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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.
Subject(s)
Animals , Mice , Epithelial Cells , Lung , Mucin 5AC/metabolism , Mycoplasma pneumoniae/metabolism , Signal TransductionABSTRACT
Edible bird's nest (EBN) is a kind of natural invigorant with a long history of consumption in Asia, especially in China. EBN is formed by mixing the saliva of swiftlets (Aerodramus) with feathers and other components during the breeding season. Proteins are the most important nutrient in EBN. By studying proteins in EBN, we can not only elucidate their components at the molecular level, but also study their bioactivities. Therefore, it is of great significance to study the proteins in EBN. Previous research on the proteins in EBN was preliminary and cursory, and no one has summarized and analyzed the proteins in EBN and correlated the bioactivities of these proteins with the biological functions of EBN. This article focused on the proteins in EBN, listed the proteins identified in different proteomic studies, and introduced the sources, structures and bioactivities of the most frequently identified proteins, including acidic mammalian chitinase, lysyl oxidase homolog 3, mucin-5AC, ovoinhibitor, nucleobindin-2, calcium-binding protein (MW: 4.5 × 104) and glucose-regulated protein (MW: 7.8 × 104). The properties of these proteins are closely related to the bioactivities of EBN. Therefore, this article can provide inspiration for further research on the efficacy of EBN.
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Objective:To observe the effects of Xiaoqinglong Decoction and Qingqi Huatan Pills on interleukin-1β(IL-1β)-induced mucushypersecretion model of human airway epithelial H292 cellsand related molecules of nuclear factor-κB/microRNA-494 (NF-κB/miR-494) signaling pathway, and to explore the mechanism of the two medicines in improving pathological airway mucus.Methods:Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to detect the effects of different concentrations of Xiaoqinglong Decoction and Qingqi Huatan Pills on the activity of H292 cellsinduced by IL-1β, and the appropriate concentration was selected for subsequent experiments. Cells were randomly divided into blank group, IL-1β model group (5 μg/L IL-1β), NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) group (5 μg/L IL-1β+100 μmol/L PDTC), Xiaoqinglong Decoction (5 μg/L IL-1β+1 000 mg/L Xiaoqinglong Decoction) and Qingqi Huatan Pill group (5 μg/L IL-1β+1 000 mg/L Qingqi Huatan Pills). 5 μg/L IL-1β was used to induce H292 cells for 24 hours to establish a model of airway epithelial mucus hypersecretion. Enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α) and IL-8 and the synthesis of intracellular MUC5AC and cystic fibrosis transmembrane conductance regulator (CFTR). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of MUC5AC mRNA, CFTR mRNA, miR-494. Western blotting was used to detect protein expression of key proteins (p65) and NF-κB inhibitors (IκB) in NF-κB signaling pathway.Results:Xiaoqinglong Decoction and Qingqi Huatan Pills with the concentration of 1 000 mg/L were selected for the follow-up experiment. Compared with the blank group, the levels of MUC5AC, TNF-α and IL-8 were significantly increased in the model group, intracellular MUC5AC protein content and mRNA expression were also significantly increased, intracellular CFTR protein content and mRNA expression were significantly decreased, and intracellular p65 protein expression was significantly up-regulated, the expression of IκB protein was significantly down-regulated, and the expression of miR-494 was significantly increased. Compared with the model group, the levels of MUC5AC, TNF-α and IL-8 were significantly reduced in PDTC group, Xiaoqinglong Decoction group and Qingqi Huatan Pill group, intracellular MUC5AC protein content and mRNA expression were also significantly decreased, and intracellular p65 protein expression was significantly down-regulated, and IκB protein expression was significantly up-regulated, miR-494 expression was significantly reduced. Intracellular CFTR protein content and mRNA expression were significantly increased in both PDTC group and Qingqi Huatan Pill group. Compared with the PDTC group, the level of TNF-α in the Xiaoqinglong Decoction group was significantly increased (ng/L: 22.77±3.14 vs. 11.09±3.37, P < 0.05), the content and mRNA expression of CFTR and IκB protein expression was significantly decreased [CFTR protein (ng/L): 97.38±6.62 vs. 227.04±19.48, CFTR mRNA (2 -ΔΔCt): 0.99±0.08 vs. 1.21±0.08, IκB/β-actin: 1.69±0.11 vs. 2.00±0.18, all P < 0.05], the level of TNF-α in Qingqi Huatan Pill group was significantly higher (ng/L: 19.08±3.71 vs. 11.09±3.37, P < 0.05). Compared with Xiaoqinglong Decoction group, the protein content and mRNA expression of CFTR and IκB protein expression in Qingqi Huatan Pill group were significantly increased [CFTR protein (ng/L) : 235.01±22.71 vs. 97.38±6.62, CFTR mRNA(2 -ΔΔCt): 1.32±0.15 vs. 0.99±0.08, IκB/β-actin: 1.94±0.16 vs. 1.69±0.11, all P < 0.05]. Conclusions:The effect of Xiaoqinglong Decoctionin improving the hypersecretion of mucus in the airway epithelium may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion, while the effect of Qingqi Huatan Pills may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion and CFTR dysfunction. Therefore, the difference in the mechanism of the two treatments of airway pathological mucus is mainly in the regulation of CFTR mRNA and protein.
