Human Bronchial Epithelial BEAS-2B Research Articles

TRIM47 promotes HDM-induced bronchial epithelial pyroptosis by regulating NEMO ubiquitination to activate NF-κB/NLRP3 signaling.

Asthma is an inflammatory disease. Airway epithelial cell pyroptosis and cytokine secretion promote asthma progression. Tripartite motif 47 (TRIM47)belongs to the E3 ubiquitin ligase family and is associated with apoptosis and inflammation in a range of diseases. However, the role of TRIM47 in asthma has not been explored. In this study, the human bronchial epithelial cell line BEAS-2B was treated with house dust mite (HDM) and TRIM47 expression was detected by RT-qPCR and Western blot. After transfection with TRIM47 interfering and overexpressing plasmids, the synthesis and secretion of cytokines, as well as pyroptosis-related indicators, were examined.Nuclear factor kappa-B (NF-κB)pathway proteins and nod-like receptor protein 3 (NLRP3)inflammasome were measured to explore the mechanism of TRIM47 action. In addition, the effect of TRIM47 on the level of NF-κB essential modulator (NEMO) ubiquitination was detected by an immunoprecipitation assay. The results showed that TRIM47 was upregulated in HDM-induced BEAS-2B cells and that TRIM47 mediated HDM-induced BEAS-2B cell pyroptosis and cytokine secretion. Mechanistically, TRIM47 promoted the K63-linked ubiquitination of NEMO and facilitated NF-κB/NLRP3 pathway activation. In conclusion, TRIM47 may promote cytokine secretion mediating inflammation and pyroptosis in bronchial epithelial cells by activating the NF-κB/NLRP3 pathway. Therefore, TRIM47 may be a potential therapeutic target for HDM-induced asthma.

Polyhydroxylated Spirostanol Saponins from the Rhizomes of Paris dulongensis.

Three new polyhydroxylated spirostanol steroidal saponins, dulongenosides B-D (2-4), along with 14 known compounds, dulongenoside A (1), padelaoside B (5), parisyunnanoside G (6), polyphyllin D (7), ophiopogonin C' (8), formosanin C (9), dioscin (10), paris saponin VII (11), paris H (12), parisyunnanoside I (13), protodioscin (14), proprotogracillin (15), crustecdysone (16), and stigmasterol-3-O-β-d-glucopyranoside (17), were isolated from the rhizomes of Paris dulongensis (Melanthiaceae). Their chemical structures were elucidated based on extensive analyses of NMR and MS data and acidic hydrolyses. The isolates were evaluated for their cytotoxicity to five human cancer cell lines (HL-60, SW480, MDA-MB-231, A549, and A549/Taxol) and the normal human bronchial epithelial cell line BEAS-2B by the MTS test. Compounds 7-12 and 14 showed cytotoxic activity, with IC50 values ranging from 0.20 to 4.35 μM. Proprotogracillin selectively inhibited A549 (IC50=0.58 μM) and A549/Taxol (IC50=0.74 μM) cells, with no significant cytotoxic activity against HL-60, SW480, MDA-MB-231, or BEAS-2B cells, with IC50 values greater than 40 μM.

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

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

German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 invivo.

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

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

Silencing KLF6 Alleviates Cigarette Smoke Extract-Induced Mitochondrial Dysfunction in Bronchial Epithelial Cells by SIRT4 Upregulation.

