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Acute or chronic cold exposure exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. Cold-inducible RNA-binding protein (CIRP) is a cold-shock protein and is induced by various environmental stressors, such as hypothermia and hypoxia. In this study, we showed that CIRP gene and protein levels were significantly increased in patients with COPD and in rats with chronic airway inflammation compared with healthy subjects. Similarly, inflammatory cytokine production and MUC5AC secretion were up-regulated in rats following cigarette smoke inhalation. Cold temperature-induced CIRP overexpression and translocation were shown to be dependent on arginine methylation in vitro. CIRP overexpression promoted stress granule (SG) assembly. In the cytoplasm, the stability of pro-inflammatory cytokine mRNAs was increased through specific interactions between CIRP and mediator mRNA 3'-UTRs; these interactions increased the mRNA translation, resulting in MUC5AC overproduction in response to cold stress. Conversely, CIRP silencing and a methyltransferase inhibitor (adenosine dialdehyde) promoted cytokine mRNA degradation and inhibited the inflammatory response and mucus hypersecretion. These findings indicate that cold temperature can induce an airway inflammatory response and excess mucus production via a CIRP-mediated increase in mRNA stability and protein translation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aims: Secretoneurin(SN), a neuropeptide, has been considered a reliable marker of allergenic stimulation. However, the relationship between SN and the secretion of airway mucin remains unclear. In this study, we aimed to examine the in vitro relationship between SN and airway mucin over synthesis, as well as the signaling pathways involved., Methods and Results: Exogenous SN was added to two human airway epithelial cell lines (16HBE and NCI-H292). Measured by real-time quantitative polymerase chain reaction (qPCR) and enzyme-linked immuno sorbent assay (ELISA) respectively, the intracellular mucin(MUC)5AC mRNA and MUC5AC protein of culture supernates exhibited a time- and dose-dependent increase after stimulation of SN. Based on the evidence of an increased phosphorylation of ERK1/2 induced by exogenous SN, we performed the radioactive binding assay. We failed to find direct binding of SN to either epidermal growth factor receptor (EGFR) or Neuropilin-1(NRP1), the co-receptor of EGFR. But we detected an enhanced binding of EGF to NRP1 in the two airway epithelial cell lines induced by exogenous SN. Either EGF neutralizing antibody or MEK specific inhibitor (PD-98059) could attenuate the over synthesis of MUC5AC induced by exogenous SN, indicating an endogenous EGF dependent mechanism in MUC5AC over synthesis induced by SN., Conclusions: We conclude that SN induces MUC5AC hypersecretion in a dose- and time-dependent manner; moreover, the MUC5AC over synthesis induced by SN is strongly associated with the enhanced binding of EGF to NRP1 and the activation of EGFR and ERK1/2 subsequently., (© 2014 S. Karger AG, Basel.)

Objective: To explore the effects of glycyrrhizin on airway mucus hypersecretion induced by interleukin-13 (IL-13) in rats., Methods: A total of 50 SD rats were divided randomly into 5 groups with a random digit table: control group, IL-13 group, and different dosage (25, 50, 75 mg/kg) glycyrrhizin groups. The integral of expression intensity in positive cells of airway epithelium under mucus histochemical stain was calculated with modality-quantitative method. HBE-16 cells were divided into 6 groups: negative control (physiological saline), IL-13 control (10 µg/L IL-13+physiological saline), different concentration glycyrrhizin interference (10 µg/L IL-13+25, 50 and 75 µmol/L glycyrrhizin, respectively) and positive control (10 µg/L IL-13+25 µmol/L zopolrestat). The expression of mucin (MUC) 5AC mRNA, MUC5AC protein, aldose reductase (AR) activity and reactive oxygen species (ROS) content were detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, fluorometric method and fluorescence intensity with General Oxidative Stress Indicator (CM-H2DFDA) catheter respectively., Results: In vivo, the integral of expression intensity in positive stain cells of airway epithelium were 0.12 ± 0.03, 0.87 ± 0.13, 0.56 ± 0.08, 0.46 ± 0.06 and 0.35 ± 0.04 respectively while the integral of different dosage glycyrrhizin groups was significantly lower than that of IL-13 group (all P < 0.05) with dose depentency and the IL-13 group was stronger than control group (P < 0.05). In vitro, the index of AR activity and ROS at 48 h of HBE16 cells in every group were 0.156 ± 0.021, 0.692 ± 0.039, 0.436 ± 0.019, 0.323 ± 0.042 and 0.290 ± 0.027; 5.127 ± 0.033, 24.257 ± 3.263, 11.966 ± 0.283, 8.892 ± 0.521 and 6.426 ± 0.173 respectively. The indices of IL-13 control group were higher than those of negative control group (P < 0.05) while those of different concentration glycyrrhizin interference groups were lower than those of IL-13 control group (all P < 0.05). The expressions of MUC5AC mRNA and protein of HBE16 cells in every group were 0.82 ± 0.05, 3.22 ± 0.12, 2.57 ± 0.34, 2.09 ± 0.54 and 1.58 ± 0.22; 0.18 ± 0.04, 0.65 ± 0.15, 0.48 ± 0.11, 0.33 ± 0.19 and 0.26 ± 0.06 respectively. The indices of IL-13 control group were higher than those of negative control group (P < 0.05) and those of different concentration glycyrrhizin interference groups were lower than those of IL-13 control group (P < 0.05)., Conclusion: Glycyrrhizin may inhibit the expression of MUC5AC mRNA and MUC5AC protein induced by IL-13 and control the hypersecretion of airway mucus.

