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Objective: To screen the differential methylation sites, genes and pathways of air pollution fine particles (PM(2.5)) on human bronchial epithelial (HBE) cells by methylation chip and bioinformation technology, so as to provide scientific basis for further study of the toxicological mechanism of PM(2.5) on HBE cells. Methods: In August 2020, HBE cells were infected with 10 μg/ml and 50 μg/ml PM(2.5) aqueous solution for 24 h, namely PM(2.5) 10 μg/ml exposure group (low dose group) and PM(2.5) 50 μg/ml exposure group (high dose group) ; uninfected HBE cells were used as control group. The DNA fragments were hybridized with the chip, the chip scanned and read the data, analyzed the data, screened the differential methylation sites, carried out GO analysis and KEGG analysis of the differential methylation sites, and analyzed the interaction relationship of the overall differential methylation sites by functional epigenetic modules (FEMs). Results: Compared with the control group, 127 differential methylation sites were screened in the low-dose group, including 89 genes, including 55 sites with increased methylation level and 72 sites with decreased methylation level. The differential methylation sites were mainly concentrated in the Body region and UTR region. Compared with the control group, 238 differential methylation sites were screened in the high-dose group, including 168 genes, of which 127 sites had increased methylation level and 111 sites had decreased methylation level. The differential heterotopic sites were mainly concentrated in the Body region and UTR region. Through FEMs analysis, 8 genes with the most interaction were screened, of which 6 genes had significant changes in methylation level. MALT1 gene related to apoptosis was found in the heterotopic site of methylation difference in low-dose group; PIK3CA and ARID1A genes related to carcinogenesis were found in the heterotopic sites of methylation difference in high-dose group; TNF genes related to tumor inhibition were found in the results of FEMs analysis. Conclusion: After PM(2.5) exposure to HBE cells, the DNA methylation level is significantly changed, and genes related to apoptosis and carcinogenesis are screened out, suggesting that the carcinogenic mutagenic effect of PM(2.5) may be related to DNA methylation.
Subject(s)
Humans , Air Pollutants/toxicity , Basic Helix-Loop-Helix Transcription Factors/analysis , Carcinogenesis , DNA Methylation , Particulate Matter/toxicity , TechnologyABSTRACT
Objective:To research the metabolomic alterations of human lung bronchial epithelial cells infected with human rhinovirus 1B (HRV1B).Methods:Untargeted metabolomics was used to determine the metabolomic alterations in human lung bronchial epithelial cells (BEAS-2B) 6 h, 12 h and during the dynamic process (6 h∶12 h) after HRV1B infection.Results:A total of 93 differentially significant metabolites (DSMs) (47 DSMs were up-regulated and 46 DSMs were down-regulated) and 88 DSMs (37 DSMs were up-regulated and 51 DSMs were down-regulated) at post infection of HRV1B in BEAS-2B at 6 h or 12 h, respectively. A total of 30 DSMs (12 DSMs were up-regulated and 18 DSMs were down-regulated) in a dynamic process (6 h∶12 h) after HRV1B infection. Unknown metabolites took up most proportions. The trends of fatty acid, lipid, amino acid, nucleotide and carbohydrate were increased along with the prolonging of HRV1B infection. DSMs such as Diisononyl phthalate was co-detected DSMs among three groups.Conclusions:Metabolites such as fatty acid, lipid, amino acid, nucleotide and carbohydrate of BEAS-2B cells are changed induced by HRV1B infection.