The incidence of chronic obstructive pulmonary disease (COPD) is increasing year by year. Kruppel-like factor 6 (KLF6) plays an important role in inflammatory diseases. However, the regulatory role of KLF6 in COPD has not been reported so far. The viability of human bronchial epithelial cells BEAS-2B induced by cigarette smoke extract (CSE) was detected by CCK-8 assay. The protein expression of KLF6 and sirtuin 4 (SIRT4) was appraised with Western blot. RT-qPCR and Western blot were applied to examine the transfection efficacy of sh-KLF6 and Oe-KLF6. Cell apoptosis was detected using flow cytometry. The levels of inflammatory factors IL-6, TNF-α and IL-1β were assessed with ELISA assay. DCFH-DA staining was employed for the detection of ROS activity and the levels of oxidative stress markers SOD, CAT and MDA were estimated with corresponding assay kits. The mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content and Complex I activity were evaluated with JC-1 staining, ATP colorimetric/fluorometric assay kit and Complex I enzyme activity microplate assay kit. With the application of mitochondrial permeability transition pore detection kit, mPTP opening was measured. Luciferase report assay was employed to evaluate the activity of SIRT4 promoter and chromatin immunoprecipitation (ChIP) to verify the binding ability of KLF6 and SIRT4 promoter. KLF6 expression was significantly elevated in CSE-induced cells. KLF6 was confirmed to suppress SIRT4 transcription. Interference with KLF6 expression significantly inhibited cell viability damage, cell apoptosis, inflammatory response, oxidative stress and mitochondrial dysfunction in CSE-induced BEAS-2B cells, which were all reversed by SIRT4 overexpression. Silencing KLF6 alleviated CSE-induced mitochondrial dysfunction in bronchial epithelial cells by SIRT4 upregulation.

In vitro EVALUATION of THE EFFECTS of POTENTIAL GSK-3β INHIBITORS TERBUTALINE and ORCIPRENALINE

Lung cancer is a type of cancer that is mostly diagnosed at an advanced stage and has a short survival time despite standard chemotherapy and targeted therapies. Terbutaline and Orciprenaline are bronchodilator agent that are potent and selective β2 receptor agonists. The purpose of this study was to investigate by continuous monitoring to assay the effects of Terbutaline and Orciprenaline on human lung carcinoma cell line. Cells were treated 1, 10, 100, 200, 400 µM concentrations with Terbutaline/Orciprenaline. MTT assay and xCELLigence real-time cell analyzer were used to determine these compounds effect on cell viability. The cell index was monitored continuously by visualizing the impedance of the E-plate wells. Because of GSK3β roles in a diverse range of cellular processes like metabolism, cell proliferation, differentiation and survival and its key position at several signaling pathways, GSK3β inhibition was investigated of these molecules. It has been determined that Terbutaline and Orciprenaline inhibits GSK-3β.
 This is the first known study to demonstrate the effects of Terbutaline and Orciprenaline on A549 and the human bronchial epithelial cell line Beas-2b in real time. In this study, it was detected that Terbutaline and especially Orciprenaline may have potential therapeutic effects in lung carcinoma.

Construction of a three-dimensional inflammation model of human bronchial epithelial cells BEAS-2B by using the self-assembling D-form peptide Sciobio-Ⅲ

Human bronchial epithelial cells in the airway system, as the primary barrier between humans and the surrounding environment, assume a crucial function in orchestrating the processes of airway inflammation. Target to develop a new three-dimensional (3D) inflammatory model to airway system, and here we report a strategy by using self-assembling D-form peptide to cover the process. By testing physicochemical properties and biocompatibility of Sciobio-Ⅲ, we confirmed that it can rapidly self-assembles under the trigger of ions to form a 3D nanonetwork-like scaffold, which supports 3D cell culture including the cell strains like BEAS-2B cells. Subsequently, inflammation model was established by lipopolysaccharide (LPS), the expression of some markers of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8), the levels of relevant inflammatory factors were measured by RT-qPCR and the secretion profile of inflammatory cytokines by ELISA, are obtained the quite difference effects in 2D and 3D microenvironment, which suggested Sciobio-Ⅲ hydrogel is an ideal scaffold that create the microenvironment for 3D cell culture. Here we are success to establish a 3D inflammation model for airway system. This innovative model allows for rapid and accurate evaluation of drug metabolism and toxicological side effects, hope to use in drug screening for airway inflammatory diseases and beyond.

Metabolomics Provides Novel Insights into the Potential Toxicity Associated with Heated Tobacco Products, Electronic Cigarettes, and Tobacco Cigarettes on Human Bronchial Epithelial BEAS-2B Cells.