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ObjectiveTo explore the effect of Yifei Huatan decoction on relieving airway hyperviscosity in asthmatic rats with spleen deficiency syndrome and its mechanism. MethodFifty-five SPF level SD rats at 8-9 week of age were used to induce asthma with spleen deficiency syndrome by animal modeling of traditional Chinese medicine combined with asthma of western medicine. After successful modeling， the rats were divided into model group， dexamethasone group， low， medium， and high-dose Yifei Huatan decoction groups by random number table method， and 11 clean SD rats at 8-9 week of age were recorded as a normal group. Rats in the dexamethasone group were given 0.087 5 mg kg-1 dexamethasone acetate by gavage. Rats in the low， medium， and high-dose Yifei Huatan decoction groups were given 0.8， 1.6， 3.2 g kg-1 Yifei Huatan decoction liquid extract by gavage， respectively. Rats in the model group and the normal group were given 10 mL kg-1 distilled water. The medicine were given once per day for 8 w， and the general situation of each group was observed. The levels of interleukin-4 （IL-4）， IL-13， and interferon-γ （IFN-γ） in bronchoalveolar lavage fluid （BALF） were determined by enzyme-linked immunosorbent assay （ELISA）. The pathological changes in lung tissues of rats were observed by hematoxylin-eosin （HE） staining. Alcian blue-periodic acid Schiff （AB-PAS） staining was used to detect the hyperplasia of airway goblet cells and mucus secretion in rats. The mRNA expressions of transforming growth factor-β1 （TGF-β1）， Smad2， Smad3， mucin 5AC （MUC5AC）， and mucin 5B （MUC5B） in the lung tissues of rats were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expressions of TGF-β1， Smad2， Smad3， MUC5AC， and MUC5B in the lung tissues of rats were detected by Western blot. ResultAs compared with the normal group， rats in the model group showed the symptoms of spleen deficiency syndrome， such as decreased body weight， muscle emaciation， decreased food intake， increased water intake， increased anal temperature， tiredness， and decreased swimming endurance， accompanied by dyspnea symptoms such as wheezing and nodding. As compared with the normal group， IL-4 and IL-13 levels in the BALF of the model group were significantly increased （P<0.01）， while the IFN-γ level was significantly decreased （P<0.01）. In the model group， a large number of inflammatory cells were observed in the mucosa and submucosa of the airway， and the smooth muscle of the trachea was significantly thickened. The hyperplasia， deformation， and exfoliation of various epithelial cells were observed in the mucosa， and the pathological scores of lung tissue increased significantly （P<0.01） in the model group. A large number of goblet cells were observed in the airway with the formation of plenty of mucous thrombus in the model group， and the positive relative staining area of airway， and mRNA and protein expressions of TGF-β1， Smad2， Smad3， MUC5AC， and MUC5B were significantly increased （P<0.01）. As compared with the model group， IL-4 and IL-13 levels in BALF of the dexamethasone group and the Yifei Huatan decoction groups decreased， while the IFN-γ level increased. The inflammatory cell infiltration in airway mucosa and submucosa， the thickening of tracheal smooth muscle， the hyperplasia， deformation， and exfoliation of epithelial cells in mucosa were gradually decreased， and the pathological scores of lung tissues decreased significantly （P<0.01） in the dexamethasone group and the Yifei Huatan decoction groups. Goblet cell proliferation gradually decreased， and the positive relative staining area of airway， and mRNA and protein relative expressions of TGF-β1， Smad2， Smad3， MUC5AC， and MUC5B decreased with statistically significant difference （P<0.05， P<0.01）. There was no significant difference in the above indexes in the dexamethasone group and the Yifei Huatan decoction low-dose group. The above indexes were dose-dependent in the low， medium， and high-dose Yifei Huatan decoction groups. ConclusionYifei Huatan decoction reduces airway hyperviscosity in asthmatic rats with spleen deficiency syndrome， which may be related to the inhibition of TGF-β1， Smad2， Smad3， MUC5AC， and MUC5B expressions， down-regulation of IL-4 and IL-13 levels， and up-regulation of IFN-γ level.
