Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.
Animals ; Mice ; Epithelial Cells ; Lung ; Mucin 5AC/metabolism* ; Mycoplasma pneumoniae/metabolism* ; Signal Transduction

BACKGROUND/AIMS: Gallbladder (GB) mucin is one of the key factors in the gallstone formation. However, there is little information about the diversity of mucin secretion according to the stone composition. Epidermal growth factor receptor (EGFR) functions in proliferation including mucin secreting goblet cell hyperplasia. We compared the expressions of MUC3, MUC5AC, MUC6 and EGFR in the GB epithelium with cholesterol gallstones (GB-chol) group and pigment gallstones (GB-pig group). METHODS: GBs from elective laparoscopic cholecystectomy for the gallstone disease were studied. Stone composition was analyzed by the spectrophotometer. Immunohistochemical stain was performed using each monoclonal antibody. The percentage of stained proportion was scored by the NIH image program and the results were compared between both groups. RESULTS: Total 20 patients were enrolled (10 patients with cholesterol gallstones, 10 patients with pigment gallstones). The percentages of stained proportion for MUC3, MUC5AC, and MUC6 were 42+/-27%, 31+/-15%, and 17+/-9%, respectively in GB-chol group and 32+/-22%, 33+/-23%, and 15+/-10%, respectively in GB-pig group (p>0.05). The expression of EGFR was 50% (5/10) in the GB-chol group and 80% (8/10) in the GB-pig group respectively. CONCLUSIONS: There was no difference in the expressions of MUC3, MUC5AC, and MUC6 between the two groups. Further studies are needed to elucidate the role of EGFR in the gallstore formation.
Bile Pigments/analysis ; Cholelithiasis/chemistry/*metabolism ; Cholesterol/analysis ; Epithelium/metabolism ; Gallbladder/*metabolism ; Humans ; Immunohistochemistry ; Mucin 5AC ; Mucin-3 ; Mucin-6 ; Mucins/*analysis ; Receptor, Epidermal Growth Factor/*analysis

Cell Line ; Glucocorticoids ; pharmacology ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Mucin 5AC ; metabolism ; Respiratory System ; drug effects ; metabolism

OBJECTIVE: To detect the mucin gene (MUC2, MUC5AC, MUC5B, MUC18 and MUC19) expression in the nasal polyps, allergic rhinitis (AR) and the normal nasal mucosa in human. To investigate the role and clinical significance of mucin gene in the pathogenesis of nasal polyps and AR patients. METHOD: We obtained samples from 35 cases of nasal polyps, 18 cases of AR inferior turbinate and 18 cases of simple nasal septum deviation inferior turbinate. Specimens were analyzed with RT-PCR and Real-time FQ-RT-PCR. RESULT: The results of RT-PCR and FQ-RT-PCR showed that the expression of MUC5AC, MUC5B in nasal polyps and AR patients was significantly higher than that in normal mucosa (P<0.05). The expression of MUC5AC, MUC5B in nasal polyps was not significantly different from that in AR patients (P>0.05). The expression of MUC2, MUC18 in nasal polyps and AR was not significantly different from that in normal mucosa (P>0.05). And the results of RT-PCR for MUC19 expression in AR was higher than that in nasal polyps group and normal group (P<0.05 or P<0.01). CONCLUSION MUC5AC and MUC5B are highly expressed in epithelium of human nasal polyps and AR, and they take part in mucus over-secretion in nasal polyps and AR. The expression of MUC19 in AR was higher than that in nasal polyps group and normal group. It indicates that the secretion of MUC19 in allergic rhinitis was on high level. There was no difference of the expression of MUC2 and MUC18 in nasal polyps group, AR group and in normal group.
Adolescent ; Adult ; Female ; Gene Expression ; Humans ; Male ; Middle Aged ; Mucin 5AC ; genetics ; Mucin-2 ; genetics ; Mucin-5B ; genetics ; Mucins ; genetics ; metabolism ; Nasal Mucosa ; metabolism ; pathology ; Nasal Polyps ; genetics ; metabolism ; Rhinitis ; genetics ; metabolism ; Young Adult

