronchial epithelial cells to explore the role of chrysene-mediated TRPA1 activation in the regulation of TJ protein expression through the extracellular signal-regulated protein kinase (ERK) pathway. Animal tests were conducted to determine whether chrysene could enhance airway hyperresponsiveness (AHR) induced by house dust mites (HDMs) and disrupt barrier function, thereby contributing to asthma. ResultsThe cell-based assay revealed that chrysene could disrupt the function of the bronchial epithelial barrier and decrease the expression levels of E-cadherin, zonula occludens-1 (ZO-1), occludin, and claudin-5 through the ERK pathway. Chrysene induced airway epithelial barrier dysfunction primarily through TRPA1 instead of transient receptor potential vanilloid 1. TRPA1 knockdown was able to attenuate chrysene-induced downregulation of TJ protein expression and downregulate ERK activation (p-ERK). Compared with exposure to HDM alone, coexposure to chrysene and HDM resulted in an increased incidence of AHR, disruption of barrier function, and eosinophilic inflammatory responses in a mouse model of asthma. Coexposure to chrysene and HDM increased TRPA1 expression. The animal test verified that the TRPA1 inhibitor HC030031 could suppress chrysene and HDM-induced asthma in mice. ConclusionsOur findings showed that chrysene contributed to the breakdown of the function of the bronchial epithelial barrier through the TRPA1–ERK axis and therefore acted as an adjuvant to contribute to asthma.

Upregulated DNMT3a coupling with inhibiting p62-dependent autophagy contributes to NNK tumorigenicity in human bronchial epithelial cells

NNK, formally known as 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanoe, is a potent chemical carcinogen prevalent in cigarette smoke and is a key contributor to the development of human lung adenocarcinomas. On the other hand, autophagy plays a complex role in cancer development, acting as a "double-edged sword" whose impact varies depending on the cancer type and stage. Despite this, the relationship between autophagy and NNK-induced lung carcinogenesis remains largely unexplored. Our current study uncovers a marked reduction in p62 protein expression in both lung adenocarcinomas and lung tissues of mice exposed to cigarette smoke. Interestingly, this reduction appears to be contingent upon the activity of extrahepatic cytochrome P450 (CYP450), revealing that NNK metabolic activation by CYP450 enzyme escalates its potential to induce p62 downregulation. Further mechanistic investigations reveal that NNK suppresses autophagy by accelerating the degradation of p62 mRNA, thereby promoting the malignant transformation of human bronchial epithelial cells. This degradation process is facilitated by the hypermethylation of the Human antigen R (HuR) promoter, resulting in the transcriptional repression of HuR - a key regulator responsible for stabilizing p62 mRNA through direct binding. This hypermethylation is triggered by the activation of ribosomal protein S6, which is influenced by NNK exposure and subsequently amplifies the translation of DNA methyltransferase 3 alpha (DNMT3a). These findings provide crucial insights into the nature of p62 in both the development and potential treatment of tobacco-related lung cancer.

An added value of Azithromycin: mitigation of Doxorubicin associated oxidative damage and genotoxicity in normal human bronchial epithelium cells.

Doxorubicin, a well-known and widely used antineoplastic agent with direct ROS-accumulating activity, has proven effective in treating various cancer types. However, its non-specific cytotoxicity towards non-cancerous cells prompts concerns regarding potential adverse effects. Azithromycin is an antibiotic for treating bacterial infections and an anti-inflammatory agent, particularly beneficial in managing respiratory conditions like bronchitis and sinusitis. Despite azithromycin's well-documented antibacterial properties, its potential cellular/genomic protective effects remain unexplored. As an in vitro model, BEAS-2B cells (normal human bronchial epithelium cells) were employed in the present study to assess whether azithromycin possesses any protective properties against doxorubicin-induced cellular toxicity. Cells in pre-treatment culture were treated to various amounts of azithromycin (3.125, 6.25, 12.5, 25, and 50 μg/mL) in combination with doxorubicin at IC50 (0.08 μg/mL). Doxorubicin at 0.08 μg/mL highlighted cytotoxicity, oxidative stress, and genotoxicity. Azithromycin at 25 and 50 μg/mL markedly modulated oxidative stress and genomic damage by decreasing the ROS and LPO amounts, and suppressing DNA fragmentation in the comet assay parameters. Consequently, azithromycin may be regarded as a cytomodulating, antigenotoxic, and antioxidant agent.

Androgens have therapeutic potential in T2 asthma by mediating METTL3 in bronchial epithelial cells

Studies have shown that androgens can alleviate the symptoms of T2 asthma and are inversely correlated with the severity of allergic asthma. METTL3, a crucial component of m6A modification, mitigates the development of T2 asthma by inhibiting Th2 cell differentiation. However, the impact of androgens, such as dihydrotestosterone (DHT), on the progression of T2 asthma through METTL3 has yet to be investigated. At the clinical level, patients with T2 asthma exhibited reduced levels of DHT and METTL3 mRNA, along with increased levels of 17β-estradiol (E2). DHT and METTL3 were found to be negatively associated with the severity of T2 asthma, while E2 was positively associated with it. Administration of DHT and E2 in induced T2 asthma mouse models showed that DHT improved lung function, reduced airway inflammation, and inhibited Th2 cell differentiation. Interestingly, DHT reversed the damage to METTL3, whereas E2 had the opposite effect. In vitro studies of mouse bronchial epithelial cells (BECs) confirmed that METTL3-dependent m6A modification inhibited the T2 inflammatory response, and DHT inhibited Th2 cell differentiation in T2 asthma by promoting METTL3 expression in BECs. In conclusion, our study suggests that DHT has therapeutic potential for T2 asthma by regulating METTL3 in BECs.

The Punicalagin Compound Mitigates Bronchial Epithelial Cell Senescence Induced by Cigarette Smoke Extract through the PAR2/mTOR Pathway.

Tobacco smoke is an important inducer of airway epithelial cell aging. Punicalagin(PCG) is a natural anti-aging compound. The effect of PCG on tobacco smoke-induced airway epithelial cell senescence is unknown. Our study investigated whether PCG can treat the human bronchial epithelial cell line (BEAS-2B) aging by inhibiting the protease-activated receptor 2 (PAR2)/m- TOR pathway. Bioinformatics techniques were used to analyze the potential biological functions of PAR2. Molecular dynamics evaluated the binding ability of PCG and PAR2. The CCK8 assay was used to detect the cytotoxicity of CSE and PCG. The activity of the PAR2/mTOR pathway and the expression of the characteristic aging markers p16, p21, and SIRT1 are detected by qRT-PCR and Western blotting. Cell senescence was observed by Senescence-associated β-galactosidase (SA-β-gal) staining. The senescence-associated secretory phenotype (SASP): concentrations of interleukin IL-6, IL-8, and TNF- α were detected by ELISA. The GSE57148 bioinformatics analysis dataset showed that PAR2 regulates lung senescence through the mTOR signaling pathway. Molecular dynamics results found that PCG and PAR2 had a strong and stable binding force. CSE induces BEAS-2B cell senescence and activates the PAR2/mTOR pathway. Inhibition of PAR2 mitigated the senescence changes. In addition, PCG's pretreatment can significantly alleviate CSE-induced BEAS-2B cell senescence while inhibiting the PAR2/mTOR pathway. PCG has a therapeutic effect on the senescence of airway epithelial cells.