Smoking is an established risk factor for various pathologies including lung cancer. Electronic cigarettes (e-cigs) and heated tobacco products (HTPs) have appeared on the market in recent years, but their safety or, conversely, their toxicity has not yet been demonstrated. This study aimed to compare the metabolome of human lung epithelial cells exposed to emissions of e-cigs, HTPs, or 3R4F cigarettes in order to highlight potential early markers of toxicity. BEAS-2B cells were cultured at the air-liquid interface and exposed to short-term emissions from e-cigs set up at low or medium power, HTPs, or 3R4F cigarettes. Untargeted metabolomic analyses were performed using liquid chromatography coupled with mass spectrometry. Compared to unexposed cells, both 3R4F cigarette and HTP emissions affected the profiles of exogenous compounds, one of which is carcinogenic, as well as those of endogenous metabolites from various pathways including oxidative stress, energy metabolism, and lipid metabolism. However, these effects were observed at lower doses for cigarettes (2 and 4 puffs) than for HTPs (60 and 120 puffs). No difference was observed after e-cig exposure, regardless of the power conditions. These results suggest a lower acute toxicity of e-cig emissions compared to cigarettes and HTPs in BEAS-2B cells. The pathways deregulated by HTP emissions are also described to be altered in respiratory diseases, emphasizing that the toxicity of HTPs should not be underestimated.

Berberine improved the microbiota in lung tissue of colon cancer and reversed the bronchial epithelial cell changes caused by cancer cells

ObjectiveThe lung is a common organ for colon cancer metastasis, and the objective of this experiment was to explore the protective effect of berberine on lung tissue or alveolar epithelial cells induced by colon cancer. MethodsThirty-six BALB/c nude mice were used to establish a xenograft model of colon cancer with the HT29 cell line and were treated with berberine and probiotics. Human bronchial epithelial BEAS-2B cells were induced by conditioned medium (CM) from the colon cancer cell lines HT29 and RKO and were treated with berberine. Lung tissues were collected to detect the changes in the microbiota using 16S rDNA sequencing and the expression of inflammatory cytokines. The expression of E-cadherin and N-cadherin in BEAS-2B cells was detected by cellular immunofluorescence. The changes in cell proliferation were detected by the CCK-8 assay. Western blotting was used to detect E-cadherin, N-cadherin, collagen I, fibronectin, PDGF-β, and RAD51 expression in BEAS-2B cells. ResultsThe richness and evenness of the microbiota in the lung tissues of mice with colon cancer were significantly lower than those of the control group. Berberine significantly increased the abundances of Bacteroidetes, Bacteroidia, Bacteroidales, Lactobacillaceae, Lactobacillus and Acinetobacter in the lung tissue of mice with colon cancer, with reduced abundances of Actinobacteria, Bacillales, Staphylococcaceae and Staphylococcus. Berberine or probiotics significantly increased the alpha diversity of the lung microbiota. Compared with probiotics, berberine significantly enhanced the abundance of microbiota involved in the metabolism of lysosomes, flavone and flavonol biosynthesis, glycosaminoglycan degradation, and glycosphingolipid biosynthesis-ganglio. Berberine increased IL-6 and IL-10 and decreased IL-17 and IFN-γ expression in lung tissue (P > 0.05), but berberine-probiotics significantly decreased IL-17 and IFN-γ and increased IL-10 expression (P < 0.05). Colon cancer cells could not induce BEAS-2B proliferation but decreased the expression of the epithelial marker E-cadherin and altered the expression of extracellular matrix-related proteins (collagen I, fibronectin, and PDGF-β), which were reversed by berberine. Berberine increased RAD51 expression in BEAS-2B cells, which had been decreased by HT29 and RKO CM treatment. ConclusionBerberine can selectively regulate the abundance of some microbiomes of lung tissue in colon cancer, improve the inflammatory response in lung tissue, and antagonize the cancerous stimulation of colon cancer cells to lung tissue cells by regulating the bronchial epithelial cell phenotype, extracellular matrix remodelling and the expression of the repair gene RAD51.

Silk Fibroin-Modified Liposome/Gene Editing System Knocks out the PLK1 Gene to Suppress the Growth of Lung Cancer Cells.

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR-Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR-Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy.