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Objective: Many studies have shown that respiratory syncytial virus persistent infection may be the main cause of chronic respiratory pathology. However, the mechanism is unclear. Cystic fibrosis transmembrane conduction regulator (CFTR) is an apical membrane chloride channel, which is very important for the regulation of epithelial fluid, chloride ion, and bicarbonate transport. CFTR dysfunction will lead to changes in bronchial secretions and impair mucus clearance, which is related to airway inflammation. In our previous study, we observed the down-regulation of CFTR in airway epithelial cells in respiratory syncytial virus (RSV) infected mouse model. In this study, we further investigated the expression and function of CFTR by constructing an airway epithelial cell model of RSV persistent infection. Methods: 16HBE14o- cells were infected with RSV at 0.01 multiplicity of infection (MOI). The expression of CFTR was detected by real-time RT-PCR, immunofluorescence, and Western blotting. The intracellular chloride concentration was measured by N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE) and the chloride current was measured by whole-cell patch clamp recording. Results:16HBE14o-cells infected with RSV were survived to successive passages of the third generation (G3), while the expression and function of CFTR was progressively decreased upon RSV infection from the first generation (G1) to G3. Exposure of 16HBE14o-cells to RSV led to the gradual increase of TGF-β1 as well as phosphorylation of Smad2 following progressive RSV infection. Disruption of TGF-β1 signaling by SB431542 prevented Smad2 phosphorylation and rescued the expression of CFTR. Conclusion:RSV infection can lead to defective CFTR function in airway epithelial cells, which may be mediated via activation of TGF-β1 signaling pathway.
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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 @*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 @*Conclusion@#These results indicate that MEPs exposure
Subject(s)
Humans , Benchmarking/statistics & numerical data , Bronchi/physiology , Cell Line , Cell Survival/drug effects , Epithelial Cells/physiology , Motorcycles , Particulate Matter/adverse effects , Vehicle Emissions/analysisABSTRACT
Objective:To explore the effect of a Chinese medicinal composition ( Xiadanqi) on the prevention of radon exposure induced injuries of lung in vitro and in vivo. Methods:Mice were randomly divided into three groups of blank control group, radon-exposed group alone and radon-exposed group intervened with Chinese medicinal composition. The pathological changes of lung tissues in each group after 120 WLM were observed by HE and Masson staining, and the expressions of α-SMA protein and Vimentin protein in lung tissues were detected by immunohistochemistry staining. The levels of oxidative stress in lung tissue of each group were detected with SOD and MDA kits. At the same time, a radon exposed cell model and a radon exposure + Xiadanqi intervention cell model were constructed using an ecological radon chamber. The cell adhesion abilities of different groups were detected by an adhesion kit. The cell migration ability of each group was determined by the transwell migration experiment. The expression of E-cadherin and Vimentin protein was detected by Western blot. Results:Compared with the radon exposure group, the concentration of MDA was decreased ( t=4.43, P<0.05), the activity of SOD was increased ( t=3.22, P<0.05), and α-SMA and Vimentin protein expressions were decreased ( t=3.08, 7.57, P<0.05) in lung tissue of mice intervened with 2 mg/g Xiadanqi. In vitro, compared with radon exposure group, the migration ability was reduced ( t=4.78, 13.01, P<0.05), the cell adhesion property was enhanced ( t=3.41, 12.55, P<0.05), the expression of E-cadherin protein was increased ( t=2.96, 19.57, P<0.05), and the expression of Vimentin protein was obviously reduced ( t=21.00, 33.32, P<0.05) in radon-exposed cells with the treatment of Chinese medicine (150 μg/ml and 200 μg/ml). Conclusions:The Chinese medicinal composition ( Xiadanqi) has a certain radioprotective effect on radon exposure induced injury by reducing oxidative stress, attenuating EMT and fibrosis, and thus it may be applied as a protective agent for radon induced injury.
