Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Roles of Rho/Rock Signaling Pathway in Silica-induced Epithelial-mesenchymal Transition in Human Bronchial Epithelial Cells

Objective To investigate the roles of Rho/Rock signaling pathway in silica-induced Epithelial-mesenchymal transition （EMT） in human bronchial epithelial cells （BEC） in vitro. Methods Human BEC were incubated with silica with various concentrations for indicated times. Cell viability was assayed by MTT test. Morphologic Changes were observed by microscope. Mesenchymal marker a-smooth muscle actin （a-SMA）, vimentin （Vim）, and epithelial marker E-cadherin （E-cad） were analyzed by Western Blot. The pull-down assay was used to measure Rho activity. In the prevention experiments, the specific inhibitor for Rho effector ROCK （Y27632） was used to inhibit the activity of Rho. Results Human BEC stimulated with silica were converted from a ＂cobblestone＂ epithelial structure into an elongated fibroblast-like shape structure. Incubation of human BEC with silica induced de novo expression of a-SMA and Vim, and loss of E-cad. Also, silica treatment resulted in Rho activation in human BEC. Y27632 up-regulated the E-cad expression but attenuated a-SMA and Vim expression in silica-stimulated cells. Conclusion The activation of Rho/ROCK signaling pathways is most likely involved in Silica-induced EMT in human bronchial epithelial cells.

Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

Objective To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. Methods DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone （ranged from 10 μmol/L to 120 μmol/L） for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis （SCGE）] were performed respectively to detect the toxicity of hydroquinone. Results assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. Conclusions Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.

Transcriptional Factor Snail Mediates Epithelial-Mesenchymal Transition in Human Bronchial Epithelial Cells Induced by Silica

Epithelial-mesenchymal transition （EMT） plays an important role in fibrotic diseases. We have previously showed that silica induces EMT in human bronchial epithelial cells （BECs）; however, the underlying mechanism of silica-induced EMT is poorly understood. In the present study, we investigated the role of Snail in silica-induced EMT in human BECs in vitro. Human BECs were treated with silica at various concentrations and incubation times. Then MTr assay, western blot, electrophoretic mobility shift assay （EMSA）, and small interfering RNA （siRNA） transfection were performed. We found that silica increased the expression and DNA binding activity of Snail in human BECs. SNAI silica-induced expression siRNA upregulated the siRNA inhibited the of Snail. Moreover, SNAI expression of epithelial marker E-cadherin, but attenuated the expression of mesenchymal marker a-smooth muscle actin and vimentin in silica-stimulated cells. These results suggest that Snail mediates the silica-induced EMT in human BECs.

Exosomes from PM^(2.5)-treated Human Bronchial Epithelial Cells Increase Lung Cancer Metastatic Potential

Objective Fine particulate matter(PM^(2.5))is an air pollutant that has become of great concern in recent years.Numerous studies have found that PM^(2.5)may contribute to lung cancer,but the pathogenesis has not yet been fully elucidated.In this study,we explored the roles of exosomes from bronchial epithelial cells in PM^(2.5)-promoted lung cancer metastasis.Methods Exosomes were isolated from cell supernatants.An animal model of lung metastasis(established by tail vein injection of A549-luc)and in vitro studies with lung cancer cell lines were used to investigate the effects of exosomes derived from PM^(2.5)-treated human bronchial epithelial cells(PHBE-exo).Results The animal experiments revealed that PHBE-exo-treated mice showed stronger luciferase activity and a larger relative metastatic region in the lungs,thus indicating that PHBE-exo promoted the metastatic potential of lung cancer.Additionally,PHBE-exo promoted the migration,invasion and epithelial-to-mesenchymal transition of lung cancer cells,in a manner mediated by activation of c-Jun Nterminal kinase.Conclusion These results implied that PM^(2.5)may promote the development of lung cancer through exosomes derived from bronchial epithelial cells,thus providing a potential interventional target for lung cancer.These findings broadened our understanding of cancer-promoting mechanisms of environmental pollutants.