Apoptosis as a mechanism of human respiratory cell death upon exposure to carbon nanotubes

Introduction. Carbon nanotubes (CNTs) are a group of promising nanomaterials for industrial and biomedical applications. There has been shown influence of the physicochemical characteristics of CNTs on the toxic effects, including the ability to cause DNA damage and induce apoptosis. In this study, there was carried out a comparative assessment of pro-apoptotic effects under exposure to single-walled and multi-walled CNTs produced in Russia on human respiratory cells.&#x0D; Materials and methods. Human bronchial epithelial cells BEAS-2B, alveolar epithelial cells A549, and lung fibroblasts MRC5-SV40 were exposed to pristine and purified TUBALLTM SWCNTs and Taunit-M MWCNTs. In cells exposed to 4 concentrations (100, 50, 0.03, 0.0006 μg/ml) of all types of CNTs for 72 hours, the level of mRNA of the P53, BAX and BCL2 genes, as well as the level of reactive oxygen species were assessed.&#x0D; Results. All types of CNTs initiated apoptosis in human respiratory epithelial cells BEAS-2B and A549, but not in MRC5-SV40 lung fibroblasts. BEAS-2B were more sensitive to the effects of MWCNTs, while A549 were more sensitive to pristine SWCNTs. Apoptosis was initiated at low concentrations, including those corresponding to industrial exposures. The mechanism of oxidative stress could act as a factor in triggering apoptosis in lung epithelial cells.&#x0D; Limitations. Relatively short (72 hours) cell incubation time and the use of 2D cell models that do not consider real cell interactions.&#x0D; Conclusion. There were revealed differences in the mechanisms of initiation of the internal pathway of apoptosis and sensitivity to different types of CNTs depending on the type of epithelial cells. Comparative analysis of the initiation of apoptosis by different types of CNTs has shown that there are differences in potential target cells and toxic mechanisms, which should be considered in further studies.

Epigenetic downregulation of O6-methylguanine-DNA methyltransferase contributes to chronic hexavalent chromium exposure-caused genotoxic effect and cell transformation

Hexavalent chromium [Cr(VI)] is a common environmental pollutant and chronic exposure to Cr(VI) causes lung cancer and other types of cancer in humans, although the mechanism of Cr(VI) carcinogenesis remains elusive. Cr(VI) has been considered as a genotoxic carcinogen, but accumulating evidence indicates that Cr(VI) also causes various epigenetic toxic effects that play important roles in Cr(VI) carcinogenesis. However, it is not clear how Cr(VI)-caused epigenetic dysregulations contributes to Cr(VI) carcinogenesis. This study investigates whether Cr(VI) epigenetic toxic effect has an impact on its genotoxic effect. It was found that chronic low dose of Cr(VI) exposure time-dependently down-regulates the expression of a critical DNA damage repair protein O6-methylguanine-DNA methyltransferase (MGMT), leading to the increases of the levels of the highly mutagenic and carcinogenic DNA lesion O6-methylguanine (O6-MeG) in human bronchial epithelial BEAS-2B cells. Moreover, the levels of MGMT and O6-MeG in chronic Cr(VI) exposure-caused human lung cancer tissues are also significantly lower and higher than that in the adjacent normal lung tissues, respectively. It was further determined that chronic low dose of Cr(VI) exposure-transformed BEAS-2B cells display impaired DNA damage repair capacity and a high sensitivity to the toxicity of the alkylating chemotherapeutic drug Temozolomide. In contrast, stably overexpressing MGMT in parental BEAS-2B cells reverses chronic low dose of Cr(VI) exposure-caused DNA damage repair deficiency and significantly reduces cell transformation by Cr(VI). Further mechanistical studies revealed that chronic low dose of Cr(VI) exposure down-regulates MGMT expression through epigenetic mechanisms by increasing DNA methylation and histone H3 repressive modifications. Taken together, these findings suggest that epigenetic down-regulation of a crucial DNA damage repair protein MGMT contributes significantly to the genotoxic effect and cell transformation caused by chronic low dose of Cr(VI) exposure.

