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This paper reports an experimental study on the compatibility of human bronchial epithelial (HBE) cells in a collagen–alginate bioink. The compatibility was assessed using the culture well method with three bioink compositions prepared from a 10% alginate solution and neutralized TeloCol-10 mg/mL collagen stock solution. Cell viability, quantified by (live cell count—dead cell count)/live cell count within the HBE cell-laden hydrogel, was evaluated using the live/dead assay method from Day 0 to Day 6. Experimental results demonstrated that the collagen–alginate 4:1 bioink composition exhibited the highest cell viability on Day 6 (85%), outperforming the collagen–alginate 1:4 bioink composition and the alginate bioink composition, which showed cell viability of 75% and 45%, respectively. Additionally, the live cell count was highest for the collagen–alginate 4:1 bioink composition on Day 0, a trend that persisted through Days 1 to 6, underscoring its superior performance in maintaining cell viability and promoting cell proliferation. These findings show that the compatibility of HBE cells with the collagen–alginate 4:1 bioink composition was higher compared with the other two bioink compositions. [ABSTRACT FROM AUTHOR]

Objective To explore the impact of herpes simplex virus type 1 (HSV-1) mutant strain M6 on macrophage-mediated immune responses after infecting human bronchial epithelial cells (16HBE cells). Methods, HSV-1 infection of 16HBE cells was conducted to assess cytokine alterations in the culture medium; Macrophages were co-cultured with supernatants from HSV-1-infected 16HBE cells and subsequently reintroduced into mice via tail vein infusion. Cytokine expression levels in mouse lymph nodes, changes in spleen T cell proportions, mouse neutralizing antibody levels, and specific T cell responses were measured on days 1, 3, 7, 28, 56, and 90., Results Following infection of 16HBE cells with the HSV-1 mutant strain, cytokine expression in the supernatant, associated with macrophage recruitment and activation, was elevated, albeit slightly lower than that observed in the wild-type strain group. Following the tail vein infusion experiment, the mutant strain group exhibited temporal changes in the proportions of inflammatory factors, chemokines, and T cells within the mouse lymph nodes, resulting in weaker humoral immunity compared to the wild-type strain group, yet eliciting stronger specific T cell responses, with only a few parameters showing significant differences from the wild-type strain group (P < 0.05). Conclusion 16HBE cells infected with the HSV-1 mutant strain M6 can release cytokines that recruit and activate macrophages, allowing macrophages to carry specific activation information of the HSV-1 mutant strain, activating the host's immune system and inducing the host's Humoral immunity and cellular immunity. [ABSTRACT FROM AUTHOR]

Waterpipe smoking is increasingly popular and understanding how chemicals found in hookah smoke may be harmful to human bronchial epithelial cells is of great importance. 4,4'-Oxydianiline (ODA), is an aromatic amine which is present at comparatively high levels in hookah smoke. The metabolism and the subsequent toxicity of ODA in human bronchial epithelial cells remains unknown. Given that ODA is an aromatic amine, we hypothesized that ODA is N -acetylated and induces DNA damage following exposure to immortalized human bronchial epithelial cells (BEP2D cells). We measured the N -acetylation of ODA to mono-acetyl-ODA and the N -acetylation of mono-acetyl-ODA to diacetyl-ODA by BEP2D cells following separation and quantitation by high performance liquid chromatography. For ODA, the apparent K M in cells was 12.4 ± 3.7 µM with a V max of 0.69 ± 0.03 nmol/min/106 cells, while for mono-acetyl-ODA, the apparent K M was 111.2 ± 48.3 µM with a V max of 17.8 ± 5.7 nmol/min/106 cells ODA exposure for 24 h resulted in DNA damage to BEP2D cells following concentrations as low as 0.1 µM as measured by yH2Ax protein expression These results demonstrate that ODA, the most prevalent aromatic amine identified in hookah smoke, is N -acetylated and induces DNA damage in human bronchial epithelial cells. • 4,4'-Oxydianiline (ODA) is present at comparatively high levels in hookah smoke. • N -acetylation of ODA was determined in human bronchial epithelial cells. • ODA exposure resulted in DNA damage to human bronchial epithelial cells. [ABSTRACT FROM AUTHOR]

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin β1, and integrin β4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 pathway.

High concentrations of H2O2, indicative of increased oxidative stress in the lung, are observed in the exhaled breath of individuals affected by different respiratory diseases. Therefore, measuring H2O2 in exhaled breath represents a promising and non-invasive approach for monitoring the onset and progression of these diseases. Herein, we have developed an innovative, inexpensive, and easy-to-use device for the measurement of H2O2 in exhaled breath. The device is based on a silver layer covered with an electrodeposited thin film of chitosan, that ensures the wettability of the sensor in a humid atmosphere. The s-ensor was calibrated in the aerosol phase using both phosphate buffer solution and cell culture medium. In the buffer, a sensitivity of 0.110 ± 0.0042 μA μM−1 cm−2 (RSD: 4%) and a limit of detection of 30 μM were calculated, while in the cell culture medium, a sensitivity of 0.098 ± 0.0022 μA μM−1 cm−2 (RSD 2%) and a limit of detection of 40 μM were obtained. High selectivity to different interfering species was also verified. The sensor was further tested versus an aerosol phase obtained by nebulizing the culture medium derived from human bronchial epithelial cells that had been exposed to pro-oxidant and antioxidant treatments. The results were comparable with those obtained using the conventional cytofluorimetric method. Finally, sensor was tested in real exhaled breath samples and even after undergoing physical deformations. Data herein presented support that in future applications this device can be integrated into face masks allowing for easy breath monitoring.

ABSTRACTAirborne particulate matter (PM) is a major health hazard worldwide and is a key factor in lung cancer, which remains the most common type of malignancy and the leading cause of cancer-related deaths. DNA methylation is a critical mechanism underlying the detrimental effects of PM, however, the molecular link between PM exposure and lung cancer remains to be elucidated. N-α-acetyltransferase 10 (NAA10) is involved in the cell cycle, migration, apoptosis, differentiation, and proliferation. In order to investigate the role of NAA10 in PM-induced pathogenesis processes leading to lung cancer, we determined the expression and methylation of NAA10 in normal human bronchial epithelial (NHBE) cells treated with PM10, PM10-polycyclic aromatic hydrocarbons (PAH), and PM2.5 and evaluated the prognostic value of the NAA10 methylation status in lung cancer patients. Exposure to all PM types significantly increased the expression of NAA10 mRNA and decreased the methylation of the NAA10 promoter in NHBE cells compared with the mock-treated control. NAA10 hypomethylation was observed in 9.3% (13/140) of lung cancer tissue samples and correlated with NAA10 transcriptional upregulation. Univariate and multivariate analyses revealed that NAA10 hypomethylation was associated with decreased survival of patients with lung cancer. Therefore, these results suggest that PM-induced hypomethylation of the NAA10 may play an important role in the pathogenesis of lung cancer and may be used as a potential prognostic biomarker for lung cancer progression. Further studies with large numbers of patients are warranted to confirm our findings.

Macrophages play a role in preventing inflammation in the respiratory tract. To investigate the mechanisms that lead to tolerance in macrophages, we examined the crosstalk between airway epithelial cells (AECs) and macrophages using a 2D coculture model. Culture of macrophages with AECs led to a significant inhibition of LPS induced pro-inflammatory responses. More importantly, AECs induced the secretion of TGF-β and IL-10 from macrophages even in the absence of LPS stimulation. In addition, the expression of inhibitory molecule, CD200R was also upregulated on AEC exposed macrophages. Furthermore, the AECs exposed macrophages induced significantly increased level of T regulatory cells. Investigation into the possible mechanisms indicated that a combination of growth factor, G-CSF, and metabolites, Kynurenine and lactic acid produced by AECs is responsible for inducing tolerance in macrophages. Interestingly, all these molecules had differential effect on macrophages with G-CSF inducing TGF-β, Kynurenine elevating IL-10, and lactic acid upregulating CD200R. Furthermore, a cocktail of these factors/metabolites induced similar changes in macrophages as AEC exposure. Altogether, these data identify factors secreted by AECs that enhance tolerance in the respiratory tract. These mediators thus have the potential to be used for therapeutic purposes to modulate respiratory inflammation following local viral infections and lung diseases.