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Objective:To observe the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating apoptosis during malignant transformation of human bronchial epithelial cells (HBE cells) induced by sodium arsenite (NaAsO 2). Methods:HBE cells were treated with 0.0 and 1.0 μmol/L NaAsO 2, which were control group and arsenic exposed group respectively. HBE cells were treated with 1.0 μmol/L NaAsO 2 for 43 passages to establish a malignant transformation model. The dynamic changes of indexes in different passages (0, 1st, 8th, 15th, 22nd, 29th, 36th, and 43rd) after exposure to NaAsO 2 were monitored, including the apoptosis rate detected by flow cytometry and apoptosis-related proteins and Nrf2 protein detected by Western blotting. Nrf2 siRNA was transfected into malignant transformed HBE cells (T-HBE cells) to silence Nrf2. The silencing effect of Nrf2 protein was verified. And, the apoptosis rate and apoptosis-related proteins were detected. Results:With the increase of arsenic exposure, the apoptosis rates of HBE cells decreased (0, 1, 8, 15, 22, 29, 36 and 43 passages were 0.370 ± 0.029, 0.443 ± 0.069, 0.357 ± 0.046, 0.330 ± 0.016, 0.273 ± 0.050, 0.160 ± 0.024, 0.110 ± 0.022, 0.097 ± 0.012, respectively, Ftrend = 22.981, P < 0.05). Compared with the 0 passage cells, the apoptosis rates of the 22nd, 29th, 36th and 43rd passages in the arsenite group were lower. The differences between them were statistically significant ( P < 0.05). With the increase of arsenic exposure, the expressions of pro-apoptotic proteins caspase-3, cleaved-caspase-3, C/EBP-homologous protein (CHOP) and B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) showed downward trends ( Ftrend = 22.356, 3.738, 6.130, 8.061, P < 0.05), while the anti-apoptotic proteins myeloid cell leukemia 1 protein (Mcl-1) and Bcl-2 showed upward trends ( Ftrend = 58.201, 7.691, P < 0.05). Compared with the 0 passage and the control group of the same passage, from the 22nd passage of caspase-3, cleaved-caspase-3, from the 15th passage of CHOP, Mcl-1, and Bcl-2, from the 29th passage of Bax in the arsenite group, the differences of protein were statistically significant ( P < 0.05). However, there were no significant differences in caspase-8, cleaved-caspase-8, caspase-12 and cleaved-caspase-12 protein expressions in the arsenic group ( P > 0.05). Compared with the 0 passage and the control group of the same passage, from the 8th passage of Nrf2 proteins in the arsenite group, the differences of expressions were statistically significant ( P < 0.05). Compared with T-HBE cells transfected with Con siRNA (control), the apoptosis rate of T-HBE cells transfected with Nrf2 siRNA was higher ( P < 0.05). Compared with T-HBE cells transfected with Con siRNA, the expression levels of Nrf2, Bcl-2 and Mcl-1 in T-HBE cells transfected with Nrf2 siRNA were lower ( P < 0.05), while the expression levels of cleaved-caspase-3/caspase-3, caspase-3, cleaved-caspase-3, CHOP, and Bax were higher ( P < 0.05). Conclusion:Nrf2 may regulate mitochondrial apoptotic pathway through Bcl-2, Mcl-1 and Bax, and endoplasmic reticulum apoptotic pathway through CHOP, so as to inhibit the apoptosis of HBE cells and participate in the process of malignant transformation of HBE cells induced by NaAsO 2.
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OBJECTIVE: To investigate the role of high mobility group protein 1(HMGB1) in toluene diisocyanate(TDI) induced nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3(NLRP3) inflammasome activation in human bronchial epithelial cells(HBECs). METHODS: i) The TDI-human serum albumin(HSA) stimulation experiment: the HBECs in logarithmic growth phase were randomly divided into control group, low-, medium-and high-dose groups that were pretreated with TDI-HSA with the final concentration of 0.00, 40.00, 80.00 and 120.00 mg/L for 12 hours. ii) The HMGB1 expression inhibition experiment: the HBECs in logarithmic growth phase were divided into control group, TDI-HSA group, TDI-HAS+negative-siRNA group, and TDI-HAS+HMGB1-siRNA group. The cells in TDI-HAS+negative-siRNA group and TDI-HAS+HMGB1-siRNA group were infected with HBECs with negative-siRNA lentivirus and HMGB1-siRNA lentivirus, respectively. Cells in these two groups and the TDI-HSA group were treated with 120.00 mg/L of TDI-HSA for 12 hours. The cells in the control group were not treated with TDI-HAS. iii) The expression of HMGB1, NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), pro-caspase-1 and caspase-1 p20 proteins in all groups were detected by Western blot. The number of NLRP3 and caspase-1 inflammasome in TDI-HSA stimulation experiment was observed by immunofluorescence method. RESULTS: i) TDI-HSA stimulation experiment: the relative protein expression of HMGB1 and ASC was higher in HBECs of medium-and high-dose groups than that of control group(all P values were <0.01). The relative protein expression of