Dendrobium Polysaccharides Mitigate CSE-induced Inflammatory Damage of Human Bronchial Epithelial Cells via TLR4/NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Dendrobium polysaccharides on the secretion of inflammatory cytokines and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB pathway in 16HBE cells exposed to cigarette smoke extract （CSE）. MethodThe 16HBE cells were classified into the control， CSE， and CSE+ Dendrobium polysaccharides （100， 200， 400 mg·L-1） groups. The cell-counting kit-8 （CCK-8） assay was employed to measure the cell viability， and a microscope was used to observe the cell morphology. The enzyme-linked immunosorbent assay was employed to measure the levels of interleukin （IL）-8， IL-1β， IL-4， IL-13， and transforming growth factor （TGF）-β in cell culture supernatants. Real-time PCR was carried out to determine the mRNA levels of Toll-like receptor 4 （TLR4）， nuclear factor-κB （NF-κB）， and IL-4. Western blot was employed to determine the protein levels of interleukin-4 receptor （IL-4R）， TLR4， myeloid differentiation primary response protein 88 （MyD88）， NF-κB， phosphorylated nuclear factor-κB （p-NF-κB）， and nucleoproteins nuclear factor-κB （NEs-NF-κB）. The immunofluorescence assay was employed to measure the nuclear translocation of NF-κB. ResultCompared with the control group， the CSE group showed elevated levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β in the cell culture supernatants （P<0.05， P<0.01）， up-regulated expression levels of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.01）， and significant nuclear translocation of NF-κB. Compared with the CSE group， Dendrobium polysaccharides increased the cell survival rate， recovered the cell activity， lowered the levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β， down-regulated the expression of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.05， P<0.01）， and reduced the nuclear translocation of NF-κB. ConclusionDendrobium polysaccharides showed significant protective effects on the 16HBE cells exposed to CSE by inhibiting the TLR4/NF-κB signaling pathway.

Effects of arsenic and its metabolites on expressions of BCL-2α and BCL-2β transcripts / 环境与职业医学