Pneumonia caused by multi-drug-resistant Klebsiella pneumoniae (MDR-Kpneu) poses a major public health threat, especially to immunocompromised or hospitalized patients. This study aimed to determine the immunostimulatory effect of the Toll-like receptor 5 ligand flagellin on primary human lung epithelial cells during infection with MDR-Kpneu. Human bronchial epithelial (HBE) cells, grown on an air–liquid interface, were inoculated with MDR-Kpneu on the apical side and treated during ongoing infection with antibiotics (meropenem) and/or flagellin on the basolateral and apical side, respectively; the antimicrobial and inflammatory effects of flagellin were determined in the presence or absence of meropenem. In the absence of meropenem, flagellin treatment of MDR-Kpneu-infected HBE cells increased the expression of antibacterial defense genes and the secretion of chemokines; moreover, supernatants of flagellin-exposed HBE cells activated blood neutrophils and monocytes. However, in the presence of meropenem, flagellin did not augment these responses compared to meropenem alone. Flagellin did not impact the outgrowth of MDR-Kpneu. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-Kpneu-infected primary human bronchial epithelium, which is associated with the release of mediators that activate neutrophils and monocytes. Topical flagellin therapy may have potential to boost immune responses in the lung during pneumonia. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of DNA damage protein targeting VCP (DVC1) in DNA protein crosslink repair induced by nitrogen mustard in human bronchial epithelial cells (16HBE). Methods DVC1 siRNA was used to interfere its expression in 16HBE. Then the cells were exposed to 50 μmol/L nitrogen mustard, and the expression levels of DNA damage repair proteins were detected by Western blotting. The total content of DNA-protein crosslink was determined by fluorescence microplate analyzer from 1 to 24 h. The crosslink content of DNA with O6-methylguanine-DNA methyltransferase (MGMT), a methyltransferase, was detected by Slot blotting. The level of ubiquitinated MGMT was detected by immunoprecipitation. After GST-DVC1 fusion protein was constructed, GST pull-down was used to detect the binding of DVC1 with MGMT and ubquitin. Results After si-DVC1 intervention, the total content of DNA-protein crosslink was increased, and exposure to nitrogen mustard decreased the level of prototype of MGMT, but increased the level of ubiquitinated MGMT. DVC1 could bind to ubquitin, and si-DVC1 intervention further enhanced the ubiquitination of MGMT, as well as the crosslink between MGMT and DNA, which was similar with NSC697923 (an E2 ubiquitin-conjugating inhibitor) and MG132 (a proteasome inhibitor) treatment. The expression of Rad51 and Ku70, 2 DNA repair proteins, were increased and decreased respectively by si-DVC1 interference. Conclusion Clearance of DNA-protein crossslink is depend on ubiquitination of MGMT. DVC1 promotes the clearance of nitrogen mustard induced DNA-protein crossslink by interacting with ubiquitin, and maintain the ability of homologous recombination repair.

This paper reports an experimental study on the compatibility of human bronchial epithelial (HBE) cells in a collagen–alginate bioink. The compatibility was assessed using the culture well method with three bioink compositions prepared from a 10% alginate solution and neutralized TeloCol-10 mg/mL collagen stock solution. Cell viability, quantified by (live cell count—dead cell count)/live cell count within the HBE cell-laden hydrogel, was evaluated using the live/dead assay method from Day 0 to Day 6. Experimental results demonstrated that the collagen–alginate 4:1 bioink composition exhibited the highest cell viability on Day 6 (85%), outperforming the collagen–alginate 1:4 bioink composition and the alginate bioink composition, which showed cell viability of 75% and 45%, respectively. Additionally, the live cell count was highest for the collagen–alginate 4:1 bioink composition on Day 0, a trend that persisted through Days 1 to 6, underscoring its superior performance in maintaining cell viability and promoting cell proliferation. These findings show that the compatibility of HBE cells with the collagen–alginate 4:1 bioink composition was higher compared with the other two bioink compositions.

Background: Studies have shown that fine particulate matter (PM2.5) remains a significant problem in developing countries and plays a critical role in the onset and progression of respiratory illnesses. Circular RNAs (circRNAs) are involved in many pathophysiological processes,but their relationship to PM2.5 pollution is largely unexplored. Objectives: To elucidate the functional role of hsa_circ_0000992 in PM2.5-induced inflammation in a human bronchial epithelial cell line (16HBE) and to clarify whether the competing endogenous RNA (ceRNA) mechanism is involved in the interrelationships between hsa_circ_0000992 and hsa-miR-936 and the inflammatory signaling pathways. Methods: Detection of inflammatory factors in 16HBE cells exposed to PM2.5 by RT-qPCR and ELISA.High throughput sequencing and bioinformatics analysis methods were used to screen circRNA.The bioinformatics analysis method western blotting and dual-luciferase reporter gene system were used to verify mechanisms associated with circRNA. Results: PM2.5 cause inflammation in the 16HBE cells. High throughput sequencing and RT-qPCR result revealed that the expression of hsa_circ_0000992 was markedly up-regulated in 16HBE exposed to PM2.5. The binding sites between hsa_circ_0000992 and hsa-miR-936 was confirmed by dual-luciferase reporter gene system.Western blotting and RT-qPCR showed that hsa_circ_0000992 can interact with hsa-miR-936 to regulate AKT serine/threonine kinase 3(AKT3),thereby activating the PI3K/AKT pathway and ultimately promoting the expression of interleukin (IL)− 1β and IL-8. Conclusion: PM2.5 can induce the inflammatory response in 16HBE cells by activating the PI3K/AKT pathway. The expression of hsa_circ_0000992 increased when PM2.5 stimulated 16HBE cells,and the circRNA could then regulate the inflammatory response.Hsa_circ_0000992 regulates the hsa-miR-936/AKT3 axis through the ceRNA mechanism,thereby activating the PI3K/AKT signaling pathway,increasing the expression of cellular inflammatory factors,and promoting PM2.5-induced respiratory inflammation.

Fine particulate matter (PM2.5) pollution increases the risk of respiratory diseases and death, and apoptosis is an important factor in the occurrence of respiratory diseases caused by PM2.5 exposure. In addition, circular RNAs (circRNAs) can interact with proteins and widely participate in physiological and pathological processes in the body. The aim of this study was to investigate the mechanism of circRNA and protein interaction on PM2.5-induced apoptosis of human bronchial epithelial cells (16HBE) in vitro. In this study, we exposed human bronchial epithelial cells to a PM2.5 suspension with different concentration gradients for 24 h. The results showed that apoptosis of 16HBE cells after PM2.5 treatment was accompanied by cell proliferation. After exposure of PM2.5 to 16HBE cells, circRNAs related to apoptosis were abnormally expressed. We further found that the expression of hsa_circ_0002854 increased with the increase in exposure concentration. Functional analysis showed that knocking down the expression of hsa_circ_0002854 could inhibit apoptosis induced by PM2.5 exposure. We then found that hsa_circ_0002854 could interact with MAPK1 protein and inhibit MAPK1 phosphorylation, thus promoting apoptosis. Our results suggest that hsa_circ_0002854 can promote 16HBE apoptosis due to PM2.5 exposure, which may provide a gene therapy target and scientific basis for PM2.5-induced respiratory diseases. [ABSTRACT FROM AUTHOR]