NLRP3 and casepase-1 p20 and the number of NLRP3-caspase-1 inflammasome were higher in HBECs of 3 dose groups than that of control group(all P values were <0.01). The number of NLRP3-caspase-1 inflammasome in HBECs increased obviously in low-, medium-and high-dose groups as compared to the control group(all P values were <0.05). The number of NLRP3-caspase-1 inflammasome in HBECs increased with the increase of TDI-HSA dose(all P values were <0.01). ii) The HMGB1 expression inhibition experiment: the relative protein expression of HMGB1, NLRP3, ASC, pro caspase-1 and caspase-1 p20 in HBECs were higher in the TDI-HSA group and TDI-HSA + negative-siRNA group than those of the control group(all P values were <0.01). The above indexes of HBECs were lower in the TDI-HAS + HMGB1-siRNA group than those in the TDI-HSA group and TDI-HSA + negative-siRNA group(all P values were <0.01).CONCLUSION: TDI treatment in HBECS can induce the increase of HMGB1 protein expression and activate NLPR3 inflammasome. Inhibition of HMGB1 expression can down-regulate the expression of NLPR3 and its related proteins.
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OBJECTIVE: To identify the differentially expressed long non-coding RNAs (IncRNAs) in γ-rays irradiated p53 knockout HBE (HBEp53-/-) cells, and to provide more baseline information for further investigation of p53 related IncRNAs in irradiation-induced injury, such as epithelial-mesenchymal transition in lung tissue epithelial cells. METHODS: HBEp53-/-cells were established with CRISPR-cas9. Western blotting was used to detect the knockout efficacy of P53 protein, while real time RT-PCR was used to verify the selected expressions of IncRNAs. Bioinformatic analysis including GO and KEGG analysis was used to predict the IncRNA function and signaling pathway. RESULTS: Compared to the irradiated HBE cells, 239 IncRNAs were up-regulated and 289 down-regulated in irradiated HBEp53-/-cells. The top five of the up-regulated or down-regulated differentially expressed IncRNAs were further confirmed by RT-PCR analysis. Through bioinformatic prediction GO analysis, the targeted genes of IncRNAs with changed expressions were related to multiple biological processes and molecular functions, including β3adrenergic receptor binding, protein tyrosine kinase activity, DNA binding, zic ion transmembrane transporter activity, ubiquitin binding and syntaxin binding. KEGG analysis revealed that the functional organismal systems involved included the sensory, nervous, immune, endocrine, environmental adaption, developmental, and circulatory systems. CONCLUSION: p53-related radiation-inducible IncRNAs have been identified in HBEp53-/-cells. These identified IncRNAs may play critical roles in various biological processes and functional signaling pathways in cellular response to radiation injury.
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Objective@#To study the effect of particulate matter 2.5 (PM2.5) on oncogene expression in human bronchial epithelial (HBE) cells.@*Methods@#HBE cells were selected as the study subjects, and PM2.5 treatment group (10 μg/ml and 50 μg/ml) , negative control group and positive control group (10 μmol/L Cr6+) were set. CCK8 assay was used to test the IC50 value of PM2.5. HBE cells were treated with PM2.5 for 24 h at 10 μg/ml and 50 μg/ml, additionally, cells were treated with blank as negative control, 10 μmol/L Cr6+ as a positive control for 24 h. After the treatment, mRNA expression of oncogenes including c-myc, c-fos, k-ras and p53 were detected by fluorescent quantitative RT-PCR, the protein expression of oncogenes were detected with western blot.@*Results@#The IC50 value of PM2.5 in HBE cells is 70.12 μg/ml. The qRT-PCR data showed that compared with the control group, the expression level of c-myc gene increased by respectively 500.1%、780.7%、305.3% after exposure to 10、50 μg/ml PM2.5 and positive control group; c-fos gene increased respectively 34.0%、76.7%、131.3% after exposure to 10、50 μg/ml PM2.5 and positive control group; k-ras gene increased respectively 50.3%、107.0%、49.7% after exposure to 10、50 μg/ml PM2.5 and positive control group; p53 gene decreased by 28.3%、28.7%、59.7% after exposure to 10、50 μg/ml PM2.5 and positive control group. The western blot results showed that compared with the control group, c-myc protein increased respectively 29.7%、77.3% after exposure to 50 μg/ml PM2.5 and positive control group; c-fos protein increased respectively 200.3%、137.0% after exposure to 50 μg/ml PM2.5 and positive control group; k-ras protein increased respectively 106.3%、130.3%、116.7% after exposure to 10、50 μg/ml PM2.5 and positive control group; p53 protein decreased by 43.7%、53.3%、52.1% after exposure to 10、50 μg/ml PM2.5 and positive control group.@*Conclusion@#PM2.5 could promote the expression of oncogenes in HBE cells, the carcinogenicity of haze might be related to promotion of oncogenes expression induced by PM2.5.