Objective: To investigate the effect of different pressure on mucin (MUC) secretion in dog airway mucus layer and explore the participation mechanisms., Methods: Totally 24 healthy dogs were randomly divided into 4 groups (n=6) after double-lumen endobronchial tube intubation. In group A the dogs were ventilated bilaterally with normal breathing frequency and pressure; In group B: one side of the dog lung did not ventilate, while the other side was excessively ventilated. In group C: the tension-sensitive cation channel (TRPV) 4 blocker Ruthenium Red (RR) was injected in advance, and ventilated as in group A. In group D: TRPV4 blocker RR was injected firstly, and then ventilated as in group B. After 12 h ventilation, we collected bronchoalveolar lavage fluid (BALF), tested MUC (2, 5AC, 5B) protein content by ELISA, and detected the transcription of MUC (2, 5AC, 5B) mRNA of bronchial lung tissue by RTPCR., Results: Compared with the normal ventilation (A2) group, the protein level of MUC of excessive ventilation (B2) group was significantly higher, mainly MUC5AC (P<0.05); compared with the normal ventilation (A1) group, the protein level of MUC of no ventilation (B1) group was significantly decreased (P<0.05). After pretreatment with RR, the MUC protein level of group C1, C2 were significantly lower than that of group A1, A2 (P<0.05); the MUC protein level of group B2were significantly lower than that of group D2 (P<0.05). The MUC (2, 5AC and 5B) mRNA level was higher in excessive ventilation group (B2) than in normal ventilation group (A2). After pretreatment with RR, in comparison with group A1, A2, the MUC (2, 5AC and 5B) mRNA level of group C1, C2 declined; in comparison with group B2, the MUC (2, 5AC and 5B) mRNA level of group D2 declined., Conclusion: Rhythmic pressure waves may regulate and balance basic airway mucin secretion through activating the TRPV4 channel in dogs.