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

Organic dust, as found in agricultural farm work, is ranked among the highest occupational exposure hazards by the CDC. Agricultural dust containing endotoxins, pesticides, mold, and other chemicals, contributes to increased rates of respiratory diseases among these workers. Human bronchial epithelial cells (HBEC), which line upper airways, are frequently exposed to pathogens. Understanding the role of HBEC in inflammation following dust exposure (DE) is necessary to understand the mechanisms underlying inflammatory diseases. The complement system, a nonspecific and non-adaptable defense mechanism, is composed of circulating proteins that promote inflammation by attacking the cell membranes of pathogens and recruiting immune cells that secrete mediators of inflammation. We characterized complement protein expression in DE-treated HBEC using previously generated SWATH proteomics data and Western blotting. Western blotting identified that DE treatment in HBEC mediates the release and activation of C3, while data identified via SWATH-MS proteomics indicated significant upregulation of CD59—a regulator of complement activation. These data suggest that DE-HBEC may regulate complement activation and aim to elucidate the mechanisms by which HBEC promotes the complement system, and thus induce pulmonary inflammation in the presence of organic dust.

Objective To investigate the effects of cigarette smoke extracts (CSE) on lipid metabolism in human bronchial epithelial cells (HBECs) based on liquid chromatography-mass spectrometry (LC-MS) technology. Methods A cell model of HBECs treated with CSE was established in vitro, with 5% CSE intervention bronchial epithelial cells as the experimental group and no CSE intervention cells as the control group. LC-MS was used to quantitatively detect lipids in HBECs. The data was analyzed by PCA analysis and OPLS-DA analysis, the differential lipids were screened out from 728 lipids (11 categories). Results Compared with the control group, CSE-induced HBECs showed 191 differentially expressed lipids, of which 185 lipids were up-regulated and six lipids were down-regulated. Among them, the lipids with the highest proportion of increased numbers were sphingolipid pathway lipids, and with the highest increase ratio was triglycerides. Conclusion CSE causing the lipid metabolism disorders in HBECs, especially the accumulation of lipids in the sphingolipid pathway, may be associated with the chronic obstructive pulmonary disease process. [ABSTRACT FROM AUTHOR]

Background: Coal dust exposure has caused a variety of lung diseases. In addition to genotoxicity and cytotoxicity, other biological changes caused by coal dust (CD) exposure need further study. Objective: To observe the cellular transformation effects of CD exposure and explore its underlying molecular mechanism, human bronchial epithelial cells (BEAS-2B) were cultured with continuous CD exposure. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, colony formation assay, wound healing assay, next-generation sequencing (NGS) and western blotting were performed to observe the cell proliferation, migration, genomic transcription and pathological signaling pathways. Results: We demonstrated that BEAS-2B cells with long-term chronic CD exposure show accelerated proliferation rate and enhanced migration ability, and have altered gene expression profiles and aberrant activation of EGFR/Raf/ERK and PI3K/AKT/mTOR pathways. Conclusions: The results indicate that chronic CD exposure could induce abnormal proliferation and migration of BEAS-2B cells, lead to the transformation potential of human bronchial epithelial cells. [ABSTRACT FROM AUTHOR]

Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell–cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae. This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte- macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein- 1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway. [ABSTRACT FROM AUTHOR]

Pneumonia caused by multi-drug-resistant Klebsiella pneumoniae (MDR-Kpneu) poses a major public health threat, especially to immunocompromised or hospitalized patients. This study aimed to determine the immunostimulatory effect of the Toll-like receptor 5 ligand flagellin on primary human lung epithelial cells during infection with MDR-Kpneu. Human bronchial epithelial (HBE) cells, grown on an air–liquid interface, were inoculated with MDR-Kpneu on the apical side and treated during ongoing infection with antibiotics (meropenem) and/or flagellin on the basolateral and apical side, respectively; the antimicrobial and inflammatory effects of flagellin were determined in the presence or absence of meropenem. In the absence of meropenem, flagellin treatment of MDR-Kpneu-infected HBE cells increased the expression of antibacterial defense genes and the secretion of chemokines; moreover, supernatants of flagellin-exposed HBE cells activated blood neutrophils and monocytes. However, in the presence of meropenem, flagellin did not augment these responses compared to meropenem alone. Flagellin did not impact the outgrowth of MDR-Kpneu. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-Kpneu-infected primary human bronchial epithelium, which is associated with the release of mediators that activate neutrophils and monocytes. Topical flagellin therapy may have potential to boost immune responses in the lung during pneumonia.

Objective To observe the effect of cigarette smoke exposure on ferroptosis of human bronchial epithelial cells in vitro and to investigate its mechanism. Methods BEAS-2B cells were divided into 0%,0. 5%,1. 0% or 2. 0% CSE groups, which were treated with 0%,0. 5%,1. 0% or 2. 0% CSE (dissolved in DMEM) media for 24 h, respectively. CCK-8 assay was used to observe cell viability; EdU staining was used to detect cell proliferation ability; Fe2+ and GSH levels were observed by colorimetric method; flow cytometry was used to detect ROS level; the mRNA and protein expression levels of ferroptosis-related glutathione peroxidase 4(GPx4), ferritin heavy chain 1(FTH1) and prostaglandinendoperoxide synthase 2(Ptgs2) were detected by q-PCR and Western blotting; necrotic cell death rate was indicated by PI staining; and the structure of mitochondria was observed by transmission electron microscope. Next, BEAS-2B cells were divided into the Fer-1, Nec-1, Z-VAD-FMK and 2% CSE groups, which were treated with 2% CSE in the presence or absence of Fer-1, Nec-1 or Z-VAD-FMK; cell viability was determined by CCK-8 assay; Fe2+ and GSH levels were observed by colorimetric method; flow cytometry was used to detect ROS level; the expression levels of ferroptosis-related mRNAs and proteins GPx4, FTH1 and Ptgs2 were detected by q-PCR and Western blotting; and necrotic cell death rate was indicated by PI staining. Results EdU positive cells and OD values in different concentrations of CSE groups were lower than those in the control group, which in the 1. 0% or 2. 0% CSE groups were lower than those in the 0. 5% CSE group, and those in the 2. 0% CSE group were lower than those in the 1. 0% CSE group (all P<0. 05). Fe2+ and ROS levels in the 0. 5%,1. 0% or 2. 0% CSE groups were higher than those in the control group, which in the 1. 0% or 2. 0% CSE groups were higher than those in the 0. 5% CSE group, and those in the 2. 0% CSE group were higher than those in the 1. 0% CSE group (all P<0. 05). GSH levels in different concentrations of CSE groups were lower than that in the control group, which in the 1. 0% or 2. 0% CSE groups were lower than that in the 0. 5% CSE group, and that in the 2. 0% CSE group was lower than that in the 1. 0% CSE group (all P<0. 05); the mRNA and protein expression levels of FTH1, GPx4 in different concentrations of CSE groups were lower than those in the control group, which in the 1. 0% or 2. 0% CSE groups were lower than those in the 0. 5% CSE group, and those in the 2. 0% CSE group were lower than those in the 1. 0% CSE group (all P<0. 05); the mRNA and protein expression levels of Ptgs2 in the 0. 5%,1. 0% or 2. 0% CSE groups were higher than those in the control group, which in the 1. 0% or 2. 0% CSE groups were higher than those in the 0. 5% CSE group, and those in the 2. 0% CSE group were higher than those in the 1. 0% CSE group (all P<0. 05); necrotic cell death rates in the 0. 5%,1. 0% or 2. 0% CSE groups were higher than that in the control group, which in the 1. 0% or 2. 0% CSE groups was higher than that in the 0. 5% CSE group, and that in the 2. 0% CSE group was higher than that in the 1. 0% CSE group (all P<0. 05); transmission electron microscopy showed that BEAS-2B cells treated with 2. 0% CSE had typical ultrastructural characteristic changes in mitochondria, including mitochondrial shrinkage, increased bilayer membrane density, reduced or even removed mitochondrial cristae. OD value in Fer-1 group was higher than those in Nec-1, Z-VADFMK and 2% CSE groups; cellular Fe2+ and ROS levels in Fer-1 group were lower than those in 2% CSE group; GSH level in Fer-1 group was higher than that in 2% CSE group; the mRNA and protein expression levels of FTH1, GPx4 in Fer-1 group were higher than those in 2% CSE group, however, the mRNA and protein expression levels of Ptgs2 in Fer-1 group were lower than those in 2% CSE group; cell death rate in Fer-1 group was lower than that in 2% CSE group (all P< 0. 05). Conclusion Cigarette smoke exposure can induce the ferroptosis of BEAS-2B cells, which might be mediated by iron overload and redox disorder in BEAS-2B cells. [ABSTRACT FROM AUTHOR]