Knockdown of JMJD3 ameliorates cigarette smoke extract-triggered bronchial epithelial cell injury via ACSL4-dependent ferroptosis

Chronic obstructive pulmonary disease (COPD), a heterogeneity of chronic respiratory disease pattern, presents considerable prevalence and mortality. We aimed to explore the role and mechanisms of Jumonji domain-containing protein-3 (JMJD3) in COPD. The viability and JMJD3 expression in human bronchial epithelial cell line BEAS-2B were respectively assayed by CCK-8 assay and Western blot following stepwise exposure to increasing concentrations of cigarette smoke extract (CSE). After JMJD3 was silenced and acyl-CoA synthetase long-chain family member 4 (ACSL4) was overexpressed in CSE-treated BEAS-2B cells, cell viability, cytotoxicity, oxidative stress and total iron level were estimated using kits. ELISA estimated inflammatory levels. DCFH-DA probe and BODIPY 581/591 C11 probe were exposed to assess ROS production and lipid peroxidation. Western blot tested the expressions of ferroptosis-associated proteins. Besides, H3K27me3 and ACSL4 expressions were tested by Western blot and immunofluorescence staining. In CSE-induced BEAS-2B cells, JMJD3 expression was increased and deletion of JMJD3 improved cell viability, reduced LDH release, mitigated inflammation, oxidative stress and inhibited ferroptosis. Moreover, JMJD3 interference raised H3K27me3 expression whereas lessened ACSL4 expression in CSE-treated BEAS-2B cells. CSE exposure reduced the abundance of ACSL4 in H3K27me3 antibody. Further ACSL4 elevation reversed the impacts of JMJD3 silencing on the damage of CSE-induced BEAS-2B cells. Collectively, JMJD3 depletion might suppress ferroptosis mediated by ACSL4 to alleviate CSE-triggered inflammation and oxidative stress in BEAS-2B cells.

Protective effects of myricetin on airway inflammation and oxidative stress in ovalbumin-induced asthma mice

Myricetin, a flavonoid isolated from many edible vegetables and fruits, has multiple biological effects, including anti-inflammatory and anti-tumor effects. Myricetin could inhibit mast cell degranulation in vitro, and it reduced the eosinophil content in bronchoalveolar lavage fluid (BALF) of ovalbumin (OVA)-sensitized mice. However, it remains unclear whether myricetin alleviates airway hyperresponsiveness (AHR), airway inflammation, and oxidative stress in asthma. Here, we investigated whether myricetin attenuated AHR, airway inflammation, and eosinophil infiltration in lungs of asthmatic mice. Mice were sensitized with OVA, then injected intraperitoneally with myricetin to investigate anti-inflammatory and antioxidant effects of myricetin. Moreover, we examined its effects on human bronchial epithelial BEAS-2B cells stimulated with TNF-α and IL-4, in vitro. Myricetin effectively mitigated eosinophil infiltration, AHR, and goblet cell hyperplasia in lung, and it reduced Th2 cytokine expression in BALF from asthmatic mice. Myricetin effectively promoted glutathione and superoxide dismutase productions and mitigated malondialdehyde expressions in mice by promoting Nrf2/HO-1 expression. Myricetin also reduced the production of proinflammatory cytokines, eotaxins, and reactive oxygen species in BEAS-2B cells. Myricetin effectively suppressed ICAM-1 expression in inflammatory BEAS-2B cells, which suppressed monocyte cell adherence. These results suggested that myricetin could effectively improve asthma symptoms, mainly through blocking Th2-cell activation, which reduced oxidative stress, AHR, and airway inflammation.

Impact of Electronic Cigarettes, Heated Tobacco Products and Conventional Cigarettes on the Generation of Oxidative Stress and Genetic and Epigenetic Lesions in Human Bronchial Epithelial BEAS-2B Cells.