Background Arsenic is a toxicant that can affect the expressions of the cellular anti-apoptotic gene BCL-2 and its protein, but the effects of arsenic on BCL-2α and BCL-2\begin{document}$\beta $\end{document} transcripts have not been reported. Objective To investigate the potential effects of arsenic and its metabolites, methylarsonic acid (MMA) and dimethylarsonic acid (DMA), on BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T (total of α and \begin{document}$\beta $\end{document} transcripts) in human bronchial epithelial cells (16HBE) and human lung adenocarcinoma cells (A549). Methods 16HBE cells and A549 cells were randomly divided into three categories of exposure after in vitro culture: single-selected arsenic compound exposure groups with isoconcentration (16HBE cells were treated with 4.5 μmol·L−1 of MMA, DMA, and sodium arsenite, respectively, while A549 cells were treated with 60 μmol·L−1 of MMA, DMA, and sodium arsenite, respectively), sodium arsenite exposure groups with different concentrations (16HBE cells were treated with 1.5, 3.0, and 4.5 μmol·L−1 of sodium arsenite respectively, while A549 cells were treated with 20, 40, and 60 μmol·L−1 of sodium arsenite respectively), and combined exposure groups (i.e. MMA+sodium arsenite, and DMA+sodium arsenite; the exposure concentrations of 16HBE cells were both 1.5 μmol·L−1 and both 4.5 μmol·L−1 respectively, and those of A549 cells were both 20 μmol·L−1 and both 60 μmol·L−1 respectively). Meanwhile, a blank control group was also set up in each exposure category. After 48 h of continuous exposure, the relative expressions of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T in both cells were detected by real-time PCR. Results Regarding the single-selected arsenic compound exposure, in 16HBE cells, the expression levels of BCL-2α and BCL-2T under 4.5 μmol·L−1 MMA treatment were lower than those in their control groups (q=3.27, 2.93, both P<0.05), and the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T under 4.5 μmol·L−1 sodium arsenite were lower than those in their respective control groups (q=11.06, 3.65, 10.70, all P<0.05). In A549 cells, the expression level of BCL-2T treated with 60 μmol·L−1 DMA was lower than that in the control group (q=3.12, P<0.05), and the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T treated with 60 μmol·L−1 sodium arsenite were lower than those in their respective control groups (q=7.59, 7.27, 8.06, all P<0.05). Regarding the sodium arsenite exposure: 16HBE cells treated with 1.5 μmol·L−1 sodium arsenite had a lower expression level of BCL-2α and a higher expression level of BCL-2\begin{document}$\beta $\end{document} than those in their respective control groups (q=6.06, 11.92, both P<0.05); the expression level of BCL-2α under 3.0 μmol·L−1 sodium arsenite was lower than that in the control group (q=12.72, P<0.05); and under 4.5 μmol·L−1 sodium arsenite treatment, the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T were lower than those in their respective control groups (q=15.72, 6.79, 6.62, all P<0.05). The expression levels of BCL-2α gradually decreased with increasing concentrations of sodium arsenite (Fα trend=144.80, P<0.001), while BCL-2\begin{document}$\beta $\end{document} and BCL-2T decreased in a dose-dependent manner in the range of 1.5-4.5 μmol·L−1 (F\begin{document}${}_{\beta } $\end{document} trend=135.40, FT trend=38.24, both P<0.001). In A549 cells, the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T under each concentration of sodium arsenite treatments were lower than those in their respective control groups (all P<0.05); the results of further trend tests showed that their expression levels gradually decreased with increasing concentrations of sodium arsenite (Fα trend =31.97, F\begin{document}${}_{\beta} $\end{document} trend=549.50, FT trend=252.40, all P<0.001). Regarding the combined exposure, under MMA+sodium arsenite treatment at both 60 μmol·L−1, the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T in A549 cells were higher than those in their respective control groups (q=6.37, 14.91, 5.33, all P<0.05); under DMA+sodium arsenite treatment at both 60 μmol·L−1, their expression levels in A549 cells were also higher than those in their respective control group (q=8.60, 17.29, 6.91, all P<0.05). Conclusion Exposure to a high concentration (16HBE: 4.5 μmol·L−1, A549: 60 μmol·L−1) of a single arsenic metabolite has no effect on BCL-2 transcripts in 16HBE cells and A549 cells. Exposure to a low concentration (1.5 μmol·L−1) of sodium arsenite alone would decrease the expression level of BCL-2α and increase the expression level of BCL-2\begin{document}$\beta $\end{document} in 16HBE cells, and exposure to all designed concentrations of sodium arsenite alone would decrease the expressions of all transcripts in A549 cells. The combined exposure to high concentrations (both 60 μmol·L−1) of MMA plus sodium arsenite or high concentrations (both 60 μmol·L−1) of DMA plus sodium arsenite would increase the expressions of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T in A549 cells, which are different from the effects presented by single exposure.

Analysis of the potential role of SET in chromium-induced malignant transformation cells based on quantitative proteomics / 中华劳动卫生职业病杂志

Objective@#To investigate alteration of proteins profile in malignant transformation bronchial epithelial cells(16HBE-T) induced by hexavalent chromium[(Cr(VI))] and analyze the expression level of SET protein, then to provide some new insights for the carcinogenesis mechanism of Cr(VI).@*Methods@#Total protein was extracted from 16HBE cells and was alkylated and desalinated before digested into peptides. The products were labeled with Tandem Mass Tag (TMT) and identified using LC-ESI-MS/MS.@*Results@#A total of 3 517 proteins were found, expression differences greater than 1.5 or less 0.67 times were to found have 185 and 201 proteins, respectively. Gene enrichment analysis revealed that differential proteins were mainly involved in autophagy, DNA damage repair, RNA processing and other biological processes. Western blot results showed the expression level of SET was significantly increased while downregulated in histone H3K18/27 acetylation and p53 protein.@*Conclusion@#Proteins involved in multiple biological processes altered in 16HBE-T cells and regulation mode of SET inhibiting histone H3K18/27 acetylation regulating transcriptional activity of p53 may paly an important role in Cr(VI)-association carcinogenesis.