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@#AIM: To observe the effects of butterflybush flower eye drops at different concentrations on expression of inflammatory crtokines IL-1β, Mucin 5AC(MUC5AC)and P38MAPK in castrated male rabbits, and to explore the therapeutic effect of that drops on dry eyes. <p>METHODS: Thirty-six male rabbits were randomly divided into blank group(A), model group(B), low concentrations butterflybush flower eye drops group(C, 1mg/mL), the medium concentrations drops group(D, 1.5mg/mL), the high concentrations drops group(E, 3mg/mL), and testosterone group(F). In addition to group A, the testes and epididymis were removed from each group to establish a dry eye animal model. After successful modeling, groups A and B remain unchanged. Groups C, D, and E were given different concentrations of butterflybush flower eye drops, 3 times/d. In group F, testosterone propionate was injected into the muscles of the thigh at a dose of 0.5mL/kg once every 3d. Fluorescein staining, Schirmer I test(SⅠt)and tear film break time(BUT)were measured under general anesthesia in each group, eatment. After 4wk of treatment, the rabbits were sacrificed and the conjunctival tissues of the eyes were taken. The expression of IL-1β, mucin 5AC and P38MAPK in the conjunctiva was detected by immunohistochemical staining.<p>RESULTS: Among low concentrations butterflybush flower eye drops group, the medium concentrations drops group and the high concentrations drops group, the SⅠt value was significantly higher than that of model group, and BUT was significantly longer than model group. The positive staining of corneal fluorescein was significantly improved compared with model group, which was statistically significant(<i>P</i><0.01). Among IL-1β and P38MAPK in the conjunctiva of high concentrations butterflybush flower eye drops group, the medium concentrations drops group and the low concentrations drops group, the positive expressions were lower than those in model group, and the expression of MUC5AC was higher than that in group model group(<i>P</i><0.01). In addition, the high concentrations drops group was superior to the low and the medium concentrations drops group.<p>CONCLUSION: Butterflybush flower eye drops have androgen-like effect. For castrated dry eyes of male rabbits, they can down-regulate the expression of IL-1beta and P38MAPK in dry conjunctival tissue and increase the expression of MUC5AC, thus reducing inflammation infiltration in dry conjunctival tissue and maintaining tear film stability, but their effect is weaker than that of androgen. To the treatment of dry eyes, the middle and high concentration groups of the drops had stronger effects than the low one, and the high concentration group was better than the medium one.
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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.
Subject(s)
Humans , Bronchi , Epithelial Cells/drug effects , Interleukin-13 , Menthol/pharmacology , Mucin 5ACABSTRACT
Objective: To observe the effect of Pinelliae Rhizoma Praeparatum Cum Alumine polysaccharides(PRPCAP)on airway mucus secretion in rats with allergic asthma, in order to study the material basis of the "macromolecule" component of the polysaccharides as the original medicinal materials. Method: The 60 SPF-grade Wistar rats were induced by intraperitoneal injection of chicken ovalbumin (OVA) and aluminum-magnesium adjuvant, except for the normal control group. The OVA solution was aerosolized to establish a rat model of allergic asthma. After successful modeling, the rats were randomly divided into 5 groups, namely allergic asthma model group, positive drug group (montalurast sodium,5 mg·kg-1), high-dose PRPCAP group (400 mg·kg-1), middle-dose PRPCAP group (200 mg·kg-1) and low-dose PRPCAP group (100 mg·kg-1). The contents of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum and bronchoalveolar lavage fluid (BALF) supernatant were determined by enzyme-linked immunosorbent assay (ELISA), and the count of eosinophils (EOS) was detected by BALF sediment. The histopathological changes were observed by hematoxylin-eosin (HE) staining in lung tissue. The mRNA expression of mucin 5AC (MUC5AC) was detected by Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR). Result: Compared with the normal control group, serum IL-4 level in the allergic asthma model group was significantly increased (Pγ level was significantly decreased (PPPPPγ in serum (PPPPConclusion: The "macromolecule" component of polysaccharides in the Pinelliae Rhizoma Praeparatum Cum Alumine may be the material basis for the efficacy of eliminating dampness and eliminating phlegm.