Airborne particulate matter (PM) is a major health hazard worldwide and is a key factor in lung cancer, which remains the most common type of malignancy and the leading cause of cancer-related deaths. DNA methylation is a critical mechanism underlying the detrimental effects of PM, however, the molecular link between PM exposure and lung cancer remains to be elucidated. N-α-acetyltransferase 10 (NAA10) is involved in the cell cycle, migration, apoptosis, differentiation, and proliferation. In order to investigate the role of NAA10 in PM-induced pathogenesis processes leading to lung cancer, we determined the expression and methylation of NAA10 in normal human bronchial epithelial (NHBE) cells treated with PM10, PM10-polycyclic aromatic hydrocarbons (PAH), and PM2.5 and evaluated the prognostic value of the NAA10 methylation status in lung cancer patients. Exposure to all PM types significantly increased the expression of NAA10 mRNA and decreased the methylation of the NAA10 promoter in NHBE cells compared with the mock-treated control. NAA10 hypomethylation was observed in 9.3% (13/140) of lung cancer tissue samples and correlated with NAA10 transcriptional upregulation. Univariate and multivariate analyses revealed that NAA10 hypomethylation was associated with decreased survival of patients with lung cancer. Therefore, these results suggest that PM-induced hypomethylation of the NAA10 may play an important role in the pathogenesis of lung cancer and may be used as a potential prognostic biomarker for lung cancer progression. Further studies with large numbers of patients are warranted to confirm our findings. [ABSTRACT FROM AUTHOR]

Thymic stromal lymphopoietin (TSLP) is an epithelium-derived pro-inflammatory cytokine involved in lung inflammatory responses. Previous studies show conflicting observations in blood TSLP in COVID-19, while none report SARS-CoV-2 inducing TSLP expression in bronchial epithelial cells. Our objective in this study was to determine whether TSLP levels increase in COVID-19 patients and if SARS-CoV-2 induces TSLP expression in bronchial epithelial cells. Plasma cytokine levels were measured in patients hospitalized with confirmed COVID-19 and age- and sex-matched healthy controls. Demographic and clinical information from COVID-19 patients was collected. We determined associations between plasma TSLP and clinical parameters using Poisson regression. Cultured human nasal (HNEpC) and bronchial epithelial cells (NHBEs), Caco-2 cells, and patient-derived bronchial epithelial cells (HBECs) obtained from elective bronchoscopy were infected in vitro with SARS-CoV-2, and secretion as well as intracellular expression of TSLP was detected by immunofluorescence. Increased TSLP levels were detected in the plasma of hospitalized COVID-19 patients (603.4 ± 75.4 vs 997.6 ± 241.4 fg/mL, mean ± SEM), the levels of which correlated with duration of stay in hospital (β: 0.11; 95% confidence interval (CI): 0.01–0.21). In cultured NHBE and HBECs but not HNEpCs or Caco-2 cells, TSLP levels were significantly elevated after 24 h post-infection with SARS-CoV-2 (p < 0.001) in a dose-dependent manner. Plasma TSLP in COVID-19 patients significantly correlated with duration of hospitalization, while SARS-CoV-2 induced TSLP secretion from bronchial epithelial cells in vitro. Based on our findings, TSLP may be considered an important therapeutic target for COVID-19 treatment. [ABSTRACT FROM AUTHOR]

Objective To investigate the inhibition of house dust mite induced oxidative stress with human bronchial epithelial cell line BEAS-2B by aldose reductase inhibitor (ARIs). Methods BEAS-2B was divided into control group, model group, NF-κB inhibitor group (PDTC group) and ARIs intervention group (ARIs group). Oxidative stress reaction (MDA and ROS content, SOD activity) was detected by ELISA. The contents of IL-5, IL-13 and IFN-γ were measured by ELISA. MTT was used to detect cell viability. Cell apoptosis was detected by flow cytometry. The expressions of NF-κB, NF-κB P65 and P38 MAPK proteins were detected by Western blot. Results SOD activity, IFN-γ content and cell survival rate in model group were lower than those in control group (P＜0.05), MDA, IL-5, IL-13 content and cell apoptosis rate were higher than those in control group (P＜0.05). Compared with model group, SOD activity, IFN-γ content and cell survival rate in PDTC and ARIs groups were increased(P＜0.05), MDA, IL-5, IL-13 content and cell apoptosis rate were decreased (P＜0.05). Compared with control group, the expression of NF-κB, NF-κB P65 and P38 MAPK protein in model group was significantly increased(P＜0.05). The expression of NF-κB, NF-κB P65 and P38 MAPK protein in PDTC group and ARIs group were significantly decreased (P＜0.05). Conclusions Aldose reductase inhibitors improve the oxidative stress response of human bronchial epithelial cells induced by house dust mites, promote the proliferation of BEAS-2B cells and inhibit apoptosis.

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. [Display omitted] • Circ_0004664 was downregulated in cadmium-transformed BEAS-2B (Cd–B2B) cells. • Circ_0004664 was transferred from normal BEAS-2B cells to Cd–B2B cells via exosomes. • Exosomal transfer of circ_0004664 inhibited migration and invasion of Cd–B2B cells. • Circ_0004664 promoted PTEN expression via sponging miR-942-5p in Cd–B2B cells. • Exosomal circ_0004664 is a promising target to prevent cadmium-induced lung cancer. [ABSTRACT FROM AUTHOR]

• Hemagglutinin inhibits 16-HBE proliferation and migration. • Hemagglutinin promotes the expression of PAI-1, uPA and tPA in 16-HBE and mice lung tissue. • Hemagglutinin promotes apoptosis of 16-HBE cells. • Hemagglutinin promotes pathological injury of lung tissue in mice. The most prevalent glycoprotein on the influenza virus envelope is called hemagglutinin (HA), yet little is known about its involvement in the pathophysiology and etiology of severe influenza pneumonia. Here, after stimulating human bronchial epithelial cells (16-HBE) and mice with HA of H1N1 for 12 h, we investigated the proliferation, migration, inflammatory cytokines expression, and apoptosis in 16-HBE and the pathological damage in mouse lung tissue. The expression of inflammatory cytokines plasminogen activator inhibitor 1(PAI-1), urokinase‐type (uPA) and tissue‐type (tPA) plasminogen activators, and apoptosis were all enhanced by HA, which also prevented the proliferation and migration of bronchial epithelial cells. HA enhanced up-regulated PAI-1, uPA, and tPA protein expression within mouse lung tissue and caused lung injury. In conclusion, HA alone, but not the whole H1N1 virus, induces lung tissue injury by inhibiting cell proliferation and migration, while promoting the expression of inflammatory cytokines and apoptosis. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • PM2.5 impairs mitochondrial bioenergetics and induces hypoxia in lung epithelium. • Calcitriol/VDR axis restores mitochondrial dysfunction via inactivating HIF-1α. • Protective role of calcitriol/VDR axis involves regulation of HIF-1α/PGC-1α signaling. [ABSTRACT FROM AUTHOR]