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OBJECTIVE: To investigate the effect of toluene diisocyanate(TDI) on the activation of autophagy and expression of inflammatory cytokines interleukin(IL)-4 and IL-6 in normal human bronchial epithelial cells(16 HBE). METHODS: i) We prepared TDI-human serum albumin(HSA) and determined the mass concentration of TDI in TDI-HSA. ii) The cells were treated with TDI-HSA and HSA at concentrations of 0.00-400.00 mg/L for 12 hours. CCK-8 assay was used to determinate the cell viability, and TDI-HSA and HSA doses were selected for subsequent experiments. iii) The cells were treated with TDI-HSA and HSA at doses of 0.00-120.00 mg/L for 12 hours, and the levels of reactive oxygen species(ROS) in the cells were detected by flow cytometry. The levels of IL-4 and IL-6 in the cell supernatant were measured by enzyme-linked immunosorbent assay. iv) The cells were treated with TDI-HSA at doses of 0.00-120.00 mg/L for 12 hours, and the autophagy activity was observed under transmission electron microscope. Western blot was utilized to detect the expression of Beclin1, microtubule-associated protein 1 light chain(LC3β) and P62. RESULTS: i) The mass concentrations of TDI in 40.00, 80.00 and 120.00 mg/L TDI-HSA groups were 0.44, 0.89 and 1.33 mg/L respectively. ii) The results of CCK-8 showed that TDI-HSA and HSA at doses below 120.00 mg/L did not affect cell viability, and 0.00-120.00 mg/L was selected as the TDI-HSA and HSA treatment doses for subsequent experiments. iii) The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells in the TDI-HSA group at 40.00, 80.00, and 120.00 mg/L were higher than that in HSA group at the same dose(P<0.01). The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells increased with the increase of TDI-HSA doses(P<0.01). iv) Transmission electron microscopy showed that the number of autophagic lysosomes in 16 HBE cells increased significantly, and the number of mitochondrial vacuoles increased in 40.00, 80.00, 120.00 mg/L TDI-HSA group compared with 0.00 mg/L group. With the increase of TDI-HSA dose, the relative expression of Beclin1 protein and LC3β-Ⅱ/Ⅰ ratio in 16 HBE cell supernatant increased(P<0.05), and the relative expression of P62 protein decreased(P<0.05). CONCLUSION: TDI-HSA induces increased expression of ROS and inflammatory factors and induces autophagy activation in 16 HBE cells. Autophagy may be an important factor for the development of airway inflammation in TDI-induced occupational asthma.