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Objective@#To evaluate the effect of penehyclidine hydrochloride on the expression of airway mucin 5AC (MUC5AC) during ventilator-induced lung injury (VILI) and the relationship with Toll-like receptor 4/myeloid differentiation factor 88 (TLR4/MyD88) signaling pathway in rats.@*Methods@#Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), VILI group (group VILI), and penehyclidine hydrochloride group (group P). The rats were tracheotomized in group S. The rats were tracheotomized, connected to a small animal ventilator and mechanically ventilated for 4 h with the tidal volume of 20 ml/kg, respiratory rate 80 breaths/min, inspiratory/expiratory ratio 1∶1, and inspired oxygen fraction ratio 21% in VILI and P groups.At 30 min before mechanical ventilation, penehyclidine hydrochloride 2 mg/kg was injected via the tail vein in group P, and the equal volume of normal saline was given instead in S and VILI groups.At 4 h of mechanical ventilation, the arterial blood samples were taken for measurement of PaO2.The rats were then sacrificed, and broncho-alveolar lavage fluid (BALF) was collected for determination of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) concentrations by enzyme-linked immunosorbent assay.The lung specimens were collected for calculation of the wet/dry weight ratio (W/D ratio), for examination of pathological changes which were scored after haematoxylin and eosin staining (under a light microscope), and for determination of the expression of MUC5AC (by immunohistochemistry), expression of TLR4, MyD88, p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-kappa B (NF-κB) in lung tissues (by Western blot), and expression of MUC5AC mRNA in lung tissues (by real-time polymerase chain reaction).@*Results@#Compared with group S, PaO2 was significantly decreased, the W/D ratio and lung injury score were increased, the expression of MUC5AC protein and mRNA was up-regulated, the concentrations of IL-1β, IL-6 and TNF-α in BALF were increased, and the expression of TLR4, p38MAPK, MyD88 and NF-κB was up-regulated in VILI and P groups (P<0.01). Compared with group VILI, PaO2 was significantly increased, the W/D ratio and lung injury score were decreased, the expression of MUC5AC protein and mRNA was down-regulated, the concentrations of IL-1β, IL-6 and TNF-α in BALF were decreased, and the expression of TLR4, p38MAPK, MyD88 and NF-κB was down-regulated in group P (P<0.05).@*Conclusion@#Penehyclidine hydrochloride can decrease the expression of airway MUC5AC during VILI, and the mechanism may be related to inhibiting activation of TLR4/MyD88 signaling pathway in rats.
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Objective To evaluate the effect of penehyclidine hydrochloride on the expression of airway mucin 5AC(MUC5AC)during ventilator-induced lung injury(VILI)and the relationship with Toll-like receptor 4/myeloid differentiation factor 88(TLR4/MyD88)signaling pathway in rats.Methods Thir-ty-six clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 200-250 g,were divided into 3 groups(n＝12 each)using a random number table method: sham operation group(group S),VILI group(group VILI),and penehyclidine hydrochloride group(group P).The rats were tracheotomized in group S.The rats were tracheotomized,connected to a small animal ventilator and mechanically ventilated for 4 h with the tidal volume of 20 ml/kg,respiratory rate 80 breaths/min,inspiratory/expiratory ratio 1 ∶1,and inspired oxygen fraction ratio 21％in VILI and P groups.At 30 min before mechanical ventilation,penehyclidine hydrochloride 2 mg/kg was injected via the tail vein in group P,and the equal volume of nor-mal saline was given instead in S and VILI groups.At 4 h of mechanical ventilation,the arterial blood sam-ples were taken for measurement of PaO2.The rats were then sacrificed,and broncho-alveolar lavage fluid(BALF)was collected for determination of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)concentrations by enzyme-linked immunosorbent assay.The lung specimens were collected for calculation of the wet/dry weight ratio(W/D ratio),for examination of pathological changes which were scored after haematoxylin and eosin staining(under a light microscope),and for determination of the ex-pression of MUC5AC(by immunohistochemistry),expression of TLR4,MyD88,p38 mitogen-activated protein kinase(p38MAPK)and nuclear factor-kappa B(NF-κB)in lung tissues(by Western blot),and expression of MUC5AC mRNA in lung tissues(by real-time polymerase chain reaction).Results Com-pared with group S,PaO2 was significantly decreased,the W/D ratio and lung injury score were increased,the expression of MUC5AC protein and mRNA was up-regulated,the concentrations of IL-1β,IL-6 and TNF-α in BALF were increased,and the expression of TLR4,p38MAPK,MyD88 and NF-κB was up-reg-ulated in VILI and P groups(P＜0.01).Compared with group VILI,PaO2 was significantly increased,the W/D ratio and lung injury score were decreased,the expression of MUC5AC protein and mRNA was down-regulated,the concentrations of IL-1β,IL-6 and TNF-α in BALF were decreased,and the expression of TLR4,p38MAPK,MyD88 and NF-κB was down-regulated in group P(P＜0.05).Conclusion Penehy-clidine hydrochloride can decrease the expression of airway MUC5AC during VILI,and the mechanism may be related to inhibiting activation of TLR4/MyD88 signaling pathway in rats.
