OBJECTIVES: To evaluate the protective effect of Bufei Yishen Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. METHODS: BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. RESULTS: CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. CONCLUSIONS BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.
Humans ; Epithelial Cells/cytology* ; Drugs, Chinese Herbal/pharmacology* ; NF-kappa B/metabolism* ; Bronchi/cytology* ; Smoke/adverse effects* ; Apoptosis/drug effects* ; Mucin 5AC/metabolism* ; Cell Line ; Toll-Like Receptor 4/metabolism* ; Mucin-5B/metabolism* ; Signal Transduction/drug effects* ; Nicotiana ; Rats ; Thiocarbamates/pharmacology* ; Animals

OBJECTIVES: To investigate the protective effect of the probiotic bacterium Streptococcus salivarius K12 (K12) against Mycoplasma pneumoniae (Mp) infection in mice. METHODS: Forty male BALB/c mice were randomized into normal control group, K12 treatment group, Mp infection group, and K12 pretreatment prior to Mp infection group. The probiotic K12 was administered daily by gavage for 14 days before Mp infection induced by intranasal instillation of Mp. Three days after Mp infection, the mice were euthanized for analysis of bronchoalveolar lavage fluid (BALF) cell counts and serum levels of secretory immunoglobulin A (sIgA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). RT-qPCR was performed to detect the P1 and community-acquired respiratory distress syndrome ( CARDS ) toxin of Mp in the lung tissues and the mRNA expressions of TNF-α, IL-6, chemokine 1 (CXCL1), matrix metalloproteinase 9 (MMP9), mucin 5ac (MUC5ac), collagen 3a1 (Col3a1), Toll-like receptor 2 (TLR2) and TLR4; the protein expressions of TLR2 and TLR4 in the lung tissue were detected using Western blotting. Pathological changes in the lung tissue and airway remodeling were examined with HE staining and AB/PAS staining. RESULTS: Compared with the Mp-infected mice with PBS treatment, the infected mice with K12 treatment showed significantly lowered mRNA levels of P1 and CARDS in the lung tissue and reduced white blood cell counts in the BALF (P<0.05). In spite of the absence of significant differences in serum levels of inflammatory factors between the two groups, the mRNA expressions of TNF‑α, IL-6, CXCL1, MMP9, MUC5ac and COL3A1 and the mRNA and protein levels of TLR2 and TLR4 in the lung tissues were significantly lower in K12-treated mice, in which AB/PAS staining showed obviously decreased mucus secretion. CONCLUSIONS K12 pretreatment can effectively reduce pulmonary inflammatory responses, improve airway remodeling and alleviate lung injury in Mp-infected mice.
Animals ; Mice ; Pneumonia, Mycoplasma/metabolism* ; Mice, Inbred BALB C ; Toll-Like Receptor 2/metabolism* ; Mycoplasma pneumoniae ; Male ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lung/microbiology* ; Toll-Like Receptor 4/metabolism* ; Streptococcus salivarius ; Probiotics/administration & dosage* ; Bronchoalveolar Lavage Fluid ; Matrix Metalloproteinase 9/metabolism* ; Mucin 5AC/metabolism* ; Chemokine CXCL1/metabolism* ; Immunoglobulin A, Secretory/metabolism* ; Bacterial Toxins ; Bacterial Proteins

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

Objective To evaluate the antagonistic effects of N-acetylcysteine (NAC) on mitogen-activated protein kinases (MAPK) pathway activation, oxidative stress and inflammatory responses in rats with lung injury induced by fine particulate matter (PM_2.5). Methods Forty eight male Wistar rats were randomly divided into six groups: blank control group (C1), water drip control group (C2), PM_2.5 exposed group (P), low-dose NAC treated and PM_2.5 exposed group (L), middle-dose NAC treated and PM_2.5 exposed group (M), and high-dose NAC treated and PM_2.5 exposed group (H). PM_2.5 suspension (7.5 mg/kg) was administered tracheally once a week for four times. NAC of 125 mg/kg, 250 mg/kg and 500 mg/kg was delivered intragastrically to L, M and H group respectively by gavage (10 ml/kg) for six days before PM_2.5 exposure. The histopathological changes and human mucin 5 subtype AC (MUC5AC) content in lung tissue of rats were evaluated. We investigated IL-6 in serum and bronchoalveolar lavage fluid (BALF) by Enzyme-linked immunosorbent assay (ELISA), MUC5AC in lung tissue homogenate by ELISA, glutathione peroxidase (GSH-PX) in serum and BALF by spectrophotometry, and the expression of p-ERK1/2, p-JNK1/2 and p-p38 proteins by Western blot. All the measurements were analyzed and compared statistically. Results Lung tissue of rats exposed to PM_2.5 showed histological destruction and increased mucus secretion of bronchial epithelial cells. Rats receiving NAC treatment showed less histological destruction and mucus secretion. Of P, L, M and H group, MUC5AC in lung tissue, IL-6 in serum and BALF were higher than controls (C1 and C2) (all P<0.05), with the highest levels found in the P group and a decreasing trend with increase of NAC dose. The activity of GSH-PX in serum and BALF of PM_2.5 exposed rats (P, L, M and H) was lower than that of controls (all P<0.05), with higher activities found in NAC treated rats (L, M, and H), and an increasing trend with increase of NAC dose. The expressions of p-ERK1/2, p-JNK1/2 and p-p38 proteins in PM_2.5 exposed lung tissue (P, L, M and H) was higher than controls (all P<0.05), with decreased levels and dose dependent downregulation found in NAC treated rats. Conclusion NAC can antagonize major MAPK pathway activation, lung oxidative stress and inflammatory injury induced by PM_2.5 in rats.
Acetylcysteine/pharmacology* ; Animals ; Bronchoalveolar Lavage Fluid ; Enzyme Activation/drug effects* ; Glutathione Peroxidase/metabolism* ; Inflammation/pathology* ; Interleukin-6/metabolism* ; Lung/pathology* ; Lung Injury/pathology* ; Male ; Mitogen-Activated Protein Kinases/metabolism* ; Mucin 5AC/metabolism* ; Mucus/metabolism* ; Oxidative Stress/drug effects* ; Particle Size ; Particulate Matter/toxicity* ; Phosphorylation/drug effects* ; Rats, Wistar