Fine particulate matter (PM2.5) pollution increases the risk of respiratory diseases and death, and apoptosis is an important factor in the occurrence of respiratory diseases caused by PM2.5 exposure. In addition, circular RNAs (circRNAs) can interact with proteins and widely participate in physiological and pathological processes in the body. The aim of this study was to investigate the mechanism of circRNA and protein interaction on PM2.5-induced apoptosis of human bronchial epithelial cells (16HBE) in vitro. In this study, we exposed human bronchial epithelial cells to a PM2.5 suspension with different concentration gradients for 24 h. The results showed that apoptosis of 16HBE cells after PM2.5 treatment was accompanied by cell proliferation. After exposure of PM2.5 to 16HBE cells, circRNAs related to apoptosis were abnormally expressed. We further found that the expression of hsa_circ_0002854 increased with the increase in exposure concentration. Functional analysis showed that knocking down the expression of hsa_circ_0002854 could inhibit apoptosis induced by PM2.5 exposure. We then found that hsa_circ_0002854 could interact with MAPK1 protein and inhibit MAPK1 phosphorylation, thus promoting apoptosis. Our results suggest that hsa_circ_0002854 can promote 16HBE apoptosis due to PM2.5 exposure, which may provide a gene therapy target and scientific basis for PM2.5-induced respiratory diseases.

Feng-jia Chen,1,2,&ast; Li-juan Du,1,2,&ast; Zhimin Zeng,1,2,&ast; Xin-yan Huang,1,2 Chang-yi Xu,1,2 Wei-ping Tan,1,2 Can-mao Xie,1,2 Yu-xia Liang,1,2 Yu-biao Guo1,2 1Division of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 2Institute of Pulmonary Diseases, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yu-biao Guo; Yu-xia Liang, Tel +86 20 8775 5766, Email guoyb@mail.sysu.edu.cn; liangyx69@mail.sysu.edu.cnPurpose: PTPRH inhibits EGFR activity directly in cancer patients and activated EGFR induces goblet cell hyperplasia and mucus hypersecretion in asthma. However, the function of PTPRH in asthma remains unknown. The purpose of this study was to access the association of PTPRH with asthma and its underlying mechanism.Patients and Methods: We examined the PTPRH level in asthma patients (n = 108) and healthy controls (n = 35), and analyzed the correlations between PTPRH and asthma-related indicators. Human bronchial epithelial cell (HBECs) transfected with PTPRH and asthma mouse model were set up to investigate the function of PTPRH.Results: The expression of PTPRH was significantly increased and correlated with pulmonary function parameters, including airway obstruction, and T-helper2 (Th2) associated markers in asthma patients. PTPRH increased in the house dust mite (HDM)-induced asthmatic mice, while Th2 airway inflammation and Muc5ac suppressed when treated with PTPRH. Accordingly, PTPRH expression was markedly increased in IL-13-stimulated HBECs but PTPRH over-expression suppressed MUC5AC. Moreover, HBECs transfected with over-expressed PTPRH inhibited the phosphorylation of EGFR, ERK1/2 and AKT, while induced against PTPRH in HBECs dephosphorylated of EGFR, ERK1/2 and AKT.Conclusion: PTPRH reduces MUC5AC secretion to alleviate airway obstruction in asthma via potential phosphorylating of EGFR/ERK1/2/AKT signaling pathway, which may provide possible therapeutic implications for asthma.Keywords: asthma, PTPRH, human bronchial epithelial cells, EGFR/ERK1/2/AKT, MUC5AC

This study investigated the effect of microRNA mir-548ar-3p on cigarette smoke extract (CSE)-induced chronic obstructive pulmonary disease (COPD). High-throughput sequencing was performed on peripheral blood from smoking COPD patients and non-smoking individuals with normal pulmonary function, and miR-548ar-3p RNA, possessing large differential expression was selected. Experimental groups were divided into control, experimental model (EM), EM+mimic miRNA, negative control (NC) and EM+miR-548ar-3p groups; an empty vector or miR-548ar-3p mimic was transfected into human bronchial epithelial (HBE) cells. A COPD model was established by treating HBE cells with CSE. Cell viability, apoptosis and solute carrier family 17 member 9 (SLC17A9) protein expression were examined by cell counting kit-8, flow cytometry and Western blotting, respectively. Cell viability in the EM+miR-548ar-3p group decreased significantly, and the apoptosis rate and SLC17A9 protein expression increased significantly compared with the control (P

This study investigated the inhalation toxicity of the emissions from 3-D printing with acrylonitrile butadiene styrene (ABS) filament using an air-liquid interface (ALI) in vitro model. Primary normal human-derived bronchial epithelial cells (NHBEs) were exposed to ABS filament emissions in an ALI for 4 hours. The mean and mode diameters of ABS emitted particles in the medium were 175 ± 24 and 153 ± 15 nm, respectively. The average particle deposition per surface area of the epithelium was 2.29 × 107 ± 1.47 × 107 particle/cm2, equivalent to an estimated average particle mass of 0.144 ± 0.042 μg/cm2. Results showed exposure of NHBEs to ABS emissions did not significantly affect epithelium integrity, ciliation, mucus production, nor induce cytotoxicity. At 24 hours after the exposure, significant increases in the pro-inflammatory markers IL-12p70, IL-13, IL-15, IFN-γ, TNF-α, IL-17A, VEGF, MCP-1, and MIP-1α were noted in the basolateral cell culture medium of ABS-exposed cells compared to non-exposed chamber control cells. Results obtained from this study correspond with those from our previous in vivo studies, indicating that the increase in inflammatory mediators occur without associated membrane damage. The combination of the exposure chamber and the ALI-based model is promising for assessing 3-D printer emission-induced toxicity. [ABSTRACT FROM AUTHOR]

To explore the effects of coal dust exposure on DNA damage and repair, human bronchial epithelial BEAS-2B cells were exposed to coal dust and the cellular response was investigated. It was found that γ-H2AX foci of DNA damage appeared, γ-H2AX protein level increased, and the rate of cell apoptosis was significantly elevated when BEAS-2B cells were exposed to coal dust for a short time. Phagocytized coal dust particles, swollen mitochondria, and reduced mitochondrial membrane potential were simultaneously identified. Moreover, Caspase-9, Caspase-3, and DFF45 proteins of the mitochondrial apoptotic pathway were activated. After the cells were exposed to coal dust chronically, phosphorylation levels of DNA repair kinases (ATM/ATR, DNA-PKcs) and downstream regulatory protein AKT were significantly upregulated. γ-H2AX foci and tail DNA of the cells following treatment with cisplatin were also reduced, and the colony formation rate was improved. It was concluded that coal dust could induce DNA damage, cause mitochondrial depolarization, and activate mitochondrial apoptosis pathways in BEAS-2B cells. Additionally, activated DNA repair kinases (ATM/ATR and DNA-PKcs) and their regulatory protein AKT increased DNA repair and proliferation of BEAS-2B cells chronically exposed to coal dust. [ABSTRACT FROM AUTHOR]

Abstract Background Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs). Method Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 μg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 μg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 μg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex. Results Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients. Conclusion MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.