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More and more studies have shown that the expression levels of mucin 5AC (MUC5AC) and mucin 6 (MUC6) are closely related to Helicobacter pylori (Hp) infection and occurrence of gastric cancer, among which the gene polymorphism of MUC5AC and MUC6 has become one of the research hotspots. This article reviewed the relationship between MUC5AC, MUC6 and Hp infection and gastric cancer.
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Objective To investigate the mechanism of epidermal growth factor receptor-forkhead transcription factor A2 (EGFR-FOXA2) pathway-involved high secretion of mucus in human bronchial epitheli-um (HBE) cells after respiratory syncytial virus (RSV) infection and to evaluate the effects of intervention using agonist ( rosiglitazone ) and antagonist ( GW9662 ) of peroxidase proliferation activated receptor γ( PPARγ) and EGFR inhibitor ( AG1478 ) . Methods HBE cells were randomly divided into six groups: A group ( AG1478+RSV) , B group ( rosiglitazone+RSV) , C group ( GW9662+RSV) , D group ( RSV) , E group (0. 1％ dimethyl sulfoxide DMSO) and F group (HBE cell control group). Two hours before RSV infection, A, B and C groups were respectively treated with 10 μmol/L of AG1478, rosiglitazone and GW9662. Expression of EGFR, PPARγ and FOXA2 at mRNA level in each group was detected by real-time fluorescence quantitative RT-PCR 12 h, 24 h and 48 h after HBE cells were infected with or without RSV. Expression of phosphorylated-EGFR ( p-EGFR) and EGFR at protein level was detected by Western blot. ELISA was performed to measure the expression of mucin-5AC (MUC5AC). Results Compared with F group, EGFR expression at mRNA lev-el, p-EGFR/EGFR protein ratio and MUC5AC expression at protein level were increased in a time-dependent manner in A, B, C and D groups at 12 h, 24 h and 48 h. Compared with group F, the expression of PPARγat mRNA level in A, B, and D groups increased at each time point. Moreover, PPARγ expression gradually in-creased over time in A and B groups, reaching the peaks at 48 h, but was in decline in D group. Expression of FOXA2 at mRNA level in RSV-infected HBE cells was declined at each time point compared with that in group F, especially in D group. Compared with group D, A and B groups showed significantly decreased EGFR ex-pression at mRNA level, p-EGFR/EGFR protein ratio and MUC5AC expression at protein level, but markedly increased FOXA2 expression at mRNA level. Conclusions RSV infection increased the expression of MUC5AC at protein level in HBE cells. PPARγand EGFR-FOXA2 signaling pathways were involved in the hypersecretion of airway mucus during RSV infection.
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Objective: To explore the effect of endoplasmic reticulum stress (ERS) on neutrophil elastase (NE) induced mucin 5AC (MUC5AC) production in human airway epithelial cells. Methods: HBE16 airway epithelial cells were cultured and pretreated with reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) or ERS inhibitor 4-phenylbutyrate (4-PBA), or transfected with small interfering RNA (siRNA) against inositolrequiring kinase 1α (IRE-1α) or X-box binding protein 1 (XBP-1), respectively before incubation with NE. NE group and blank control group were also set up. ROS production was assayed by detection kit; expression of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK), activating transcription factor 6 (ATF6), phosphorylated IRE-1α (pIRE-1α), and XBP-1 protein was detected by Western blotting; spliced XBP-1 (XBP-1s) mRNA was measured by real-time PCR; levels of MUC5AC protein in culture supernatant and cytoplasm were assayed by ELISA and immunofluorescence. Results: There was an obvious increase of ROS production with strong elevation of GRP78, ATF6, pPERK, and pIRE-1α protein in NE group cells after 24 h, compared with blank control group (P<0.05). The protein and mRNA of XBP-1s, and MUC5AC production also increased obviously (P<0.05). NAC and 4-PBA reduced ERS-related protein expression and MUC5AC production and secretion (P<0.05). Further studies showed that MUC5AC secretion was also blunted by IRE-1α siRNA or XBP-1 siRNA, accompanied with decreased expression of XBP-1s mRNA and protein (P<0.05). Conclusion: NE induces ERS by producing ROS, and increases MUC5AC protein production and secretion; IRE-1α/XBP-1 play a certain role in this process.