Effect of nano-SiO_2 on the survival and PARP-1 expression in 16HBE cells / 中国职业医学

OBJECTIVE: To explore the effects of nano-silicon dioxide( SiO_2) on the survival and poly( ADP-ribose)polymerase-1( PARP-1) expression in human bronchial epithelial cells( 16 HBE cells). METHODS: i) The 16 HBE cells were treated with nano-SiO_2 at concentrations ranging from 0 to 100 mg/L for 24. 0 hours,and CCK-8 assay was used to examine cell viability. ii) The 16 HBE cells were divided into 6 groups: solvent control group( equal volume solvent treatment),micro-SiO_2 control group( treated with 20 mg/L micro-SiO_2),5,10,and 20 mg/L nano-SiO_2 groups( treated with the corresponding final dose of nano-SiO_2),and curcumin group. The curcumin group was given pretreatment with curcumin at a final concentration of 10 μmol/L for 2. 0 hours followed by treatment with a final concentration of 20 mg/L of nano-SiO_2. Cells in each group were harvested at time points of 4. 0,12. 0 and 24. 0 hours after treatment. The relative expression of PARP-1 mRNA and protein in 16 HBE cells was detected by quantitative real-time polymerase chain reaction and Western blotting respectively. RESULTS: i) The survival of 16 HBE cells decreased with increasing nano-SiO_2 treatment dose,showing a dose-effect relationship( P < 0. 01). ii) The expression of PARP-1 mRNA and protein in 16 HBE cells were dose-dependently decreased after nano-SiO_2 stimulation at the 12. 0 and 24. 0 hours time points( P < 0. 01). The expression of PARP-1 mRNA and protein in 5,10,and 20 mg/L nano-SiO_2 groups decreased at the above mentioned time points( P < 0. 05),compared with the solvent control group at the same time points. The expression of PARP-1 mRNA and protein in 20 mg/L nano-SiO_2 group was lower than that in the micro-SiO_2 control group at the same 12. 0 and 24. 0 hours time point( P < 0. 05). The above two indexes of cells were higher in curcumin group than that of 20 mg/L nano-SiO_2 group at the 12. 0 hours time point( P < 0. 05). CONCLUSION: Nano-SiO_2 stimulation can lead to decrease survival of 16 HBE cells in a dose-dependent manner and down-regulation of PARP-1 expression may be one of the mechanisms of proliferation and inhibition of 16 HBE cells induced by nano-SiO_2. Curcumin has certain protective effect on nano-SiO_2-induced 16 HBE cell injury.

Curcumin suppresses cigarette smoke extract-induced oxidative stress through PPARγ/ NF-κB pathway in human bronchial epithelial cells / 南方医科大学学报

<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>

Study on the expression of epidermal growth factor receptor protein during benzo(a)pyrene induced carcinogenesis / 中华预防医学杂志

Objective@#To explore the expression of epidermal growth factor receptor(EGFR) protein during benzo(a)pyrene (BaP) induced carcinogenesis.@*Methods@#This study, we firstly utilized immunofluorescence assay and Western-blot to examine EGFR expression of the BaP which was constructed previously by project team induced malignant transformation human bronchial epithelial cell (BTC) and the control (16HBE cell). Then, we selected 36 healthy SD rats, divided into two groups according to simple random method, 18 rats each group. The constructed rat lung neoplasm model induced by pulmonary injection of BaP (10 mg/ml of BaP solution in 0.2 ml corn oil), contrast group use 0.2 ml corn oil, lung tissue was collected and the EGFR expression of lung tissue was detected by immunofluorescence assay and Western blot. T analysis was used to test the different of EGFR between two groups.@*Results@#Immunofluorescence analysis showed that the EGFR expression in BTC was significantly higher than 16HBE cell. Meanwhile, Western blot also was used to confirmed this result, the relative expression of EGFR protein in the rats of the model group the control group were 1.04±0.13 and 2.32±0.12, respectively, and the difference was statistically significance (t=12.39, P<0.001). In vivo, well-defined tumor was found in the rat with pulmonary injection of BaP, and the lung showed diffuse alveolar septal thickening, alveolar wall destruction and pulmonary alveoli fusion, which suggested that the rat lung neoplasm model was constructive successfully. Furthermore, we found the EGFR expression of lung was increased dramatically in the rat lung neoplasm model by immunofluorescence detection and Western blot. The relative expression of EGFR protein in the rats of the model group the control group were 0.21±0.03 and 1.30±0.07, respectively, and the difference was statistically significance (t=12.84, P<0.001).@*Conclusion@#Expression of EGFR protein was increased during BaP carcinogenesis, and EGFR may play an important role in the carcinogenesis of BaP.