Mutations in the CFTR gene lead to cystic fibrosis, a genetic disease associated with chronic infection and inflammation and ultimately respiratory failure. The most common CF‐causing mutation is F508del and CFTR modulators (correctors and potentiators) are being developed to rescue its trafficking and activity defects. However, there are currently no modulators that stabilize the rescued membrane F508del‐CFTR which is endocytosed and quickly degraded resulting in a shorter half‐life than wild‐type (WT). We previously reported that the extracellular signal‐regulated kinase (ERK) MAPK pathway is involved in CFTR degradation upon cigarette smoke exposure. Interestingly, we found that ERK phosphorylation was increased in CF human bronchial epithelial (HBE) cells (CF‐HBE41o‐ and primary CF‐HBE) compared to non‐CF controls, and this was likely due to signaling by the epidermal growth factor receptor (EGFR). EGFR can be activated by several ligands, and we provide evidence that amphiregulin (AREG) is important for activating this signaling axis in CF. The natural osmolyte ectoine stabilizes membrane macromolecules. We show that ectoine decreases ERK phosphorylation, increases the half‐life of rescued CFTR, and increases CFTR‐mediated chloride transport in combination with the CFTR corrector VX‐661. Additionally, ectoine reduces production of AREG and interleukin‐8 by CF primary bronchial epithelial cells. In conclusion, EGFR‐ERK signaling negatively regulates CFTR and is hyperactive in CF, and targeting this axis with ectoine may prove beneficial for CF patients. [ABSTRACT FROM AUTHOR]

Hexavalent chromium [Cr(VI)] is a common industrial pollutant, and exposure may cause toxic effects in multiple organ systems and carcinogenesis, including lung cancer. However, the toxic effect of Cr(VI) on the respiratory system is poorly understood. In the present study, it was demonstrated that Cr(VI) exposure significantly decreased the viability of human bronchial epithelial cells (16-HBE) in a dose-dependent manner. Flow cytometry demonstrated that Cr(VI) enhanced the transition of 16-HBE cells from G1 to S phase and arrested S-phase progression. Reverse transcription-quantitative polymerase chain reaction analysis revealed a significant alteration in the expression of apoptosis-associated genes in Cr(VI)-treated 16-HBE cells. In addition, using two-dimensional fluorescence differential gel electrophoresis with mass spectrometry, 15 differentially expressed proteins (1 upregulated and 14 downregulated) were identified in 16-HBE cells with Cr(VI) treatment compared with controls. Functional classification revealed that these differentially expressed proteins were involved in apoptosis, cytoskeletal structure, and energy metabolism. In conclusion, these data suggested that Cr(VI) caused toxic effects in bronchial epithelial cells and the mechanisms may involve the abnormal expression of apoptosis-associated proteins, cytoskeletal proteins, and energy metabolism-associated proteins. [ABSTRACT FROM AUTHOR]

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19. [ABSTRACT FROM AUTHOR]

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASOmediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein. [ABSTRACT FROM AUTHOR]

• Cas9 and Cas12a can successfully correct W1282X-CFTR variant in 16HBE cells. • Cas9 shows higher levels W1282X-CFTR correction than Cas12a. • CRISPR/Cas9 gene editing of W1282X-CFTR increases levels of CFTR mRNA and protein. • CFTR function is fully restored in W1282X homozygous-corrected clones. W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations. We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors. Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR. Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease. [ABSTRACT FROM AUTHOR]

This study investigated the effect of microRNA mir-548ar-3p on cigarette smoke extract (CSE)-induced chronic obstructive pulmonary disease (COPD). High-throughput sequencing was performed on peripheral blood from smoking COPD patients and non-smoking individuals with normal pulmonary function, and miR-548ar-3p RNA, possessing large differential expression was selected. Experimental groups were divided into control, experimental model (EM), EM+mimic miRNA, negative control (NC) and EM+miR-548ar-3p groups; an empty vector or miR-548ar-3p mimic was transfected into human bronchial epithelial (HBE) cells. A COPD model was established by treating HBE cells with CSE. Cell viability, apoptosis and solute carrier family 17 member 9 (SLC17A9) protein expression were examined by cell counting kit-8, flow cytometry and Western blotting, respectively. Cell viability in the EM+miR-548ar-3p group decreased significantly, and the apoptosis rate and SLC17A9 protein expression increased significantly compared with the control (P<0.05, all groups). In smoking COPD patients, interferon (IFN)-γ and interleukin (IL)-17α expression detected by ELISA was significantly higher than in normal individuals. miR-548ar-3p expression was significantly lower (P<0.05, all groups). These findings suggest that miR-548ar-3p was expressed at a lower level in COPD patients. miR-548ar-3p may increase the extent of CSE-induced COPD injury through SLC17A9. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate the protective effect of Xuanfei Pingchuan Capsules (XFPC) on autophagy and p38 phosphorylation in human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). Methods: HBE cells were divided into five groups: blank, CSE, low XFPC dose (XFPC-L), medium XFPC dose (XFPC-M), and high XFPC dose (XFPC-H). HBE cells were induced by CSE to establish a cell model for chronic obstructive pulmonary disease, and different doses of XFPC medicated serum were used to treat the cells. The Cell Counting Kit-8 was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. Fluorescence microscopy and the expression level of microtubule-associated protein light chain 3 (LC3)-II in immunohistochemical method were used to observe autophagy in cells. Western blot was used to detect the protein expression level of p38, phospho-p38 (p-p38), LC3-I, LC3-II and Beclin 1. Real-time polymerase chain reaction was used to detect the expression of LC3-I , LC3-II and Beclin 1 on mRNA level. Results: Compared with the blank group, the cell viability of the CSE group was significantly decreased, and apoptosis and the level of autophagy in cells were significantly increased. The mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and the protein level of p-p38 were significantly increased in the CSE-HBE cells. Compared to the CSE group, the different doses of XFPC medicated serum increased cell viability, decreased cell apoptosis, and inhibited mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and protein level of p-p38. These results were especially observed in the group XFPC-H. After adding a p38 agonist, the therapeutic effect of XFPC on cell viability and autophagy was suppressed. Conclusion: XFPC significantly increased cell viability in a CSE-induced HBE cell model for chronic obstructive pulmonary disease through inhibiting the level of autophagy mediated by phosphorylation of p38. [ABSTRACT FROM AUTHOR]

Thymic stromal lymphopoietin (TSLP) is an epithelium-derived pro-inflammatory cytokine involved in lung inflammatory responses. Previous studies show conflicting observations in blood TSLP in COVID-19, while none report SARS-CoV-2 inducing TSLP expression in bronchial epithelial cells. Our objective in this study was to determine whether TSLP levels increase in COVID-19 patients and if SARS-CoV-2 induces TSLP expression in bronchial epithelial cells. Plasma cytokine levels were measured in patients hospitalized with confirmed COVID-19 and age- and sex-matched healthy controls. Demographic and clinical information from COVID-19 patients was collected. We determined associations between plasma TSLP and clinical parameters using Poisson regression. Cultured human nasal (HNEpC) and bronchial epithelial cells (NHBEs), Caco-2 cells, and patient-derived bronchial epithelial cells (HBECs) obtained from elective bronchoscopy were infected in vitro with SARS-CoV-2, and secretion as well as intracellular expression of TSLP was detected by immunofluorescence. Increased TSLP levels were detected in the plasma of hospitalized COVID-19 patients (603.4 ± 75.4 vs 997.6 ± 241.4 fg/mL, mean ± SEM), the levels of which correlated with duration of stay in hospital (β: 0.11; 95% confidence interval (CI): 0.01–0.21). In cultured NHBE and HBECs but not HNEpCs or Caco-2 cells, TSLP levels were significantly elevated after 24 h post-infection with SARS-CoV-2 (p < 0.001) in a dose-dependent manner. Plasma TSLP in COVID-19 patients significantly correlated with duration of hospitalization, while SARS-CoV-2 induced TSLP secretion from bronchial epithelial cells in vitro. Based on our findings, TSLP may be considered an important therapeutic target for COVID-19 treatment.