Objective: To explore the mechanisms of mucus hypersecretion in airway of rats induced by the synergies between cold air and cigarette smoke inhalation and understand the intervention effects of saussurea and budesonide in this process., Methods: A total of 70 SD rats were randomly divided into 7 groups. Group A: control; Group B: cold stimulation group receiving cold air inhalation for 3 h daily for 40 d; Group C: cigarette smoke inhalation group receiving cigarette smoke inhalation for 0.5 h daily for 40 d; Group D: cigarette smoke inhalation + cold stimulation group; Group E: cigarette smoke inhalation + cold stimulation + saussurea (0.8 mg/kg saussurea intraperitoneally injected once daily for 40 d); Group F: cigarette smoke inhalation + cold stimulation + inhaled budesonide (0.5 mg/kg inhaled once daily for 40 d); Group G: cigarette smoke inhalation + cold stimulation + saussurea + inhaled budesonide. The relative quantities of TRPM8 mRNA within bronchial epithelium of each group were detected by real-time polymerase chain reaction (PCR) and TRPM8 protein was detected by immunohistochemical assay and Western blot. The levels of mucin (MUC) 5AC, interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay., Results: TRPM8 mRNA of groups A-G were 1.00 ± 0.00, 0.98 ± 0.07, 2.27 ± 0.29, 2.31 ± 0.30, 1.55 ± 0.38, 1.66 ± 0.40 and 1.31 ± 0.23; TRPM8 protein 0.16 ± 0.05, 0.16 ± 0.04, 0.22 ± 0.06, 0.25 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, 0.15 ± 0.05, 0.25 ± 0.04, 0.24 ± 0.03, 0.58 ± 0.06, 0.56 ± 0.09, 0.41 ± 0.09, 0.39 ± 0.07 and 0.20 ± 0.06 respectively. TRPM8 mRNA and protein of groups C and D were significantly higher than those of group A. And groups E, F and G were significantly lower than those of group D (all P < 0.05). In BALF of groups A-G, MUC5AC were (57 ± 6), (69 ± 5), (66 ± 4), (185 ± 43), (142 ± 30), (147 ± 36) and (60 ± 11) µg/mg, IL-8 (58 ± 14), (146 ± 38), (134 ± 29), (379 ± 101), (262 ± 67), (294 ± 70) and (81 ± 27) ng/L, TNF-α(153 ± 28), (208 ± 90), (274 ± 64), (600 ± 113), (458 ± 96), (498 ± 84) and (169 ± 65) ng/L respectively. The values of groups B, C and D were significantly higher than those of group A (all P < 0.05) while groups E, F and G were significantly lower than those of group D (all P < 0.05). Cigarette smoke inhalation and cold stimulation synergistically enhanced the expression of MUC5AC, IL-8 and TNF-α. Saussurea and inhaled budesonide synergistically inhibited the expression of MUC5AC, IL-8 and TNF-α., Conclusions: Cold air inhalation evokes the release of proinflammatory cytokines and MUC5AC via activated TRPM8 channel up-regulated by cigarette smoke inhalation. Saussurea and inhaled budesonide synergistically inhibits the above mentioned process.

This study investigated whether aldose reductase (AR) inhibition affects interleukin (IL)-13-induced mucus production in the human bronchial epithelial cell line-16 (HBE16) cells. The HBE16 cells were cultured with AR inhibitors (zopolrestat) or were transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with 10 ng/ml IL-13 for 2h. The levels of mucin (MUC)5AC mRNA and protein were measured by using RT-PCR or ELISA. Intracellular reactive oxygen species (ROS) were measured fluorimetrically with the CM-H2DCFDA probe. Western blotting was performed to determine the levels of AR, phosphorylated signal transducer and activator of transcription 6 (p-STAT6) and phosphorylated Janus kinase 2 (p-JAK2). The results show that treatment with zopolrestat or transfection with AR siRNA significantly suppressed IL-13-stimulated MUC5AC mRNA and protein in the HBE16 cells (P<0.05). AR inhibition could suppress IL-13-induced ROS generation, the phosphorylation of JAK2/STAT6 pathway and the activation of nuclear factor (NF)-kappa B, thereby decreasing mucus production in vitro (all P<0.05). Therefore, the inhibition of AR could be a therapeutic target for mucus hypersecretion in chronic inflammation lung disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Theaflavins isolated from black tea have been used in studies on the prevention of tumor growth. The aim of this study was to investigate whether treatment with theaflavins influences the mucus hypersecretion induced by cigarette smoke in the lungs of experimental rats. Firstly, cigarette smoke was aerosolized using a machine designed for inhalation by rats. The rats were divided into the negative control group, the cigarette smoke inhalation group, the theaflavins (TFs) treatment group, and the TFs +cigarette smoke inhalation group. The animals were sacrificed on day 60 of the experiment. Secondly, the rats were treated with theaflavins at different doses via a gastric tube and sacrificed on day 30. The changes in the levels of mucin 5AC (MUC5AC) and epidermal growth factor receptor (EGFR) in the airway were evaluated. Cigarette smoke induced a significant increase in the levels of MUC5AC and EGFR in all groups. These increases could be reversed by intragastric administration of theaflavins. The effect was more pronounced with the duration of treatment and coincided with a decrease in the expression of both targets. The rats showed various degrees of reduction in the expression of these parameters, which correlated with the theaflavin dose. TFs could inhibit the activation of EGFR, decrease the level of MUC5AC, and relieve airway mucous hypersecretion via the EGFR signaling pathway. These effects correlated directly with the duration of action and the dosage. In the future, oral theaflavins might be valuable in the treatment of chronic airway inflammation.