Autophagy Attenuates MnCl2-induced Apoptosis in Human Bronchial Epithelial Cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the role of autophagy in MnCl2-induced apoptosis in human bronchial epithelial 16HBE cells.</p><p><b>METHODS</b>Cell proliferation was measured by MTT assay. Mitochondrial membrane potential (MMP) and apoptosis were measured by flow cytometry. Autophagic vacuoles were detected by fluorescence microscopy. Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.</p><p><b>RESULTS</b>16HBE cell proliferation was inhibited by MnCl2 in a dose- and time-dependent manner. MnCl2-induced 16HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis. Our data revealed that MnCl2-induced apoptosis in 16HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3. It was observed that when we exposed 16HBE cells to MnCl2 in a dose-dependent manner, the formation of autophagic vacuoles and the levels of LC-3B-II were elevated. RNA interference of LC3B in these MnCl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced. Additionally, the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis, but did not affect the cellular levels of LC3B in MnCl2-treated 16HBE cells.</p><p><b>CONCLUSION</b>MnCl2 dose- and time-dependently inhibits 16HBE cell proliferation and induces MMP loss and apoptosis. Autophagy acts in a protective role against MnCl2-induced apoptosis in 16HBE cells.</p>

Effect and mechanism of IL-31 on the expression of VEGF, EGF and EGFR in 16HBE cells / 基础医学与临床

Objective_To explore the influence and mechanism of IL-31 on the expression of VEGF, EGF and EG-FR in 16HBE cells.Methods_16HBE cells were cultured and treated with IL-31 with or without SB203580 or SP600125, real-time PCR and Western blot were applied to determine the mRNA and protein expression of VEGF, EGF and EGFR respectively.Meanwhile, Western blot was used to examine the changes of P38 MAPK and JNK signaling pathways.Results_Compared with control group, the mRNA expression of VEGF, EGF and EGFR was increased markedly under the stimulation of IL-31 ( P<0.01 ) , the expression of p-P38 MAPK and p-JNK signifi-cantly increased ( P<0.01) .Compared with IL-31 group, the expression of p-P38 MAPK significantly decreased in IL-31 combined with SB203580 or SB203580 group ( P <0.01 ) , while the expression of p-JNK markedly decreased in IL-31 combined with SP600125 or SP600125 group( P<0.01) .Compared with IL-31 group, the expression of VEGF was significantly decreased in IL-31 combined with SB203580 or SP600125 group ( P <0.01 ) , while the expression of EGF and EGFR was markedly declined in IL-31 combined with SB203580 group ( P<0.01 ) .Conclusions_IL-31 may up-regulate the expression of VEGF through activating P38 MAPK and JNK signaling pathways and up-regulate the expression of EGF and EGFR through activating P38 MAPK signaling path-way in16 HBE cells.

Experimental study on malignant transformation of human bronchial epithelial cells induced by glycidyl methacrylate and analysis on its methylation / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish the model of human bronchial epithelial cells (16HBE) malignant transformation induced by glycidyl methacrylate (GMA) and define the different methylation genes at different stages.</p><p><b>METHODS</b>DNA was extracted at different 16HBE malignant phases and changes of genes DNA methylation at different stages were detected using Methylation chip of 'NimbleGen HG18 CpG Promoter Microarray Methylation'. Methylation-specific PCR (MSP) was used to observe the methylation status of some genes, and then compared with the control groups.</p><p><b>RESULTS</b>The result showed that GMA induced 16HBE morphorlogical transformation at the dose of 8 µg/mL, and cell exposed to GMA had 1374 genes in protophase, 825 genes in metaphase, 1149 genes in anaphase, respectively; 30 genes are all methylation in the 3 stages; 318 genes in protophase but not in metaphase and anaphase; 272 genes in metaphase but not in protophase and anaphase; 683 genes in anaphase but not in metaphase and protophase; 73 genes in protophase and metaphase but not in anaphase; 67 genes in protophase and anaphase but not in metaphase; 59 genes in metaphase and anaphase but not in protophase.</p><p><b>CONCLUSION</b>The pattern of DNA methylation could change in the process of 16HBE induced by GMA.</p>