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Purpose To investigate the effect of down-regulation of miR-92 a on the proliferation and angiogenesis of nonsmall cell lung cancer (NSCLC). Methods Human NSCLC cell A549 was divided into three groups: A549 group (non-transfected A549 cells), sc-siRNA group (A549 cells transfected with sc-siRNA) and miR-92a-siRNA group (A 5 4 9 cells trans-fected with miR-92a-siRNA). The relative expression level of miR-92 a, PTEN and vascular endothelial growth factor (VEGF) in A549 cells and human bronchial epithelial (HBE) cells were detected by RT-PCR and Western blot respectively. The proliferation ability of A549 cells in each group was detected by living cell count and crystal violet staining experiment. Results The relative expression of miR-92 a in A549 cells was significantly higher than that in HBE cells (P < 0.05), the expression level of PTEN protein in A549 cells was significantly lower than that in HBE cells (P < 0.05), and the expression level of VEGF protein was significantly higher than that in HBE cells (P < 0.05).In the miR-92a-siRNA group, the relative expression of miR-92 a decreased (P < 0.05), the expression level of PTEN protein in-creased (P < 0.05), and the expression level of VEGF protein decreased (P < 0.05). The expression levels of PI3 K and Akt in miR-92a-siRNA group decreased (P < 0.05). the number of cells and cell proliferation ability in miR-92a-siRNA group reduced. Conclusion The expression of miR-92 a in NSCLC A549 cells is up-regulated, miR-92 a gene silencing can significantly inhibit cell proliferation and inhibit cell angiogenesis, PTEN and VEGF related PI3K/Akt signaling pathways may play an important role in this process.
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<p><b>OBJECTIVE</b>To investigate the effect of curcumin against cigarette smoke extract (CSE)- induced oxidative stress in human bronchial epithelial cells and explore the underlying mechanism.</p><p><b>METHODS</b>Human bronchial epithelial cell line 16HBE was treated for 24 h with curcumin, CSE, CSE + curcumin, and CSE + curcumin with transfection by a short hairpin RNA targeting PPARγ (shPPARγ). MTT assay was used to observe the changes in the cell viability after the treatments. Quantitative real-time PCR was performed to detect the mRNA expressions of tumor necrosis factor- (TNF-), iNOS and PPARγ in the cells, and the protein expressions of iNOS, PPARγ and the phosphorylation of NF-κB p65 were detected using Western blotting.</p><p><b>RESULTS</b>The treatments did not cause significant changes in the cell viability. Exposure to CSE for 24 h significantly lowered PPARγ expression and increased TNF- and iNOS expressions and phosphorylation of NF-κB p65 in the cells. The effects of CSE were significantly suppressed by curcumin, but transfection of the cells with shRNA-PPARγ obviously abrogated the suppressive effects of curcumin.</p><p><b>CONCLUSIONS</b>Curcumin suppresses CSE-induced oxidative stress and inflammation via the PPARγ/NF-κB signaling pathway in 16HBE cells, suggesting the potential of curcumin in the treatment of chronic obstructive pulmonary disease.</p>
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Objective:To investigate the effect of Lyn on the expression of MUC5AC in human bronchial epithelial cells induced by house dust mite(HDM),and to explore its possible mechanism.Methods:The human bronchial epithelial cells(16 HBE)were divided into PBS group and HDM group(1 μg·L-1HDM).The cells were transfected by liposome.The luciferase report gene of MUC5AC promoter was constructed.The relative luciferase unit(RLU)was detected by double luciferase report gene assay.The expression of MUC5AC in the cells was detected by immunofluorescence technique and observed by confocal microscope.The expression level of STAT6 in the 16HBE cells was detected by Western blotting method.Results:The results of double luciferase report gene assay showed that the RLU in HDM group was higher than that in PBS group(P<0.05).The RLU of cells in HDM group treated with LynsiRNA intervention was higher than that of the cells without LynsiRNA intervention(P<0.05).The immunofluorescence results demonstrated that the expression level of MUC5AC in HDM group was higher than that in PBS group(P<0.05),and the expression level of MUC5AC in HDM group was increased after LynsiRNA intervention(P<0.05).The Western blotting results indicated that the expression level of STAT6 was up-regulated in HDM group when the cells were intervened with LynsiRNA(P<0.05). Conclusion:Deficiencyof Lyn can increase the expression of MUC5AC in the human bronchial epithelial cells,and its mechanism may be related to regulating the STAT6 signal pathway by Lyn.