PURPOSE: If cholesterol in the cell membrane is depleted by treating cells with methyl-beta-cyclodextrin (MbetaCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1beta is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1beta showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. MATERIALS AND METHODS: After NCI-H292 cells were pretreated with 1% MbetaCD before adding IL-1beta for 24 hours, MUC5AC mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MbetaCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. RESULTS: Cholesterol in the cell membrane was significantly depleted by treatment with MbetaCD on cells. IL-1beta-induced MUC5AC mRNA expression was decreased by MbetaCD and this decrease occurred IL-1-receptor-specifically. Moreover, we have shown that MbetaCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1beta. This result suggests that MbetaCD-mediated suppression of IL-1beta-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. CONCLUSION: Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.
Cell Membrane/drug effects/*metabolism ; Cholesterol/*metabolism ; Epithelial Cells/metabolism ; Gene Expression ; Humans ; Mucin 5AC/genetics/*metabolism ; Respiratory System/*metabolism/pathology ; beta-Cyclodextrins/pharmacology

OBJECTIVETo investigate the expression of CLCA3 and Muc5ac in nasal mucosa in allergic rhinitis rats and the effects of glucocorticoid on its expression.

METHODSThirty SD rats were randomly divided into allergic rhinitis group, dexamethasone group and control group. Expression of CLCA3 mRNA and Muc5ac protein in nasal mucosa were detected by RT-PCR and immunohistochemical assay, respectively.

RESULTSCLCA3 mRNA and Muc5ac protein in allergic rhinitis group were significantly higher than those in control group (t = 8.565, 5.317, P < 0.01, respectively). The increased expression of CLCA3 mRNA in allergic rhinitis group was well correlated with the expression of Muc5ac protein and the correlation coefficient was 0.813 (P < 0.05). After treatment with dexamethasone, the expression of CLCA3 mRNA and Muc5ac protein was notably lower than that in allergic rhinitis group (t = 3.102, 2.226, P < 0.05, respectively).

CONCLUSIONSThe stronger gene expression of CLCA3 exists, complicated with mucus overproduction in the nasal mucosa of allergic rhinitis rats. CLCA3 expression may play a pivotal role in mucus overproduction in allergic rhinitis. Dexamethasone substantially downregulates the expression of CLCA3 mRNA and Muc5ac protein.

Animals ; Calcium Channel Agonists ; metabolism ; Chloride Channels ; metabolism ; Dexamethasone ; pharmacology ; Female ; Glucocorticoids ; pharmacology ; Mucin 5AC ; metabolism ; Nasal Mucosa ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic, Perennial ; metabolism

OBJECTIVETo explore the roles of epidermal growth factor receptor (EGFR) signaling pathway on cultured human nasal epithelial cells RPMI-2650.

METHODSRPMI-2650 cells were cultured in vitro, the growth curve was measured and the ultrastructure was observed using scanning electron microscope. When the cells were significantly confluent, they were divided into 4 groups, group A: maintained in Eagle's minimum essential media (EMEM) medium without adding any stimulators; group B: added with epidermal growth factor (EGF) 25 ng/ml; group C: added with AG1478 (EGFR selective inhibitor) 10 micromol/L followed by EGF 25 ng/ml 30 minutes later; group D: added with PD98069 (p44/42MAPK selective inhibitor) 30 micromol/L followed by EGF 25 ng/ml 30 minutes later. After incubated for 24 hours, the expression of EGFR and MUC5AC proteins in the cells of these 4 groups was studied using cytoimmunity and Western blotting.