FBXW7 regulates epithelial barrier impairment in human bronchial epithelial cells in vitro by targeting apoptosis signal-regulating kinase1 via the p38 pathway

Bronchial asthma is a common chronic inflammatory disease characterized by airway hyperresponsiveness(AHR),inflammatory cell infiltration,and airway remodeling.F-box/WD repeat-containing protein 7(FBXW7),an E3 ubiquitin ligase,is required for various endothelial functions,such as cell migration,inflammation,and endothelial integrity.This study aimed to investigate the role of FBXW7 in lipopolysaccharide(LPS)-induced epithelial barrier impairment in bronchial epithelial cells in vitro.By using lentivirus-based technology,FBXW7 was overexpressed or silenced(24 h)in human bronchial epithelial(16HBE)cells,which were treated with LPS or not(24 h).Immunoprecipitation(IP)detection and Western blot analysis were used to evaluate the interaction of target proteins.Cell permeability was measured using transepithelial electrical resistance and FITC dextran flux(48 h).IL-1β,IL-18 and TNF-αin cell supernatants were measured using ELISA(48 h).The results showed that LPS stimulation suppressed FBXW7 expression in a time-and dose-dependent manner.LPS exposure decreased cell proliferation,elevated IL-1β,IL-18 and TNF-α,increased epithelial permeability,and p38 phosphorylation.These LPS-induced changes were partly compromised by FBXW7 overexpression.Similar to LPS stimulation,FBXW7 knockdown increased epithelial permeability and levels of inflammatory cytokines and p38 phosphorylation,which were,in part,blocked by apoptosis signal-regulating kinase(ASK)1 knockdown or p38 pathway inhibition.IP and Western blot analysis showed that FBXW7 interacted with ASK1.ASK1 expression was inversely associated with FBXW7 expression.FBXW7 overexpression markedly enhanced ASK1 ubiquitination.These data revealed that FBXW7 counter against inflammation and protects epithelial barrier integrity in bronchial epithelial cells by promoting ubiquitination-mediated degradation of ASK1 via the p38 pathway.

Proteasome inhibitor MG-132 regulates the expression of VEGF in human bronchial epithelial cell line, BEAS-2B

Objective： To explore the effects of MG-132 on the expression of VEGF in bronchial epithelial cell line, BEAS- 2B. Methods： Semi-quantitive RT-PCR for VEGF mRNA and enzyme-linked immunosorbent assay （ELISA） for VEGF protein were performed. Results： MG-132 increased the expression of VEGF mRNA and protein BEAS-2B cells in time-and concentration-dependent manners. After 24-h stimulation, 25 ktmol/L MG-132 increased the maximal levels of VEGF protein in cell-conditioned medium. When the ceUs were stimulated with cycloheximide（CHX） before treatment with MG-132, the MG-132-induced production of VEGF protein was inhibited compared to the unstimulated cells. Supematant of condition-medium treatment with MG-132 enhanced the growth of HUVEC. Conclusion： MG-132 induces VEGF gene expression in human bronchial epithelial cells line, BEAS-2B, and the MG-132-induced expression of VEGF may modulate lung tissue injury due to airway inflammation.

NOD-like receptors mediated activation of eosinophils interacting with bronchial epithelial cells： a link between innate immunity and allergic asthma

Key intracytosolic pattern recognition receptors of innate immunity against bacterial infections are nucleotide-binding oligomerization domain （NOD）-Iike receptors （NLRs）. We elucidated the NOD1 and NOD2-mediated activation of human eosinophils, the principal effector cells for allergic inflammation, upon interacting with human bronchial epithelial BEAS-2B cells in allergic asthma. Eosinophils constitutively expressed NOD1,2 but exhibited nonsignificant responses to release chemokines upon the stimulation by NOD1 ligand 7-D-glutamyl-meso-diaminopimelic acid （iE-DAP） and NOD2 ligand muramyl dipeptide （MDP）. However, iE-DAP and MDP could significantly upregulate cell surface expression of CD18 and intercellular adhesion molecule （ICAM）-I on eosinophils and ICAM-1 on BEAS-2B cells, as well as induce chemokines CCL2 and CXCL8 release in the coculture system （all P〈0.05）. Both eosinophils and BEAS-2B cells were the main source for CXCL8 and CCL2 release in the coculture system upon iE-DAP or MDP stimulation. Direct interaction between eosinophils and BEAS-2B cells is responsible for CCL2 release, and soluble mediators are implicated in CXCL8 release. ERK and NF-KB play regulatory roles for the expression of adhesion molecules and chemokines in coculture. Treatment with NOD1,2 ligand could induce the subepithelial fibrosis and significantly enhance the serum concentration of total IgE, chemokine CCL5 for eosinophils and T helper type 2 （Th2） cells and asthma Th2 cytokine IL-13 in bronchoalveolar lavage fluid of ovalbumin-sensitized allergic asthmatic mice （all P〈0.05）. This study provides further evidence of bacterial infection-mediated activation of NOD1,2 in triggering allergic asthma via the activation of eosinophils interacting with bronchial epithelial cells at inflammatory airway.