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Objective: To investigate the effect of Lyn on the expression of MUC5AC in human bronchial epithelial cells induced by house dust mite (HDM), and to explore its possible mechanism. Methods: The human bronchial epithelial cells 16HBE) were divided into PBS group and HDM group 1 μg · L-1 HDM). The cells were transfected by liposome. The luciferase report gene of MUC5AC promoter was constructed. The relative luciferase unit (RLU) was detected by double luciferase report gene assay. The expression of MUC5AC in the cells was detected by immunofluorescence technique and observed by confocal microscope. The expression level of STAT6 in the 16HBE cells was detected by Western blotting method. Results: The results of double luciferase report gene assay showed that the RLU in HDM group was higher than that in PBS group (P<0.05). The RLU of cells in HDM group treated with LynsiRNA intervention was higher than that of the cells without LynsiRNA intervention (P<0.05). The immunofluorescence results demonstrated that the expression level of MUC5AC in HDM group was higher than that in PBS group (P<0.05), and the expression level of MUC5AC in HDM group was increased after LynsiRNA intervention (P<0.05). The Western blotting results indicated that the expression level of STAT6 was up-regulated in HDM group when the cells were intervened with LynsiRNA (P<0.05). Conclusion: Deficiency of Lyn can increase the expression of MUC5AC in the human bronchial epithelial cells, and its mechanism may be related to regulating the STAT6 signal pathway by Lyn.
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PURPOSE: In order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls. METHODS: HBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-β and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR. RESULTS: PIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-β mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-β, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-β mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics. CONCLUSIONS: The immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status.
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Humans , Asthma , Bronchi , Bronchoscopy , Chemokine CCL5 , Chemokines , Enzyme-Linked Immunosorbent Assay , Epithelial Cells , Gene Expression , Immunity, Innate , Interferons , Paramyxoviridae Infections , Polymerase Chain Reaction , Rhinovirus , RNA, MessengerABSTRACT
AIM:To investigate the role of caveolin-1 on epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells induced by transforming growth factor beta 1 (TGF-β1).METHODS:Immunofluorescence, real-time PCR and Western blot were applied to detect the mRNA and the protein expression of caveolin-1 in the 16HBE cells during EMT.The influence of siRNA-mediated silencing of caveolin-1 on EMT in the 16HBE cells was detected by Western blot.RESULTS:Caveolin-1 was widely present on the cell membrane of the 16HBE cells.The expression of caveolin-1 at mRNA and protein levels was significantly decreased in a time-dependent manner in the 16HBE cells compared with control group (P<0.05) after stimulation with TGF-β1.The morphologic changes of the 16HBE cells induced by TGF-β1 were promoted by caveolin-1 silencing compared with TGF-β1 group.The protein expression of E-cadherin and α-SMA induced by TGF-β1 was promoted by caveolin-1 silencing compared with TGF-β1 group (P<0.05).The phosphorylation levels of AKT and Smad3 were the highest at 30 min and increased significantly compared with control group (P<0.05) after stimulated with TGF-β1.Treatment of the 16HBE cells with TGF-β1 for 30 min after silencing caveolin-1 gene for 24 h significantly increased the phosphorylation levels of AKT and Smad3 compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 down-regulates the expression of caveolin-1 in the 16HBE cells.Caveolin-1 may participate in TGF-β1/Smad pathway and PI3K-AKT pathway, which are the signal transduction pathways for TGF-β1 inducing EMT.
