OBJECTIVENaringenin has been reported to attenuate Mucin (MUC) 5AC secretion in many pathological models. Many stimuli activate MUC5AC expression through JNK/AP-1 signaling pathways. We hypothesized that naringenin may have inhibitory effects on mucous hypersecretion by modulating MUC5AC production and inhibiting JNK/AP-1 signaling pathways.

METHODThe cell model of mucous hypersecretion was made by human lung adenocarcinoma epithelial (A549) cells stimulated by RSV. A549 cells were subcultured and then randomly divided into 7 groups, which were designated as group C (cell control group), groups R1-3 (cells were infected with RSV at the multiplication of infection (MOI) of 0. 5, 1. 0, 5. 0), groups N1-2 (cells infected with viruses in presence of Nar 30 - 100 mol/L), groups N3-4 (uninfected cells treated with Nar 30 - 100 µmol/L), group D (DMSO), group S (cells infected with viruses in presence of SP600125). After incubating for 24 hrs, the expression of MUC5AC at mRNA and protein level in the groups were determined by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). The protein expression changes of JNK, p-JNK and AP-1 were measured by Western blotting.

RESULTThe expressions of MUC5AC protein and mRNA in all RSV infected groups were significantly higher than that in group C in a dose-dependent manner (all P <0. 05). Nar of 30 and 100 µmol/L significantly and dose-dependently decreased RSV-induced secretion of MUC5AC protein in cell supernatant and expression of MUC5AC mRNA (P <0. 05). The relative content of p-JNK, AP-l in R2 groups were 3. 31 ± 0. 34 and 1. 94 ± 0. 05. Theyfrweremtgnificanty increased as compared with group C (both 1. 00 ± 0. 00) (all P <0. 05). The levels of p-JNK in N2 and S groups were 2. 10 ± 0. 20. 27 and 1.±97 ± 0. 16. The levels of AP-1 in N2 and S groups were 1. 40 ± 0. 03, 1. 36 ± 0. 05. Nar and SP600125 led to a largest decrease in levels of p-JNK and AP-1 when compared with group R2 (P <0. 05). The MUC5AC protein in group R2 was (48. 19 ± 0. 47) µg/L. The protein expression of MUC5AC in group R2 was significantly higher than that in group C [(36. 67 ± 1. 50) g/L] with a statistically significant difference (P <0. 05). The protein expression of MUC5AC in groups N2 and S were(43. 17 ± 1. 06) µg/L, (44.±02 ± 0. 99) µg/L, Nar and SP600125 remarkably inhibited RSV-induced secretion of MUC5AC in supernatant of A549 cells (P < 0. 05).

CONCLUSIONSNaringenin might be able to block RSV-induced mucous

Adenocarcinoma ; Blotting, Western ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Flavanones ; pharmacology ; Humans ; Lung Neoplasms ; Mucin 5AC ; secretion ; Mucus ; secretion ; Random Allocation ; Signal Transduction ; Transcription Factor AP-1 ; drug effects

BACKGROUND/AIMS: The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner. METHODS: Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor kappaB (NF-kappaB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified. RESULTS: Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-kappaB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-kappaB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls. CONCLUSIONS: Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-kappaB activation and IL-8 expression in H. pylori-infected human epithelial cells.
Adult Stem Cells/physiology ; Caco-2 Cells ; Cell Line ; Epithelial Cells/*metabolism/microbiology ; Gene Expression ; Genomic Islands ; Helicobacter Infections/*genetics ; *Helicobacter pylori ; Humans ; Interleukin-8/genetics/secretion ; NF-kappa B/metabolism ; Neutrophils/*physiology ; Nod1 Signaling Adaptor Protein/*physiology ; RNA, Messenger/metabolism ; Signal Transduction ; Transendothelial and Transepithelial Migration/*physiology ; Up-Regulation

OBJECTIVETo observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib.

METHODSThe effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib.

RESULTSThe MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin.

CONCLUSIONSHGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.

Adenocarcinoma ; metabolism ; pathology ; Antineoplastic Agents ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Drug Resistance, Neoplasm ; Epithelial-Mesenchymal Transition ; Fibroblasts ; cytology ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; secretion ; Humans ; Lung ; cytology ; Lung Neoplasms ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-met ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; drug effects ; Transforming Growth Factor alpha ; pharmacology ; Tumor Microenvironment ; Vimentin ; metabolism

OBJECTIVETo evaluate the protective effect of licoflavone on gastric mucosa in rats with chronic superficial gastritis and explore the possible mechanism.