Background: This study aimed to investigate the protective effect of Xuanfei Pingchuan Capsules (XFPC) on autophagy and p38 phosphorylation in human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE).Methods: HBE cells were divided into five groups: blank, CSE, low XFPC dose (XFPC-L), medium XFPC dose (XFPC-M), and high XFPC dose (XFPC-H). HBE cells were induced by CSE to establish a cell model for chronic obstructive pulmonary disease, and different doses of XFPC medicated serum were used to treat the cells. The Cell Counting Kit-8 was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. Fluorescence microscopy and the expression level of microtubule-associated protein light chain 3 (LC3)-II in immunohistochemical method were used to observe autophagy in cells. Western blot was used to detect the protein expression level of p38, phospho-p38 (p-p38), LC3-I, LC3-II and Beclin 1. Real-time polymerase chain reaction was used to detect the expression of LC3-I, LC3-II and Beclin 1 on mRNA level.Results: Compared with the blank group, the cell viability of the CSE group was significantly decreased, and apoptosis and the level of autophagy in cells were significantly increased. The mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and the protein level of p-p38 were significantly increased in the CSE-HBE cells. Compared to the CSE group, the different doses of XFPC medicated serum increased cell viability, decreased cell apoptosis, and inhibited mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and protein level of p-p38. These results were especially observed in the group XFPC-H. After adding a p38 agonist, the therapeutic effect of XFPC on cell viability and autophagy was suppressed.Conclusion: XFPC significantly increased cell viability in a CSE-induced HBE cell model for chronic obstructive pulmonary disease through inhibiting the level of autophagy mediated by phosphorylation of p38.

Polycyclic aromatic hydrocarbons (PAHs) are the most common contaminants in the air pollutants. Inhalation exposure to PAHs could increase the risk of respiratory disease, cardiovascular disease and even cancer. However, the biotoxicity of multi‐component PAHs from atmospheric pollutants has been poorly studies. The main topic of this study was to investigate the PAHs mixture, which derived from atmospheric pollutants, induced toxic effects and inflammatory effects on human bronchial epithelial cells in vitro. The results showed that PAHs mixture could decrease the cell viability, increase the apoptosis rate, and induce cell cycle arrest at S‐phase. Furthermore, the expression of inflammatory factors IL‐1β and IL‐6 were increased and NF‐κB signaling pathway was activated in PAHs mixture‐treated cells. The findings of this study indicate that PAHs mixture‐induced cytotoxicity and inflammation may be related to intracellular ROS generation and to the activated NF‐κB signaling pathway. We investigated that the PAHs mixture induced toxic effects and inflammatory effects on human bronchial epithelial cells in vitro. The results showed that PAHs mixture could decrease the cell viability and increase the apoptosis rate. In addition, the expression of inflammatory factors IL‐1β and IL‐6 was increased in PAHs mixture‐treated cells. These may be related to intracellular ROS generation and NF‐κB signaling pathway activation. [ABSTRACT FROM AUTHOR]

Sepsis is one of the leading causes of morbidity and mortality and a major cause of acute lung injury (ALI). carried by exosomes play a role in a variety of diseases. However,there are not many studies of exosomal miRNAs in sepsis and sepsis lung injury.miR-1298-5p and suppressor of cytokine signaling 6 (SOCS6) were silenced or overexpressed in human bronchial epithelial cells (BEAS-2B). PKH-67 Dye was used to trace exosome endocytosis. Cell permeability was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC dextran flux. ELISA kits were used for cytokine detection. Quantitative RT-PCR and western blots were used to evaluate gene expression. miR-1298-5p was elevated in exosomes from patients with sepsis lung injury (Sepsis_exo). Treatment of BEAS-2B cells using Sepsis_exo significantly inhibited cell proliferation, and induced cell permeability and inflammatory response. miR-1298-5p directly targeted SOCS6. Overexpressing SOCS6 reversed miR-1298-5p-induced cell permeability and inflammatory response. Inhibition of STAT3 blocked SOCS6-silencing caused significant increase of cell permeability and inflammation. Exosomes isolated from patients of sepsis lung injury increased cell permeability and inflammatory response in BEAS-2B cells through exosomal miR-1298-5p which targeted SOCS6 via STAT3 pathway. The findings highlight the importance of miR-1298-5p/SOCS6/STAT3 axis in sepsis lung injury and provide new insights into therapeutic strategies for sepsis lung injury. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) have been demonstrated to play critical roles in the pathogenesis of human cancers and carcinogenesis of several environmental pollutants. Nevertheless, the function of circRNAs in cadmium carcinogenesis is unclear. circ-SHPRH is down-regulated in many cancers including non-small cell lung cancer. In our present study, during cadmium-induced transformation of human bronchial epithelial BEAS-2B cells, epithelial-mesenchymal transition (EMT) was induced. Meanwhile, at the middle and late stages of cell transformation, cadmium down-regulated the expression of circ-SHPRH, as well as QKI, a tumor suppressor protein known to prevent the proliferation and EMT during progression of human cancers, compared with passage-matched control BEAS-2B cells. Overexpression of circ-SHPRH in cadmium-transformed BEAS-2B cells promoted the expression of QKI and significantly inhibited proliferation, EMT, invasion, migration and anchorage-independent growth in soft agar of the cells. Mechanistic studies showed that circ-SHPRH functioned as a sponge of miR-224–5p to regulate QKI expression. Interestingly, QKI and circ-SHPRH could form a positive-feedback loop that perpetuated circ-SHPRH/miR-224–5p/QKI axis. Collectively, our results demonstrated that circ-SHPRH inhibited cadmium-induced transformation of BEAS-2B cells through sponging miR-224–5p to regulate QKI expression under cadmium treatment. Our study uncovered a novel molecular mechanism involved in circRNAs in the development of lung cancer due to cadmium exposure.

Background: Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs). Method: Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 μg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 μg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 μg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex. Results: Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients. Conclusion: MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase. [ABSTRACT FROM AUTHOR]

The role of benzo[ a ]pyrene (BaP), a prominent genotoxic carcinogen and aryl hydrocarbon receptor (AhR) ligand, in tumor progression remains poorly characterized. We investigated the impact of BaP on the process of epithelial-mesenchymal transition (EMT) in normal human bronchial epithelial HBEC-12KT cells. Early morphological changes after 2-week exposure were accompanied with induction of SERPINB2, IL1, CDKN1A/p21 (linked with cell cycle delay) and chemokine CXCL5. After 8-week exposure, induction of cell migration and EMT-related pattern of markers/regulators led to induction of further pro-inflammatory cytokines or non-canonical Wnt pathway ligand WNT5A. This trend of up-regulation of pro-inflammatory genes and non-canonical Wnt pathway constituents was observed also in the BaP-transformed HBEC-12KT-B1 cells. In general, transcriptional effects of BaP differed from those of TGFβ1, a prototypical EMT inducer, or a model non-genotoxic AhR ligand, TCDD. Carcinogenic polycyclic aromatic hydrocarbons could thus induce a unique set of molecular changes linked with EMT and cancer progression. [Display omitted] • BaP causes EMT-like state in human bronchial epithelial cell line HBEC-12KT. • Morphological changes occur after 2w exposure. • EMT-linked transcription factors and markers and cell migration are induced only after 8w exposure. • Identification of candidate genes/proteins responsible for gradual BaP-induced EMT was described. • Major inducers were AHR, CDKN1A, SERPINB2, IL1B, CXCL5, and WNT pathway components. [ABSTRACT FROM AUTHOR]