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AIM:To investigate the effect of signal transducer and activator of transcription 3 (STAT3) on air-way injury in asthma and its mechanism .METHODS:The expression and activation of STAT 3 in bronchial mucosa tissues of asthmatic patients were measured by Western blot .The expression and activation of STAT 3 in bronchial epithelial cells pretreated with Dermatophagoides pteronyssinus antigen P1 (Derp1) were estimated.Bronchial epithelial cells were trans-fected with STAT3 shRNA.STAT3 expression was measured by Western blot .The cell viability was detected by CCK-8 as-say.The concentrations of TNF-α, IL-1βand IL-6 were detected by ELISA .The content of malondialdehyde ( MDA) and the activity of superoxide dismutase ( SOD) and glutathione peroxidase ( GSH-Px) were also determined for evaluating the status of oxidative stress .The cell apoptosis was analyzed by flow cytometry .RESULTS:The protein level of p-STAT3 was significantly up-regulated in both bronchial mucosa of asthmatic tissues and bronchial epithelial cells pretreated with Derp 1. Knockdown of STAT3 inhibited the releases of TNF-α, IL-1βand IL-6, decreased the content of MDA and enhanced the activity of SOD and GSH-Px with the suppression of cell apoptosis ( P <0.05 ) .CONCLUSION: Down-regulation of STAT3 attenuates Derp1-induced the airway injury .The mechanism may involve that knockdown of STAT3 suppresses the activation of JAK/STAT signaling pathway , the release of asthma-related inflammatory cytokines and oxidative stress in bronchial epithelial cells , thus inhibiting cell apoptosis .
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Objective:To investigate the effects of airborne fine particle on cell viability and inflammation in human bronchial epithelial cells.Methods:Atmospheric PM2.5 samples were collected by PM2.5 sampler.PM2.5 morphology was observed by scanning electron microscope (SEM).Human bronchial epithelial cells (BEAS-2B) were treated with PM2.5 at different concentrations (0,50,100,200,400,800 μg/mL) for 12,24 or 48 hours,and the cell activity were evaluated by cell counting kit-8 (CCK-8).The mRNA expression levels of (granulocyte-macrophage colony stimulating factor,GM-CSF) and TNF-α were detected by quantitative real-time PCR (qRT-PCR).Western blot was used to detect the protein expressions of GM-CSF and TNF-α.Results:According to SEM,the shape of PM2.5 varied,and the diameter was different and mostly equal to or less than 2.5 μm.CCK-8 assay showed that different concentrations of PM2.5 exposure for 12 hours,24 hours and 48 hours resulted in loss of cell viability of BEAS-2B cells (P＜0.05).Different concentrations of PM2.5 increased the mRNA and protein expression of GM-CSF and TNF-α,and the higher concentration of PM2.5 induced higher expression,which have statistical significant difference between the groups (P＜0.05).Conclusion:Atmospheric PM2.5 can cause inflammatory response in human bronchial epithelial cells.They can reduce cell viability,which may be related to the PM2.5 trigger and aggravation of bronchopulmonary inflammatory diseases.
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AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 ( TGF-β1).METH-ODS:Silencing of TRPC1 gene expression was performed by siRNA.The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively.The migration and invasion abilities of the 16HBE cells were detected by wound-healing assay and Transwell assay.The protein expression of E-cadherin and vimentin was determined by Western blot.RESULTS:TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups ( P0.05).The results of wound-healing and Tr-answell assays showed that migration and invasion abilities in TRPC1 siRNA +TGF-β1 group were markedly suppressed compared with TGF-β1 group (P<0.01).The protein expression of E-cadherin and vimentin induced by TGF-β1 was in-hibited by TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TRPC1 is involved in the migra-tion of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vim-entin.
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Objective To explore the role of the transcriptional factor activator protein (AP)-1 in mediating vascular endothelial growth factor ( VEGF) expression in human bronchial epithelial cells exposed to PM 2.5.Methods Beas-2B cells was treated with PM2.5.Luciferase assay was used to detect the activation status of AP-1 and transcription of VEGF in the Beas-2B cells.The induced activation of c-Jun, ATF2 and VEGF expression was tested by Western blotting assay.Results PM2.5 induced transactivation of the transcriptional factor AP-1, accompanied by phosphorylation of the AP-1 components, c-Jun and ATF2 in Beas-2B cells.Moreover, when AP-1 activation was inhibited by knocking down c-Jun or ATF2 expressions, induction of VEGF expression was partially attenuated in Beas-2B cells.Conclusion AP-1 is a critical transcriptional factor in mediating PM2.5-induced VEGF expression and inflammatory responses in human bronchial epithelial cells.