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<p><b>Background</b>Excess mucus production is an important pathophysiological feature of chronic inflammatory airway diseases. Effective therapies are currently lacking. The aim of the study was to evaluate the effects of curcumin (CUR) on lipopolysaccharide (LPS)-induced mucus secretion and inflammation, and explored the underlying mechanism in vivo and in vitro.</p><p><b>Methods</b>For the in vitro study, human bronchial epithelial (NCI-H292) cells were pretreated with CUR or vehicle for 30 min, and then exposed to LPS for 24 h. Next, nuclear factor erythroid 2-related factor 2 (Nrf2) was knocked down with Nrf2 small interfering RNA (siRNA) to confirm the specific role of Nrf2 in mucin regulation of CUR in NCI-H292 cells. In vivo, C57BL/6 mice were randomly assigned to three groups (n = 7 for each group): control group, LPS group, and LPS + CUR group. Mice in LPS and LPS + CUR group were injected with saline or CUR (50 mg/kg) intraperitoneally 2 h before intratracheal instillation with LPS (100 μg/ml) for 7 days. Cell lysate and lung tissue were obtained to measured Mucin 5AC (MUC5AC) and Nrf2 mRNA and protein expression by a real-time polymerase chain reaction and Western blotting. Bronchoalveolar lavage fluid (BALF) was collected to enumerate total cells and neutrophils. Histopathological changes of the lung were observed. Data were analyzed by one-way analysis of variance. Student's t-test was used when two groups were compared.</p><p><b>Results</b>CUR significantly decreased the expression of MUC5AC mRNA and protein in NCI-H292 cells exposed to LPS. This effect was dose dependent (2.424 ± 0.318 vs. 7.169 ± 1.785, t = 4.534, and 1.060 ± 0.197 vs. 2.340 ± 0.209, t = 7.716; both P < 0.05, respectively) and accompanied by increased mRNA and protein expression of Nrf2 (1.952 ± 0.340 vs. 1.142 ± 0.176, t = -3.661, and 2.010 ± 0.209 vs. 1.089 ± 0.132, t = -6.453; both P < 0.05, respectively). Furthermore, knockdown of Nrf2 with siRNA increased MUC5AC mRNA expression by 47.7%, compared with levels observed in the siRNA-negative group (6.845 ± 1.478 vs. 3.391 ± 0.517, t = -3.821, P < 0.05). Knockdown of Nrf2 with siRNA also markedly increased MUC5AC protein expression in NCI-H292 cells. CUR also significantly decreased LPS-induced mRNA and protein expression of MUC5AC in mouse lung (1.672 ± 0.721 vs. 5.961 ± 2.452, t = 2.906, and 0.480 ± 0.191 vs. 2.290 ± 0.834, t = 3.665, respectively; both P < 0.05). Alcian blue/periodic acid-Schiff staining also showed that CUR suppressed mucin production. Compared with the LPS group, the numbers of inflammatory cells (247 ± 30 vs. 334 ± 24, t = 3.901, P < 0.05) and neutrophils (185 ± 22 vs. 246 ± 20, t = 3.566, P < 0.05) in BALF decreased in the LPS + CUR group, as well as reduced inflammatory cell infiltration in lung tissue.</p><p><b>Conclusion</b>CUR inhibits LPS-induced airway mucus hypersecretion and inflammation through activation of Nrf2 possibly.</p>
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Objective:To observe the effect of electroacupuncture (EA) on the expressions of acetylcholine (ACh) and mucin 5AC (MUC5AC) in the lungs of rats with chronic obstructive pulmonary disease (COPD),and explore the mechanism of EA in treating COPD.Methods:Thirty Sprague-Dawley (SD) rats were randomly divided into a control group,a COPD group,and an EA group,with 10 rats in each group.The control group was a group of normal rats.The COPD rat model was induced by cigarette smoke combined with lipopolysaccharide (LPS).The COPD rats were treated with EA at bilateral Feishu (BL 13) and Zusanli (ST 36) in the EA group,30 min each time,once a day,successively for 14 d.The lung function was tested.The contents of ACh and MUC5AC in lungs and bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA).Pearson method was used to analyze the correlation between pulmonary function and the content of MUC5AC in lungs.The mRNA and protein expressions of MUC5AC in lung tissues were detected by real-time polymerase chain reaction (RT-PCR) and Western blot (WB),respectively.The immune response of MUC5AC was observed by immunohistochemistry.Results:Eight rats were left in each group,and the other two died.Compared with the control group,the total airway resistance (Raw) increased significantly and dynamic compliance (Cdyn) decreased significantly in the COPD group (P＜0.01);compared with the COPD group,the Raw level declined significantly and Cdyn increased significantly in the EA group (P＜0.01).The contents of ACh and MUC5AC in the lungs and BALF were remarkably higher in the COPD group compared with those in the control group (P＜0.01,P＜0.001);compared with the COPD group,the contents of ACh and MUC5AC were significantly lower in the EA group (P＜0.05,P＜0.001).There was a negative correlation between MUC5AC content and lung function (P＜0.001).The mRNA and protein expressions of MUC5AC in the lungs were significantly higher in the COPD group than in the control group (P＜0.001);compared with the COPD group,the expressions were significantly lower in the EA group (P＜0.01).Compared with the control group,the immune response of MUC5AC in the airway epithelium significantly increased in the COPD group (P＜0.001);the immune response of MUC5AC was significantly lower in the EA group compared with that in the COPD group (P＜0.001).Conclusion:EA treatment can improve the lung function of COPD rats,which may be related to its effect in the down-regulation of ACh and MUC5AC contents in the lungs as well as the inhibition of mucus hypersecretion.