OBJECTIVE: To observe the role of aminophylline and simvastatin in preventing and curing chronic obstructive pulmonary disease (COPD), and to explore the underlying mechanisms based on airway inflammation and mucus hypersecretion.
 METHODS: The rat model of COPD was established by combination of cigarette smoking with intratracheal lipopolysaccharide (LPS) injection. Male SD rats were randomly divided into 4 groups (n=10 per group): a control group, a COPD group, an aminophylline group and a simvastatin group. The rats in the control group and the COPD group were treated with normal saline once a day via intragastric administration, while the rats in the aminophylline group and the simvastatin group were treated with aminophylline (5 g/L) and simvastatin (0.5 g/L) 1 mL/100 g once a day via intragastric administration, respectively. Pulmonary function and pathological changes in bronchus and lung were observed. The levels of IL-8, IL-17, and TNF-α in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of TLR4 and mucin 5AC (MUC5AC) in bronchi and lung tissues were detected by real-time PCR and Western blot, respectively.
 RESULTS: Pulmonary function and the pathophysiologic changes in bronchi and lung tissues in the COPD rats were consistent with typical phenotype of COPD. Compared with the control group, lung function indexes were significantly attenuated in the COPD group, while the levels of IL-8, IL-17, and TNF-α in BALF as well as the mRNA and protein levels of MUC5AC and TLR4 were significantly increased. Compared with the COPD group, lung function indexes were significantly increased in the aminophylline group and simvastatin group (P<0.01), while pulmonary pathological damages, the levels of IL-8, IL-17, and TNF-α in BALF as well as the mRNA and protein levels of MUC5AC and TLR4 were significantly decreased (P<0.01). Compared with the aminophylline group, the peak expiratory flow as well as the levels of IL-8, IL-17, and TNF-α in the simvastatin group were elevated (P<0.05). There are no significant difference in the mRNA and protein levels of MUC5AC and TLR4 between the 2 groups (P﹥0.05).
 CONCLUSION Aminophylline and simvastatin can decrease IL-8, IL-17, and TNF-α levels in BALF and inhibit the expression of MUC5AC and TLR4 in airway and lung tissues in COPD rats, suggesting that they may have a preventive and therapeutic effect on COPD through reducing the airway inflammation and mucus hypersecretion.
Aminophylline ; pharmacology ; Animals ; Bronchi ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Cytokines ; chemistry ; Inflammation ; drug therapy ; Lipopolysaccharides ; Lung ; metabolism ; physiopathology ; Male ; Mucin 5AC ; metabolism ; Mucus ; metabolism ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology ; Smoke ; adverse effects ; Smoking ; adverse effects ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo observe preventive and therapeutic effect of Yifei Jianpi Recipe (YJR) on chronic obstructive pulmonary disease (COPD) model rats and to explore its mechanism from the way of airway inflammation and airway mucus hypersecretion.

METHODSThe COPD rat model was established by using cigarette smoking combined with intratracheal injection of lipopolysaccharide (LPS). Male SD rats were randomly divided into the blank control group (control group), the model group, the YJR group, 6 in each group. Forced vital capacity (FVC), forced expiratory volume in 0. 1 second (FEV0. 1), FEVO. 1/FVC, peak expiratory flow (PEF) was tested by lung function device. Pathological changes of bronchi and lung tissues were observed by HE staining. Airway Goblet cells were observed using AB-PAS staining. Contents of IL-8, IL-17, and TNF-α in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). Protein expressions of intercellular cell adhesion molecule-1 (ICAM-1), nuclear factor KB (NF-KB), mucin 5AC (Muc5AC), and Toll-like receptor 4 (TLR4) in rat airway were detected by immunohistochemical assay. mRNA expressions of TLR4 and Muc5AC in bronchi and lung tissues were detected by real-time quantitative PCR (RT qPCR).