RESULTSRPMI-2650 cells were significantly confluent after incubated for 5 to 7 days. The shape of cells was round or oval, and a large number of microvilli covered to their surface but without cilia under scanning electron microscope. The EGFR protein was expressed in the cells of group A and D, abundantly in group B, while weakly in group C. The values of comparative absorbance had significant difference between group A, B, D and group C, respectively (P < 0.01). For the MUC5AC protein, its expression was strong in the cells of group A, abundant in group B, and weak in group C and D. Significant difference of the values of comparative absorbance was analyzed between group B and group C, D, respectively (P < 0.01), while no difference between group C and group D (P > 0.05).

CONCLUSIONSThe production of MUC5AC in human nasal epithelial cells RPMI-2650 is regulated via the expression and activation of epidermal growth factor receptor signaling pathway.

Cell Proliferation ; Epithelial Cells ; metabolism ; Humans ; Mucin 5AC ; genetics ; metabolism ; Mucins ; metabolism ; Nasal Cavity ; cytology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Tumor Cells, Cultured

OBJECTIVE: To evaluate the role of human calcium-activated chloride channel 1 (hCLCA1) and mucin MUC5AC in allergic rhinitis. METHOD: The expression of hCLCA1 and MUC5AC were determined by RT-PCR and immunohistochemical assay in nasal mucosa on 20 patients with allergic rhinitis and 7 normal persons. RESULT: The expression of hCLCA1mRNA in allergic rhinitis group was positive, whereas in normal control group was absent (P<0.01). The expression of MUC5AC mRNA and MUC5AC protein in the allergic rhinitis group were higher than that in the control, respectively (both P<0.01). The increased expression of hCLCA1mRNA in allergic rhinitis group were well correlated with the expression of MUC5ACmRNA and MUC5AC protein and the correlation coefficients were 0.752 and 0.694(both P<0.05). CONCLUSION Upregulation of hCLCA1 mRNA expression may play a pivotal role in mucus overproduction in allergic rhinitis group. The inhibition of HCLCA1 expression may provide a new strategy for the treatment of allergic rhinitis.
Adolescent ; Adult ; Chloride Channels ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Mucin 5AC ; metabolism ; Nasal Mucosa ; metabolism ; Rhinitis, Allergic, Perennial ; metabolism ; Young Adult

OBJECTIVE: To detect the effect of p38 mitogen activated protein kinase (p38MAPK) and cyclooxygenase-2 (COX-2) on the expression of mucin5AC (MUC5AC) in human nasal mucosa induced by histamine in vitro, and to investigate the pathogenesis of mucus hypersecretion in allergic rhinitis (AR). METHOD: Western blot was performed to detect the protein expressions of p38MAPK, COX-2 and MUC5AC in nasal mucosa induced by histamine or blocked by selective inhibitors of p38MAPK and COX-2 of different concentration gradient. RESULT: Weak expressions of p38MAPK. COX-2 and MUC5AC were detected in normal nasal mucosa in vitro. The protein expressions of p38MAPK. COX-2 and MUC5AC increased in nasal mucosa induced by histamine in a dose-dependent manner. The histamine induced protein expressions of COX-2 and MUC5AC were dose-dependently attenuated by selective inhibitor of COX-2, namely NS-398. No apparent influence of NS-398 on the expression of p38MAPK was observed. The histamine induced protein expressions of p38MAPK, C()X-2 and MUCbAC dose-dependently decreased after nasal mucosa was treated by selective inhibitor of p38MAPK, namely SB203580. And no significant change of MUC5AC protein expression induced by NS-398 or SB203580 was observed. CONCLUSION Our findings indicated that the histamine-induced increased expression of MUC5AC by activated p38MAPK/COX-2 may be a possible pathogenesis of mucus hypersecretion in AR.
Adult ; Female ; Humans ; Male ; Middle Aged ; Mucin 5AC ; metabolism ; Nasal Mucosa ; metabolism ; Rhinitis, Allergic, Perennial ; metabolism ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Animals ; Ear, Middle/*metabolism/pathology ; Mice ; Microscopy, Electron, Scanning ; Mucin 5AC/genetics/*metabolism ; RNA, Messenger/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Spectrophotometry