METHODSSD rat models of chronic superficial gastritis was established by intragastric administration of 0.02% ammonia and long-term irregular diet. The rat models were then randomized into model group, vitacoenzyme group and 3 licoflavone groups of high, medium, and low doses. After 30 days of treatment, the gastric histopathology, mucosal lesions, scanning electron microscopy, mucin function production by the gastric mucosa epithelial cells, serum PGE(2) level and gastric microcirculation were assessed to evaluate the protective effect of licoflavone on gastric mucosa.

RESULTSCompared with normal control rats, the rat models of chronic superficial gastritis showed significantly higher gastric mucosal injury rate, histopathological scores and gastric mucin content. Licoflavone significantly ameliorated gastric pathology and increased serum PGE(2) level, enhanced acidic mucin secretion by the epithelial cells, and improved gastric microcirculation in the rat models.

CONCLUSIONLicoflavone feeding suppresses gastric mucosa injury, protects and restores the injured mucosa in rats with chronic superficial gastritis, and these effects are related with the up-regulation of serum PGE(2) level.

Animals ; Chronic Disease ; Dinoprostone ; blood ; Epithelial Cells ; secretion ; Female ; Flavones ; pharmacology ; Gastric Mucosa ; drug effects ; pathology ; Gastritis ; pathology ; Male ; Microcirculation ; Mucins ; secretion ; Rats ; Rats, Sprague-Dawley

Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.
Animals ; Benzoates ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; metabolism ; Exocrine Glands ; secretion ; Flavones ; administration & dosage ; pharmacology ; Humans ; Mice ; Patch-Clamp Techniques ; Rats ; Rats, Inbred F344 ; Thiazolidines ; pharmacology ; Thyroid Gland ; cytology ; Time Factors ; Trachea ; secretion

OBJECTIVETo investigate the effect of puerarin on interleukin (IL)-8 mRNA expression and the protein release in the co-culture of human bronchial epithelial (BEAS-2B) cells and human neutrophils.

METHODSBEAS-2B cells and neutrophills were cultured separately and co-cultured with puerarin (50, 100, and 200 μg/mL) for a predetermined time. Cytokines in culture supernatant were evaluated by protein array and IL-8 quantified by enzyme-linked immunosorbent assay (ELISA). IL-8 mRNA expression was evaluated by real-time quantitative polymerase chain reaction (real-time qPCR).

RESULTSThe 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 supernatant 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 supernatant of the co-culture of BEAS-2B cells and neutrophils (P<0.01).

CONCLUSIONPuerarin could exhibit anti-inflammatory activity by suppressing IL-8 production from the co-culture of human bronchial epithelial cells and neutrophils.

Adult ; Bronchi ; cytology ; Cell Communication ; drug effects ; Cell Line ; Coculture Techniques ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Fluorescence ; Gene Expression Regulation ; drug effects ; Humans ; Interleukin-8 ; genetics ; secretion ; Isoflavones ; pharmacology ; Neutrophils ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo establish a method for isolating and culturing human amniotic epithelial cells (hAECs) in a serum-free medium and investigate their transdifferentiation ability into islet-like cells.

METHODSThe culture condition of hAECs was optimized using DMEM with different supplements. The genetic stability of the tenth-passage cells was assessed by chromosome analysis and G-banding method. The stem cell characteristics of the cells were identified by examination of the surface markers using immunofluorescence methods. The endocrine-related genes and hormones of the cells were tested after induced differentiation into islet-like cells.

RESULTSThe hAECs allow stable passaging in the presence of 10 ng epidermal growth factor (EGF) in the culture medium. After 10 passages, the cells maintained a normal karyotype and G-banding profile. The hAECs expressed many multi-potent stem cell markers, including SSEA4, TRA-1-60, and TRA-1-81. After induced differentiation, the endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon. Insulin secretion increased in the differentiated islet-like cells in response to high glucose exposure.

CONCLUSIONWe established a method for isolating and expanding the hAECs in a serum-free medium. hAECs possess stem cell characteristics and can be induced to differentiate into islet-like cells in vitro.