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OBJECTIVE To explore expression of IL-13 in eosinophilic chronic rhinosinusitis with nasal polyps(EOS-CRSwNP) and further investigate the correlation between IL-13 and MUC5AC in EOS-CRSwNP. METHODS MUC5AC was detected in tissues from normal nasal mucosa and EOS-CRSwNP by immunohistochemistry and ELISA. We also used ELISA to detect the expression of IL-13 in normal nasal mucosa and EOS-CRSwNP. Correlation between IL-13 and MUC5AC in EOS-CRSwNP was investigated by correlation analysis. Secretion of MUC5AC in IL-13 incubated primary air-liquid interface(ALI)-cultured nasal polyp epithelial cells was examined by ELISA. RESULTS MUC5AC is mainly expressed in the epithelium of nasal mucosa by immunohistochemistry. By ELISA, expressions of MUC5AC and IL-13 are higher in EOS-CRSwNP than those in normal nasal mucosa. Correlation analysis shows that there exists a high correlation between MUC5AC and IL-13 in EOS-CRSwNP. IL-13 upregulates expression of MUC5AC in IL-13 incubated primary ALI-cultured nasal polyp epithelial cells for 7 or 14 days. CONCLUSION Expressions of MUC5AC and IL-13 are higher in EOS-CRSwNP than those in normal nasal mucosa respectively, and MUC5AC correlates with IL-13 highly in EOS-CRSwNP.
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Objective To observe the changes of tear film stability,goblet cell and mucin 5AC expression in conjunctivochalasis patients,and explore the mechanism of conjunctivochalasis.Methods Conjunctivochalasis patients (30 cases) and single age-related cataract patients (15 cases) were collected as conjunctivochalasis group and normal control group.Eye symptom assessment (OSDI score),tear break-up time (BUT),Schirmer Ⅰ test,tear fern crystallization tests were performed for all the selected persons.Conjunctival rescent-shaped resections were made for all the conjunctivochalasis patients.Conjunctival tissue samples were stained by HE staining,AB staining,mucin 5AC immunohistochemical staining from the conjunctivochalasis group and norral control group respectively,and then statistical analysis was made.Results The OSDI score in the conjunctivochalasis group (37.80 ± 8.94) was significantly higher than that in the normal control group (11.40 ±4.08) (P ＜0.01).BUT in the conjunctivochalasis group (6.70 ± 2.76) s was significantly lower than that in the normal control group (13.67 ± 3.48) s (P ＜ 0.01).Schirmer Ⅰ test in the conjunctivochalasis group (6.23 ± 3.13) mum was significantly lower than the normal control group (13.40 ± 3.74)mm (P ＜ 0.01).Tear ferbing crystallization of the conjunctivochalasis group was decreased significantly compared with the normal control group (x2 =14.309,P =0.003).Light microscopic showed that conjunctival thickness was thinned,collagen fibers were less,elastic fiber was reduced,the lamina propria and interstitial were congestion and edema,the number of goblet cells was significantly reduced,and the positive staining of mucin 5AC staining was significantly lower in the conjunctivochalasis group than in normal control group (x2 =9.499,P =0.023).Conclusion For patients with conjunctivochalasis,the tear film function is affected,goblet cells are decreased,tear fern crystallization is decreased,mucin 5AC content is decreased,which finally leads the excessive conjunctival relaxation and abnormal ocular surface and tear.