Electronic cigarettes (e-cig) and heated tobacco products (HTP) are often used as smoking cessation aids, while the harm reduction effects of these alternatives to cigarettes are still the subject of controversial debate, in particular regarding their carcinogenic potential. The objective of this study is to compare the effects of e-cig, HTP and conventional cigarette emissions on the generation of oxidative stress and genetic and epigenetic lesions in human bronchial epithelial BEAS-2B cells. Our results show that HTP were less cytotoxic than conventional cigarettes while e-cig were not substantially cytotoxic in BEAS-2B cells. E-cig had no significant effect on the Nrf2 pathway, whereas HTP and cigarettes increased the binding activity of Nrf2 to antioxidant response elements and the expression of its downstream targets HMOX1 and NQO1. Concordantly, only HTP and cigarettes induced oxidative DNA damage and significantly increased DNA strand breaks and chromosomal aberrations. Neither histone modulations nor global DNA methylation changes were found after acute exposure, regardless of the type of emissions. In conclusion, this study reveals that HTP, unlike e-cig, elicit a biological response very similar to that of cigarettes, but only after a more intensive exposure: both tobacco products induce cytotoxicity, Nrf2-dependent oxidative stress and genetic lesions in human epithelial pulmonary cells. Therefore, the health risk of HTP should not be underestimated and animal studies are required in order to determine the tumorigenic potential of these emerging products.

Benzo[a]pyrene-induced up-regulation of circ_0003552 via ALKBH5-mediated m6A modification promotes DNA damage in human bronchial epithelial cells

Benzo [a]pyrene (B [a]P) is a widespread environmental chemical pollutant that has been linked to the development of various diseases. However, the specific mechanism of action remains unclear. In this study, human bronchial epithelial 16HBE and BEAS-2B cells were exposed to B [a]P at 0–32 μM to assess the DNA-damaging effects. B [a]P exposure resulted in elevated expression of γ-H2AX, a marker of DNA damage. The m6A RNA methylation assay showed that B [a]P exposure increased the extent of m6A modification and the demethylase ALKBH5 played an integral role in this process. Moreover, the results of the comet assay and Western blot analysis showed an increase in m6A modification mediated by ALKBH5 that promoted DNA damage. Furthermore, the participation of a novel circular RNA, circ_0003552, was assessed by high-throughput sequencing under the condition of high m6A modification induced by B [a]P exposure. In subsequent functional studies, an interference/overexpression system was created to confirm that circ_0003552 participated in regulation of DNA damage. Mechanistically, circ_0003552 had an m6A binding site that could regulate its generation. This study is the first to report that B [a]P upregulated circ_0003552 through m6A modification, thereby promoting DNA damage. These findings revealed that epigenetics played a key role in environmental carcinogen-induced DNA damage, and the quantitative changes it brought might provide an early biomarker for future medical studies of genetic-related diseases and a new platform for investigations of the interaction between epigenetics and genetics.

Polycyclic aromatic hydrocarbons (PAHs) in ambient air of Guangzhou city: Exposure levels, health effects and cytotoxicity

Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 pose potentially serious threats to human health. In this study, the distribution characteristics of 16 priority controlled, fine PM (PM2.5)-bound PAHs in the ambient air of Guangzhou city were analysed from 2016 to 2019. Four high-molecular-weight PAHs with the highest annual average concentrations were benzo[ghi]perylene (BghiP; 0.757 ng/m3), indeno(1,2,3-cd)pyrene (IcdP; 0.627 ng/m3), benzo[b]fluoranthene (BbF, 0.519 ng/m3) and 3,4-benzopyrene (BaP; 0.426 ng/m3). Increasing concentrations of BghiP, IcdP, BbF and BaP were associated with increasing numbers of outpatient visits for respiratory diseases, indicating that exposure to these PAHs potentially causes acute respiratory injury in residents. Acute exposure of the human bronchial epithelial cell line BEAS-2B cells to BghiP, IcdP, BbF and BaP in vitro resulted in acute inflammation, DNA damage and apoptosis. Further bioinformatic analysis indicated that nuclear receptor subfamily 1 group D member 1 (NR1D1) may be a key target gene involved in mediating the toxic effects of BghiP. Collectively, our results suggest that BghiP and the other PAHs represented by it can damage the respiratory system and induce lung cancer. This study provides valuable evidence regarding the potential health risks posed by local ambient PAHs pollution.