RESULTSChanges of bronchi and lung tissues in the model group rats were consistent with typical pathological manifestations of COPD. Compared with the model group, the degree of lung injury was significantly alleviated in the YJR group. Compared with the control group, FVC, FEV0. 1, FEVO. I/FVC, and PEF were decreased (P <0. 01), contents of IL-8, IL-17, and TNF-α in BALF were significantly increased (P <0. 01), protein expressions of ICAM-1, NF-KB, Muc5AC, and TLR4, mRNA expression levels of Muc5AC and TLR4 in bronchi and lung tissues were also significantly increased in the model group (P <0. 01). Compared with the model group, FVC, FEV0. 1, FEV0. 1/FVC, and PEF were significantly increased in the YJR group (P <0. 01, P <0. 05), but the rest indices were significantly lowered (P <0. 01, P <0. 05).

CONCLUSIONYJR could decrease contents of IL-8, IL-17, and TNF-α in BALF of COPD model rats, inhibit protein expression levels of ICAM-1, NF-κB, Muc5AC, and TLR4.in airway and lung tissues, thus playing preventive and therapeutic roles by reducing airway inflammation and airway mucus hypersecretion.

Animals ; Bronchi ; Bronchoalveolar Lavage Fluid ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Inflammation ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-17 ; metabolism ; Interleukin-8 ; metabolism ; Lipopolysaccharides ; Lung ; Male ; Models, Animal ; Mucin 5AC ; metabolism ; Mucus ; metabolism ; NF-kappa B ; metabolism ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; 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

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

Primary retroperitoneal mucinous cystadenoma is an extremely uncommon tumor, even though mucinous cystadenoma often develops in the ovary and less frequently in the pancreas. A 21-year-old female was admitted to our hospital due to severe abdominal pain. A well-demarcated, oval shaped cystic tumor at the retropancreatic area with displacement of the pancreas and surrounding major vessels was observed on CT and MRI. Exploratory laparotomy was performed, and complete excision of the entire cyst was performed without complication. The pathologic finding was consistent with primary retropancreatic mucinous cystadenoma. To the best of our knowledge, this report is the first to describe a case of retropancreatic mucinous cystadenoma arising from the retropancreatic area in Korea.
Antibodies/metabolism ; Cystadenoma, Mucinous/*diagnosis/pathology/surgery ; Female ; Humans ; Magnetic Resonance Imaging ; Mucin 5AC/immunology ; Mucin-2/immunology ; Ovarian Neoplasms/*diagnosis/pathology/surgery ; Retroperitoneal Neoplasms/*diagnosis/pathology/surgery ; Tomography, X-Ray Computed ; Young Adult

OBJECTIVE: To investigate the effect of glycyrrhizin (Gly) on human neutrophil elastase (HNE)- induced mucin (MUC) 5AC overproduction in human bronchial epithelial cells (16HBE), and the potential signaling pathway involved in this process. METHODS: The cultured cells were divided into 3 groups: a control group, cultured in serum-free DMEM medium; an HNE group, pretreated with HNE alone; and a Gly group, incubated with HNE and Gly. After stimulation with a variety of Gly concentrations, the cytotoxicity was assessed by methyl thiazolyl tetrazolium method. The mRNA expressions of p38, nuclear factor κB (NF-κB) p65, inhibitory κBα (IκBα) and MUC5AC were detected by real-time PCR. The phosphorylation levels of p38 (p-p38), NF-κB p65 (p-NF-κB p65) and IκBα (p-IκBα) were measured by Western blot while the levels of MUC5AC protein were analyzed by emzyme-linked immunosorbent assay and immunofluorescence. RESULTS: Compared with the control group, the expression levels of MUC5AC mRNA and protein in the HNE group were both significantly increased. There was a significant increase in p-p38 and p-NF-κB p65, while the production of IκBα was much lower than that in the control group. Gly significantly inhibited the increase of MUC5AC, p38 and NF-κB p65, but increased the activity of IκBα. CONCLUSION Glycyrrhizin can inhibit MUC5AC overproduction via p38-NF-κB p65/IκBα signaling pathway.
Bronchi ; cytology ; Cell Line ; Epithelial Cells ; metabolism ; Glycyrrhizic Acid ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Leukocyte Elastase ; metabolism ; Mucin 5AC ; biosynthesis ; NF-KappaB Inhibitor alpha ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcription Factor RelA ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism