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

Mucins,a family of heavily glycosylated proteins,present mainly in epithelial cells.They function as essential barriers for epithelium and play important roles in cellular physiological processes.Aberrant expression and glycosylation of mucins in gastric epithelium occur at pathological conditions,such as Helicobacter pylori infection,chronic atrophic gastritis,intestinal metastasis,dysplasia,and gastric cancer.This review addresses the major roles played by mucins and associated O-glycan structures in normal gastric epithelium.Further,we expound the alterations of expression patterns and glycan signatures of mucins at those pathological conditions.
Gastric Mucosa/pathology* ; Glycosylation ; Helicobacter Infections/pathology* ; Helicobacter pylori/metabolism* ; Humans ; Mucins/metabolism* ; Stomach Neoplasms/pathology*

Cold-heat combination is a common method in the treatment of ulcerative colitis, which is represented by classic drug pair, Coptidis Rhizoma and Zingiberis Rhizoma.The present study explored the synergetic effects of berberine and 6-shogaol, the primary components of Coptidis Rhizoma and Zingiberis Rhizoma, respectively, on intestinal inflammation and intestinal flora in mice with ulcerative colitis to reveal the effect and mechanism of cold-heat combination in the treatment of ulcerative colitis.The ulcerative colitis model was induced by dextran sulfate sodium(DSS) in mice.The model mice were administered with berberine(100 mg·kg~(-1)), 6-shogaol(100 mg·kg~(-1)), and berberine(50 mg·kg~(-1)) combined 6-shogaol(50 mg·kg~(-1)) by gavage, once per day.After 20 days of drug administration, mouse serum, colon tissues, and feces were sampled.Hematoxylin-eosin(HE) staining was used to observe histopathological changes in colon tissues.Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to observe the changes in the mucus layer of colon tissues.Enzyme-linked immunosorbent assay(ELISA) was employed to detect the serum content of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6).Immunohistochemical method was adopted to detect the protein expression of macrophage surface markers F4/80, mucin-2, claudin-1, and zonula occludens-1(ZO-1) in colon tissues.High-throughput Meta-amplicon library sequencing was used to detect changes in the intestinal flora of mice.The results indicated that the 6-shogaol group, the berberine group, and the combination group showed significantly relieved intestinal injury, reduced number of F4/80-labeled positive macrophages in colon tissues, increased protein expression of mucin-2, claudin-1, and ZO-1, and decreased serum le-vels of TNF-α, IL-1β, and IL-6.Shannon, Simpson, Chao, and Ace indexes of the intestinal flora of mice in the 6-shogaol group and the combination group significantly increased, and Chao and Ace indexes in the berberine group significantly increased.As revealed by the bioinformatics analysis of intestinal flora sequencing, the relative abundance of Verrucomicrobia at the phylum, class, and order levels decreased significantly in all treatment groups after drug administration, while that of Bacillibacteria gradually increased.In the 6-shogaol group and the combination group, Akkermansia muciniphila completely disappeared, but acid-producing bacillus still existed in large quantities.As concluded, both 6-shogaol and berberine can inhibit intestinal inflammation, reduce the infiltration and activation of macrophages, relieve intestinal damage, reduce intestinal permeability, improve the structure of flora, and promote intestinal microecological balance.The combined application of berberine and 6-shogaol has a significant synergistic effect.
Animals ; Berberine/therapeutic use* ; Catechols ; Claudin-1/therapeutic use* ; Colitis/metabolism* ; Colitis, Ulcerative/metabolism* ; Colon ; Dextran Sulfate/metabolism* ; Disease Models, Animal ; Drugs, Chinese Herbal/pharmacology* ; Inflammation/metabolism* ; Interleukin-6/metabolism* ; Mice ; Mice, Inbred C57BL ; Mucin-2/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: To investigate the effect of interleukin (IL) -13 combined with cold stimulation on synthesis and secretion of mucin (MUC) 5AC in human bronchial epithelial cell line 16HBE and explore the role of transient receptor potential 8 (TRPM8) and anti-apoptotic factor B-cell lymphoblast-2 (Bcl-2) in this process. METHODS: 16HBE cells were stimulated with 10 ng/mL IL-13, 1 mmol/L menthol, or both (1 mmol/L menthol was added after 6 days of IL-13 stimulation), and the changes in the expression of MUC5AC, intracellular Ca RESULTS: The mRNA and protein expressions of MUC5AC increased significantly in 16HBE cells following stimulation with IL-13, menthol, and both ( CONCLUSIONS Menthol combined with IL-13 produces a synergistic effect to promote the synthesis and secretion of MUC5AC in 16HBE cells possibly by activating TRPM8 receptor to upregulate the expression of Bcl-2.
Bronchi ; Epithelial Cells/drug effects* ; Humans ; Interleukin-13 ; Menthol/pharmacology* ; Mucin 5AC

To explore the role of cortical actin-binding protein (cortactin) in shear stress-induced mucin (MUC) 5AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites.
 Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin (Cort), pEGFP-N1-Cort-Y421A, pEGFP-N1-Cort-Y470A and pEGFP-N1-Cort-Y486A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFP-N1-Cort-Y421A group, a shear stress + pEGFP-N1-Cort-Y470A group, and a shear stress + pEGFP-N1-Cort-Y486A group. The shear stress were set at 4 dynes/cm2. The levels of MUC5AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate (FITC)-phalloidin.
 Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5AC protein production and mRNA expression in the different experiment groups (all P<0.05). Compared with single shear stress-stimulated group, MUC5AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group (both P<0.05), while there were no changes in the MUC5AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421A group and the shear stress + pEGFP-N1-Cort-Y470A group (both P<0.05), while there was no significant effect in the shear stress + pEGFP-N1-Cort-Y486A group (P>0.05).
 Conclusion: Cortactin is involved in shear stress-mediated MUC5AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.
Cortactin ; Epithelial Cells ; Humans ; Mucin 5AC ; Mucus ; Phosphorylation

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

Intraductal papillary neoplasms of the bile duct (IPNBs) are known to show various pathologic features and biological behaviors. Recently, two categories of IPNBs have been proposed based on their histologic similarities to pancreatic intraductal papillary mucinous neoplasms (IPMNs): type 1 IPNBs, which share many features with IPMNs; and type 2 IPNBs, which are variably different from IPMNs. The four IPNB subtypes were re-evaluated with respect to these two categories. Intestinal IPNBs showing a predominantly villous growth may correspond to type 1, while those showing papillay-tubular or papillay-villous growth correspond to type 2. Regarding gastric IPNB, those with regular foveolar structures with varying numbers of pyloric glands may correspond to type 1, while those with papillary-foveolar structures with gastric immunophenotypes and complicated structures may correspond to type 2. Pancreatobiliary IPNBs that show fine ramifying branching may be categorized as type 1, while others containing many complicated structures may be categorized as type 2. Oncocytic type, which displays solid growth or irregular papillary structures, may correspond to type 2, while papillary configurations with pseudostratified oncocytic lining cells correspond to type 1. Generally, type 1 IPNBs of any subtype develop in the intrahepatic bile ducts, while type 2 IPNBs develop in the extrahepatic bile duct. These findings suggest that IPNBs arising in the intrahepatic ducts are biliary counterparts of IPMNs, while those arising in the extrahepatic ducts display differences from prototypical IPMNs. The recognition of these two categories of IPNBs with reference to IPMNs and their anatomical location along the biliary tree may deepen our understanding of IPNBs.
Bile Ducts ; Bile Ducts, Extrahepatic ; Bile Ducts, Intrahepatic ; Bile ; Biliary Tract ; Cholangiocarcinoma ; Gastric Mucosa ; Mucins

OBJECTIVE: To study the effects of respiratory syncytial virus (RSV) infection on epidermal growth factor receptor (EGFR), tight junction association proteins and mucin in the human airway epithelial cells. METHODS: Human airway epithelial cells NCI-H292 were randomly treated by ultraviolet light-inactivated RSV (control group) or thawed RSV (RSV infection group). After 48 hours of treatment, the protein levels of occludin, E-cadherin, phosphorylated EGFR and EGFR in NCI-H292 cells were measured by Western blot. The distribution and expression levels of occludin and E-cadherin in NCI-H292 cells were examined by immunofluorescence technique. The expression levels of MUC5AC mRNA in NCI-H292 cells were assessed by RT-PCR. RESULTS: The protein levels of occludin and E-cadherin were significantly reduced in the RSV infection group compared with the control group (P<0.05). The protein levels of phosphorylated EGFR and EGFR increased significantly in the RSV infection group compared with the control group (P<0.05). The MUC5AC mRNA levels also increased significantly in the RSV infection group compared with the control group (P<0.05). CONCLUSIONS RSV may down-regulate the tight junction association proteins and up-regulate the expression of MUC5AC in airway epithelial cells, which contributes to epithelial barrier dysfunction. EGFR phosphorylation may play an important role in regulation of airway barrier.
Cell Line ; Epithelial Cells ; ErbB Receptors ; Humans ; Mucin 5AC ; Respiratory Syncytial Virus Infections ; Tight Junction Proteins ; Tight Junctions