Cryptotanshinone alleviates lipopolysaccharide and cigarette smoke-induced chronic obstructive pulmonary disease in mice via the Keap1/Nrf2 axis

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity worldwide. Cigarette smoking, which leads to abnormalities in the airways or alveoli and persistent obstruction of the airway's flow, is a significant risk factor of COPD. Cryptotanshinone (CTS) is the active ingredient in Salvia miltiorrhiza (Danshen) and has many pharmacological properties including anti-inflammatory, antitumor, and antioxidant properties, but its impact on COPD is uncertain. In the present study, the potential effect of CTS on COPD was investigated in a modified COPD mice model induced with cigarette smoke (CS) and lipopolysaccharide (LPS) exposure. CTS significantly reversed the decline in lung function, emphysema, inflammatory cell infiltration, small airway remodeling, pulmonary pathological damage, and airway epithelial cell proliferation in CS- and LPS-exposed mice. Additionally, CTS decreased inflammatory cytokines such as tumor necrosis factor α (TNF α), interleukins IL-6 and IL-1β, and keratinocyte chemoattractant (KC), increased the activities of superoxide dismutase (SOD), Catalase (CAT) and L-Glutathione (GSH), and repressed the expression of protein hydrolases matrix metalloprotein (MMP)− 9 and − 12 in pulmonary tissue and bronchoalveolar lavage fluid (BALF). The protective effects of CTS were also observed in human bronchial epithelial cell line BEAS-2B simulated with cigarette smoke condensate (CSC) and LPS. Mechanistically, CTS can repress the protein level of Keap1, resulting to activation of erythroid 2-related factor (Nrf2), finally alleviating COPD. In summary, the present findings demonstrated that CTS dramatically ameliorates COPD induced by CS and LPS via activating Keap1/Nrf2 pathway.

Crystalline silica-exposed human lung epithelial cells presented enhanced anchorage-independent growth with upregulated expression of BRD4 and EZH2 in autocrine and paracrine manners.

Crystalline silica-induced inflammation possibly facilitates carcinogenesis. Here, we investigated its effect on lung epithelium damage. We prepared conditioned media of immortalized human bronchial epithelial cell lines (hereinafter bronchial cell lines) NL20, BEAS-2B, and 16HBE14o- pre-exposed to crystalline silica (autocrine crystalline silica conditioned medium), a phorbol myristate acetate-differentiated THP-1 macrophage line, and VA13 fibroblast line pre-exposed to crystalline silica (paracrine crystalline silica conditioned medium). As cigarette smoking imposes a combined effect on crystalline silica-induced carcinogenesis, a conditioned medium was also prepared using the tobacco carcinogen benzo[a]pyrene diol epoxide. Crystalline silica-exposed and growth-suppressed bronchial cell lines exhibited enhanced anchorage-independent growth in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium compared with that in unexposed control conditioned medium. Crystalline silica-exposed nonadherent bronchial cell lines in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium showed increased expression of cyclin A2, cdc2, and c-Myc, and of epigenetic regulators and enhancers, BRD4 and EZH2. Paracrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium also accelerated the growth of crystalline silica-exposed nonadherent bronchial cell lines. Culture supernatants of nonadherent NL20 and BEAS-2B in crystalline silica and benzo[a]pyrene diol epoxide conditioned medium had higher EGF concentrations, whereas those of nonadherent 16HBE14o- had higher TNF-α levels. Recombinant human EGF and TNF-α promoted anchorage-independent growth in all lines. Treatment with EGF and TNF-α neutralizing antibodies inhibited cell growth in crystalline silica conditioned medium. Recombinant human TNF-α induced BRD4 and EZH2 expression in nonadherent 16HBE14o-. The expression of γH2AX occasionally increased despite PARP1 upregulation in crystalline silica-exposed nonadherent lines with crystalline silica and benzo[a]pyrene diol epoxide conditioned medium. Collectively, crystalline silica- and benzo[a]pyrene diol epoxide-induced inflammatory microenvironments comprising upregulated EGF or TNF-α expression may promote crystalline silica-damaged nonadherent bronchial cell proliferation and oncogenic protein expression despite occasional γH2AX upregulation. Thus, carcinogenesis may be cooperatively aggravated by crystalline silica-induced inflammation and genotoxicity.