Effect of Puerarin on the Release of lnterleukin-8 in Co-Culture of Human Bronchial Epithelial Cells and Neutrophils

Objective： To investigate the effect of puerarin on intedeukin （IL）-8 mRNA expression and the protein release in the co-culture of human bronchial epithelial （BEAS-2B） cells and human neutrophils. Methods： BEAS- 2B cells and neutrophills were cultured separately and co-cultured with puerarin （50, 100, and 200 ug/mL） for a predetermined time. Cytokines in culture supematant were evaluated by protein array and IL-8 quantified by enzymelinked immunosorbent assay （ELISA）. IL-8 mRNA expression was evaluated by real-time quantitative polymerase chain reaction （real-time qPCR）. Results： The co-culture of BEAS-2B cells and neutrophils exhibited synergistic effects on IL-8 mRNA expression in BEAS-2B cells, but not in neutrophils after 12 h incubation （P〈0.01）, as compared with that in BEAS-2B cells or neutrophils alone. IL-8 protein release in the culture supernatant was obviously elevated when BEAS-2B cells were co-cultured with human neutrophils as compared with that in the supematant of BEAS-2B cells or neutrophils alone after incubated for 2, 6, 12, and 18 h （P〈0.01）. Treatment with puerarin could significantly down-regulate the expression of IL-8 mRNA in BEAS-2B cells and IL-8 release in the supematant of the co-culture of BEAS-2B cells and neutrophils （P〈0.01）. Conclusion： Puerarin could exhibit anti-inflammatory activity by suppressing IL-8 production from the co-culture of human bronchial epithelial cells and neutrophils.

Induction of Adhesion Molecule Expression in Co-culture of Human Bronchial Epithelial Cells and Neutrophils Suppressed by Puerarin via Down-regulating p38 Mitogen-Activated Protein Kinase and Nuclear Factor κB Pathways

Objective： In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase （p38 MAPK） and nuclear factor kappa B （NF- K B） in modulating adhesion molecules expressions. Methods： Neutrophils and BEAS-2B cells （one human bronchial epithelial cell line） were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction （real-time qPCR）. Phosphorylated p38 MAPK and inhibitor K B were analyzed by Western blot. Results： In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrephils were enhanced significantly （P〈0.05）. Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor K B in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin （P〈0.05）. Conclusions： Co- culture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF- K B pathways, and exhibiting its anti-inflammation activity.

Human bronchial epithelial cell injuries induced by fine particulate matter from sandstorm and non-sandstorm periods：Association with particle constituents

Epidemiological studies have demonstrated the exacerbation of respiratory diseases following sandstorm-derived particulate matter（PM） exposure.The presence of anthropogenic and biological agents on the sandstorm PM and the escalation of PM 〈 2.5 μm（PM2.5）pollution in China have led to serious concerns regarding the health effects of PM2.5during Asian sandstorms.We investigated how changes in PM2.5composition,as the weather transitioned towards a sandstorm,affected human airway epithelial cells.Six PM2.5samples covering two sandstorm events and their respective background and transition periods were collected in Baotou,an industrial city near the Gobi Desert in China.PM samples from all three periods had mild cytotoxicity in human bronchial epithelial cell line BEAS-2B,which was positively correlated with the contents of polycyclic aromatic hydrocarbons and several metals.All PM samples potently increased the release of interleukin-6（IL-6） and interleukin-8（IL-8）.Endotoxin in all samples contributed significantly to the IL-6 response,with only a minor effect on IL-8.Cr was positively correlated with both IL-6 and IL-8 release,while Si was only associated with the increase of IL-6.Our study suggests that local agricultural and industrial surroundings in addition to the sandstorm play important roles in the respiratory effects of sandstorm-derived PM.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19.Our research,along with others',has demonstrated that mast cells(MCs)play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2.In previous study,we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice.Additionally,we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells,leading to subsequent lung injury.The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation,and inflammation in these regions could promote viral spread.MCs are widely distributed throughout the respiratory tract.Thus,in this study,we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium.Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2.MC degranulation caused lesions in trachea,and the formation of papillary hyperplasia was observed.Through transcriptome analysis in bronchial epithelial cells,we found that MC degranulation significantly altered multiple cellular signaling,particularly,leading to upregulated immune responses and inflammation.The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice.Taken together,our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions.Furthermore,our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation,thereby preventing tissue damage caused by hyper-inflammation.

Assessment of Benchmark Dose in BEAS-2B Cells by Evaluating the Cell Relative Viability with Particulates in Motorcycle Exhaust via the Air-liquid Interface Exposure

Objective This study aimed to use an air-liquid interface(ALI)exposure system to simulate the inhalation exposure of motorcycle exhaust particulates(MEPs)and then investigate the benchmark dose(BMD)of MEPs by evaluating cell relative viability(CRV)in lung epithelial BEAS-2B cells.Methods The MEPs dose was characterized by measuring the number concentration(NC),surface area concentration(SAC),and mass concentration(MC).BEAS-2B cells were exposed to MEPs at different concentrations via ALI and CRV was determined using Cell Counting Kit(CCK-8)assay.BMD software was applied to calculate BMD and the lower limit of benchmark dose(BMDL)according to Akaike Information Coefficient(AIC),with P-value based on Hill,Linear,Polynomial,and Power model.Results Our results reveal that BMD of NC and SAC were estimated by the best-fitting Hill model,while MC was estimated by Polynomial model.The BMDL for CRV following ALI exposure to MEPs were as follows:364.2#/cm^(3)for NC;0.662×10^(7)nm^(2)/cm^(3)for SAC;and 0.278μg/m^(3)for MC.Conclusion These results indicate that MEPs exposure via ALI system induces a dose-dependent decrease of CRV and provides the potential exposure threshold of MEPs in a lung cell model.

Proteomics Study on the Differentially Expressed Proteins in c-fos-silenced Cells Exposed to PM2.5

Objective To investigate the effect of c-fos gene silencing on differentially expressed proteins(DEPs) in human bronchial epithelial(HBE) cells after exposure to fine particulate matter(PM2.5).Methods HBE cells and c-fos-silenced HBE cells were exposed to 50 μg/mL PM2.5, LC-MS/MS and tandem mass tag(TMT) labeling methods were combined with bioinformatics methods, and DEPs and interaction networks were identified.Results In the HBE group, 414 DEPs were screened, of which 227 were up-regulated and 187 downregulated. In the c-fos silenced HBE group, 480 DEPs were screened, including 240 up-regulated proteins and 240 down-regulated proteins. KEGG annotations showed that DEPs in the HBE group are mainly concentrated in the glycolysis/gluconeogenesis pathway and those in the c-fos silenced group are concentrated mainly in endoplasmic reticulum and the processing of proteins. Additionally, the abnormal expression of GPRC5 C, DKK4, and UBE2 C was identified in top 15 DEPs. After constructing the protein interaction network, 20 Hub proteins including HNRNPA2 B1, HNRNPL, RPS15 A, and RPS25 were screened from the HBE group and the c-fos silenced HBE group.Conclusion c-fos gene affected the expression of cancer-related proteins. Our results provided a scientific basis for further study of PM2.5-induced carcinogenesis mechanism.

Lophomonas blattarum Infection or Just the Movement o Ciliated Epithelial Cells？

INTRODUCTION Lophomonas blattarum （L. blattarum） is a multiflagellated protozoan, which parasitizes in the intestinal tracts of termites and cockroaches, belonging to the Lophomonas suborder, Hypermastigida order. More than 100 cases of bronchopulmonary L. blattarum infection have been reported since 1993. However, we identified the movable cells from the bronchoalveolar lavage fluid （BALF） of six patients with pulmonary diseases under an electron microscope, and eventually lbund that these cells were actually bronchial ciliated epithelial cells, which were basically identical with reported L. blattarum in morphology under light microscope in the literature, Through careful literature review, we found that all of the reported L. blattarum infections were just diagnosed by morphology under light microscope rather than electron microscope, isolation and culture, or molecular procedures; images exhibited in these literatures were all compatible with the microscopic characteristics of bronchial ciliated epithelial cells.