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Background:Gastric cancer is one of the malignant tumors with highest morbidity and mortality,and the early change of molecular marker in gastric mucosal lesion is the hot spot of gastric cancer study. Aims:To investigate the expressions and clinical significance of trefoil factor 2(TFF2),claudin 18(CLDN18),mucin 5AC(MUC5AC)in gastric mucosal lesions. Methods:Gastroscopy biopsies and surgery specimens from Dec. 2008 to May 2009 at Inner Mongolia People’s Hospital were collected,including 20 normal gastric mucosal tissues,20 intestinal metaplasia tissues,11 dysplasia tissues and 20 gastric cancer tissues. The protein expressions of TFF2, CLDN18, MUC5AC were determined by immunohistochemical SP method. Results:The positive expression rates of TFF2,CLDN18 and MUC5AC in normal gastric mucosal tissues were all 100% ,and were gradually decreased in the order of intestinal metaplasia,dysplasia and gastric cancer tissue,the differences were statistically significant(P < 0. 01). Conclusions:Expressions of TFF2,CLDN18 and MUC5AC protein are closely related to the degree of malignancy of gastric mucosal lesions,and can be considered as a potential biological marker for predicting the development and prognosis of gastric cancer.
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Objective:To investigate the effects of CCR3 on Muc5ac in the airway of asthmatic mice.Methods: Fifty clean BALB/c mice were randomly divided into control group,asthmatic group,dexamethasone treatment group,SB328437 treatment group, vehicle-control group.Total cells and differential inflammatory cells were counted in BALF, the levels of IL-4 and TNF-αwere determined by ELISA,lung tissue HE staining the expressions of Mucin5ac(Muc5ac) and CCR3 in lung tissue were detected by immu-neohistochemical staining and RT-PCR.Results:The total cells,eosinophil,monocytes and lymphocyte cells in BALF,the levels of IL-4,TNF-αin BALF ,the goblet cell of airway wall,the expression of Muc5ac and CCR3 positive staining IOD in the airway and the expression of Muc5ac and CCR3 mRNA lung tissue obviously decreased in SB328437 treatment group and DEX treatment group which compared with asthmatic mice model group ( P<0.05 ) .Conclusion: SB328437 can inhibit the expression of CCR3 in pulmonary tissue,furtherly inhibit the expression and secretion of Muc5ac and control the airway inflammation.
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Objective To evaluate the effect of pronase on amoxicillin and metronidazole concentrations in gastric tissue. Methods C57BL/6 mice were randomly divided into experimental group ( n = 70 ) and control group ( n = 70 ) . Amoxicillin ( 28. 6 mg/kg ) , metronidazole ( 22. 5 mg/kg) and omeprazole (138.2 mg/kg) were administered orally to C57BL/6 mice, combined with pronase (110 mg/kg) or same amount of sterile PBS. Gastric tissue and blood plasma samples were taken at 10 point-in time (7 mice/time) from 15 min up to 360 min after administration. Concentrations of amoxicillin and metronidazole were detected by high performance liquid chromatography. Gastritis index of gastric mucosa ( hematoxylin-eosin staining) and the gastric tissue expressions of mucin 5 AC (Western blot) were detected at 120 min and 360 min after administration. Results The time to peak concentration of amoxicillin and metronidazole in gastric tissue appeared earlier than that in blood plasma (15 min vs 60 min). Tissue concentrations of amoxicillin and metronidazole of experimental group were significantly higher than those of control, and they were mainly at 15 min to 90 min (P ＜0. 05). Plasma concentrations of amoxicillin and metronidazole of experimental group at 15 min and 30 min were higher than those of control ( P ＜ 0. 05 ). There was no difference in gastritis index between experimental group and control at 120 min and 360 min after administration (0.28±0. 18 vs 0. 14 ±0. 14,P＞0.05; 0. 43 ±0. 20 vs 0. 28 ±0. 18,P ＞0. 05). The expressions of mucin 5 AC in experimental group were lower than those of control ( 0. 036 ± 0. 006 vs 0. 197 ± 0. 058; P ＜0. 05; 0. 039 ± 0. 008 vs 0. 208 ± 0. 072, P ＜ 0. 05 ). Conclusions Pronase can significantly enhance the drugs penetration from mucus into gastric tissue. Concentrations of amoxicillin and metronidazole of experimental group in local gastric tissue and plasma are higher than those of control, especially in improving concentrations of gastric tissue and prolongation of exposed time.
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BACKGROUND: We investigated whether curcumin and genistein affect the MUC5AC mucin production from human airway epithelial cells that is induced by phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-alpha (TNF-alpha). METHODS: Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with PMA or TNF-alpha for 24 hours. MUC5AC mucin production was measured by an ELISA. RESULTS: (1) Curcumin dose-dependently inhibited the production of MUC5AC mucin that was induced by PMA or TNF-alpha; (2) Genistein inhibited PMA-induced MUC5AC mucin production. However, it did not decrease TNF-alpha-induced MUC5AC mucin production. CONCLUSION: These results suggest that curcumin and genistein inhibit the production of airway mucin induced by PMA.