Fine atmospheric particles with a diameter of 2.5 µm or less (PM2.5) have a large specific surface area, and carry a variety of organic matter, heavy metals, minerals and bacteria. They are an important risk factor in human non-communicable disease. To explore the molecular regulatory mechanism of the airway inflammation caused by PM2.5, an in vitro human bronchial epithelial (16HBE) cells poisoning model was deployed. Results showed that PM2.5 had a strong inhibitory effect on cells viability, and induced cells to secrete high levels of IL-6 and CXCL 8. These two biomarkers of inflammation were significantly reduced in the presence of TAK 242. TLR4, MyD88, IKK, and p-p65 proteins were highly expressed on exposure to PM2.5. Pretreatment with TAK 242 interfered with the activation of the TLR4 signaling pathway. By detecting the presence of lipopolysaccharides (LPS) in PM2.5 which had been autoclaved, it was speculated that the activation of the TLR4/NF-κB signaling pathway may be mediated by LPS. It was demonstrated using gain- and loss- function experiments that miR-140-5p negatively regulated TLR4 to mediate inflammation in 16HBE cells. The dual-luciferase reporter assay confirmed that miR-140-5p directly binds to the 3ˈ untranslated region (3ˈ UTR) of TLR4 to initiate biological activity. In conclusion, this study revealed a new mechanism by which the miR-140-5p/TLR4 signaling pathway mediated the inflammatory response of 16HBE cells induced by PM2.5. Differential expression of miRNA, and the activation of the TLR4/NF-κB signaling pathway induced by PM2.5 implicates PM2.5 in the pathogenesis of airway inflammation.

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

Air pollution is a public health issue and the toxicity of ambient particulate matter (PM) is well‐recognized. Although it does not mostly contribute to the total mass of PM, increasing evidence indicates that the ultrafine fraction has generally a greater toxicity than the others do. A better knowledge of the underlying mechanisms involved in the pathological disorders related to nanoparticles (NPs) remains essential. Hence, the goal of this study was to determine better whether the exposure to a relatively low dose of well‐characterized iron‐rich NPs (Fe‐NPs) might alter some critical toxicological endpoints in a relevant primary culture model of human bronchial epithelial cells (HBECs). We sought to use Fe‐NPs representative of those frequently found in the industrial smokes of metallurgical industries. After having noticed the effective internalization of Fe‐NPs, oxidative, inflammatory, DNA repair, and apoptotic endpoints were investigated within HBECs, mainly through transcriptional screening. Taken together, these results revealed that, despite it only produced relatively low levels of reactive oxygen species without any significant oxidative damage, low‐dose Fe‐NPs quickly significantly deregulated the transcription of some target genes closely involved in the proinflammatory response. Although this inflammatory process seemed to stay under control over time in case of this acute scenario of exposure, the future study of its evolution after a scenario of repeated exposure could be very interesting to evaluate the toxicity of Fe‐NPs better. We sought to determine better whether the exposure to a relatively low dose of well‐characterized iron‐rich nanoparticles (Fe‐NPs) might alter some critical toxicological endpoints in a relevant primary culture model of human bronchial epithelial cells. Under these conditions, Fe‐NPs also produced only low levels of reactive oxygen species without any significant oxidative damage, but, in contrast, significantly deregulated the transcription of some critical target genes closely involved in the proinflammatory response, which seemed to stay under control over time. [ABSTRACT FROM AUTHOR]

Accumulating evidence reveals that exosome plays an important role in cell‐to‐cell communication in both physiological and pathological processes by transferring bioactive molecules. However, the role of exosomal secretion in the adaption of its source cells to the stimuli of environmental chemicals remains elusive. In this study, we revealed that the exposure of hydroquinone (HQ; the main bioactive metabolite of benzene) to human bronchial epithelial cells (16HBE) resulted in decreased ability of cell proliferation and migration, and simultaneously DNA damage and micronuclei formation. Interestingly, when exosomal secretion of HQ treated 16HBE cells was inhibited with the inhibitor GW4869, cellular proliferation and migration were further significantly reduced; concurrently, their DNA damage and micronuclei formation were both further significantly aggravated. Herein, we conclude that exosomal secretion of 16HBE cells may be an important self‐protective function against the toxic effects induced by HQ. The role of exosomal secretion in the adaption of its source cells to the stimuli of environmental chemicals remains elusive. In this study, we revealed that exosomal secretion of human bronchial epithelial cells might be an important self‐protective function against the toxic effects induced by hydroquinone. [ABSTRACT FROM AUTHOR]

Objective To investigate the relationship between dexamethasone resistance and mucin 1 (MUC1) expression in bronchial epithelial cells. Methods Cigarette smoke extract (CSE) was extracted. Human normal bronchial epithelial cells NHBE (hereinafter referred to as NHBE cells) were cultured in vitro, and were treated with 0, 1%, 2. 5%, 5%, and 10%CSE. MTT method was used to detect the cell survival rate, and ELISA was used to detect the inflammatory factors IL-8 and GMCSF in the culture supernatant. The minimum concentration of CSE that caused the increase of NHBE cell inflammatory factors IL-8 and GM-CSF secretion was selected for subsequent experiments. NHBE cells were intervened with dexamethasone at a final concentration of 1 × 10-8 mol/L for 1 month (set as drug-resistant cell group). CSE and dexamethasone were added to treat drugresistant cells to make the final concentration of CSE reach the minimum concentration, and the final concentration of dexamethasone be 1 × 10-8 mol/L; in addition, NHBE cells were randomly divided into the non-drug-resistant cell group 1 and non-drugresistant cell group 2. The NHBE cells in the non-drug-resistant cell group 1 were added with an appropriate amount of CSE solution to make the final concentration of CSE reach the minimum concentration, and the cells in the non-drug-resistant cell group 2 were added with appropriate amount of dexamethasone to make the final concentration of dexamethasone reach 1 × 10-8 mol/L. Each group was intervened for 48 h, the inflammatory factors IL-8 and GM-CSF in the culture supernatant were detected by ELISA, and the relative expression of MUC1 mRNA was detected by RT-q PCR. Results As the concentration of CSE solution increased, the survival rate of NHBE cells gradually decreased in a dose-dependent manner (F = 8. 520, P ＜ 0. 01). The survival rate of NHBE cells after 5% and 10% CSE intervention was lower than that after 0, 1%, and 2. 5% CSE intervention, and the survival rate of NHBE cells after 10% CSE solution intervention was lower than that after 5% CSE solution intervention (all P ＜0. 05). There were no statistical differences in the survival rate between NHBE cells which were treated with 0, 1%, and 2. 5%CSE (all P ＞ 0. 05). Compared with 0, 1%, and 2. 5% CSE, 5% and 10% CSE significantly increased the levels of the inflammatory factors IL-8 and GM-CSF secreted by NHBE cells (all P ＜ 0. 05); 5% CSE was selected for the follow-up experiment because it was the minimum concentration to increase the levels of inflammatory factors IL-8 and GM-CSF. The levels of inflammatory factors IL-8 and GM-CSF in the drug-resistant cell group were significantly higher than those in the non-resistant cell group2 (both P ＜ 0. 05), but there was no statistical difference between the non-resistant cell group 2 and the non-resistant cell group1 (both P ＞ 0. 05). The relative expression of MUC1 mRNA in the drug-resistant cell group was lower than that of the non-resistant cell group 1 and the non-resistant cell group 2 (both P ＜ 0. 01), while no statistically significant difference was fouund in the relative expression of MUC1 mRNA between the non-resistant cell group 1 and non-resistant cell group 2 (P ＞ 0. 05). Conclusion The resistance of human bronchial epithelial cells to dexamethasone may be related to the decreased expression of MUC1. [ABSTRACT FROM AUTHOR]