Amnion ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; secretion ; Humans ; Insulin ; secretion ; Islets of Langerhans ; cytology ; Stem Cells ; cytology ; secretion

OBJECTIVETo determine the effect of Porphyromonas gingivalis lipopolysaccharide (LPS) on induced secretion of inflammatory cytokines by different cell lines.

METHODSLPS of P. gingivalis strain ATCC33277 (Pg-LPS) was extracted with phenol-water method, and identified by Limulus test and infrared spectrum analysis. KB, HGF-1 and THP-1 cells were treated with Pg-LPS of different concentrations and time duration, a commercial LPS of E.coli strain O111:B4 (E-LPS) was used as the control. TNF-alpha, IL-1beta, IL-6 and IL-8 levels in culture supernatants were measured by quantitative ELISA.

RESULTThe minimal dosages of both Pg-LPS and E-LPS to solidify Limulus agents were 15 ng/ml and their infrared spectrums were similar. With the treatment of Pg-LPS or E-LPS, the TNF-alpha and IL-1beta levels secreted by HGF-1 cells were remarkably increased with a single perk (P<0.01) while a continuous enhancement of secretion by THP-1 cells was observed (P<0.01). Either Pg-LPS or E-LPS stimulated HGF-1 or THP-1 cells to continuously increase the secretion of IL-6 (P<0.01). Both Pg-LPS and E-LPS induced IL-8 secretion by THP-1 cells (P<0.01), but only Pg-LPS showed the similar effect on HGF-1 cells (P<0.01). Neither Pg-LPS nor E-LPS induced KB cells to secrete inflammatory cytokines.

CONCLUSIONPg-LPS can promote target cells to increase their secretion of inflammatory cytokines. KB cells can not be used as the target cell to determine inflammation-causing effect of LPS.

Cell Line ; Epithelial Cells ; cytology ; metabolism ; Fibroblasts ; cytology ; metabolism ; Humans ; Interleukin-1beta ; secretion ; Interleukin-6 ; secretion ; Interleukin-8 ; secretion ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; chemistry ; Tumor Necrosis Factor-alpha ; secretion

Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Mucin 5AC ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Respiratory System ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation ; drug effects

OBJECTIVETo evaluate the effect of JAK/STAT signaling pathway activation on the transdifferentiation and secretion of transforming growth factor-beta1 (TGF-beta1) induced by high glucose in renal proximal tubular epithelial cells.

METHODSHuman kidney cells (HKC) were cultured and then divided into four groups: low glucose (LG) group, high glucose (HG) group, high mannitol (LG + M) group, and HG + AG490 group. Immunoprecipitation and Western blot analysis were used to determine the expression of tryosine phosphorylated Janus kinase 2 ( p-JAK2). The protein expressions of STAT1, STAT3, p-STAT1, and p-STAT3 and the expressions of alpha-SMA and E-Cadherin were observed by Western blot. The contents of TGF-B1, fibronectin and type I collagen in the supernatants of the cultured HKC were detected by enzyme-linked immunosorbent assay (ELISA). The expression of TGF-beta1 mRNA was measured by reverse transcription and polymerase chain reaction (RT-PCR).

RESULTSCompared with LG group, the expressions of JAK2, p-STAT1, p-STAT3, and TGF-beta1, mRNA were significantly increased in HG group from 6 to 72 hours. Meanwhile, the contents of TGF-beta1 and collagen I in the supernatants and the expression of alpha-SMA increased and the expression of E-Cadherin decreased. The expressions of JAK2, p-STAT1, p-STAT3, and TGF-beta mRNA as well as the levels of TGF-beta1 and collagen I in the supernatant s in HG + AG490 group were significantly lower than in the HG group. The expressions of alpha-SMA and E-Cadherin were also decreased in HG + AG490 group.

CONCLUSIONActivation of JAK/STAT signaling pathway may be involved in the high glucose-induced transdifferentiation and overproduction of TGF-beta1, and ECM proteins in HKCs.

Cell Line ; Cell Transdifferentiation ; Epithelial Cells ; cytology ; metabolism ; Glucose ; metabolism ; pharmacology ; Humans ; Janus Kinases ; physiology ; Kidney Tubules, Proximal ; cytology ; metabolism ; STAT Transcription Factors ; physiology ; Signal Transduction ; Transforming Growth Factor beta1 ; biosynthesis ; secretion ; Urothelium ; cytology ; metabolism