Mucus hypersecretion is an important manifestation in patients with chronic inflammatory airway diseases. Mucin 5AC (MUC5AC) is a major component of airway mucus. MUC5AC expression is regulated by epidermal growth factor receptor (EGFR) which can be activated by reactive oxygen species (ROS). Hyaluronan (HA), a linear glycosaminoglycan with molecular weights ranging from 2 × 10(5) to 1 × 10(7), is expressed in airway epithelium and can be depolymerized by ROS into hyaluronan fragments. The mechanisms through which fragmented HA exerts its biologic functions have been elucidated by interactions with its receptor CD44. The aim of our study was to examine the role of HA and CD44 in ROS-induced EGFR activation and MUC5AC expression. We exposed NCI-H292 cells to ROS generated by xanthine/xanthine oxidase (X/XO). ROS-induced EGFR phosphorylation, which was activated by tissue kallekrein (TK) activation and EGF release. We found ROS promoted CD44 co-immunoprecipitation with EGFR and MUC5AC up-regulation. These effects were mimicked by hyaluronan fragments. All the effects were inhibited by blocking CD44 or EGFR, suggesting that CD44 plays a critical role in ROS-induced MUC5AC up-regulation. These results show that ROS depolymerizes hyaluronan into fragments, and these fragments bind their receptor CD44 to induce TK activation, which cleaves EGF precursors into mature EGF to activate its receptor EGFR. Furthermore, we provide evidence that hyaluronan fragments are sufficient to induce CD44/EGFR interaction and EGFR signaling which lead to MUC5AC expression. The results indicate that the regulation of ROS-induced MUC5AC expression by hyaluronan and CD44 may provide important insights in the mechanism of mucus hypersecretion.

Objective: To investigate the effect of interleukin-13 (IL-13) on mucus secretion in vivo and the possible mechanism., Methods: The SD rats were randomly divided into control group, IL-13 group and IL-13 plus SP600125 group. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and the level of MUC5AC in the lung tissues were examined using Western blotting. RT-PCR was performed to examine the mRNA level of STAT4 and STAT6, and electrophoretic mobility shift assays (EMSA) was used to detect the DNA-binding activities of Forkhead box a2 (FOXA2) and activator protein-1 (AP-1)., Results: IL-13 caused a significant increase in MUC5AC and p-JNK1/2 expression, but did not affect the phosphorylation of ERK1/2. The expression of MUC5AC was attenuated after treatment with SP600125. A significant increase in STAT6 was observed in IL-13 group compared with that in the control group, whereas the expression of STAT4 mRNA was not significantly affected. The DNA-binding activity of FOXA2 was down-regulated after IL-13 exposure, which did not affect the DNA-binding activity of AP-1., Conclusion: IL-13 down-regulates mucus secretion via STAT6-FOXA2 pathway in vitro.

Objective: The effects of stretch sensitive positive ion channel, the internal flow of Ca(2+) and myristoylated alanine-rich C kinase substrate (MARCKS) on the high externalization of MUC5AC in airway epithelial cells in mechanical stretching., Methods: Mini-type Multi-functional Bio-impact Machin was used to construct the model of mechanical stretching. The airway epithelial cells were divide into control group, stretch, stretch and gadolinium, stretch and low molecular weight heparin, stretch and nifedipine, stretch and the locked nucleic acids of MARCKS effective domain (ED), stretch and the control locked nucleic acids of MARCKS ED groups. The expression of MUC5AC and SNAP23 protein in cells were determined by immunohistochemistry method, contents of MUC5AC and SNAP23 protein were measured by Image Pro Plus 5.0. MUC5AC protein in supernatant was determined by ELISA methods. SNAP23 mRNA was determined by RT-PCR methods., Results: Mechanical stretching could increase the expression of SNAP23 and MUC5AC in cells and MUC5AC in supernatant (P < 0.05). Gadolinium, nifedipine and LNA of MARCKS ED could reduce the increase the expression of SNAP23 and MUC5AC in cells and MUC5AC in supernatant (all P < 0.05)., Conclusion: Mechanical stretching could increase the expression of MUC5AC in airway epithelial cells. This maybe is concerned with stretch sensitive positive ion channel, the internal flow of Ca(2+) and MARCKS.

The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA-AM (1,2-bis-(oaminophenoxy) ethane-N,N,N',N'-tetra-acetic acid-acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue- 2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT-PCR (reverse transcription-PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA-AM and RB-2, while EGTA did not have a suppressive effect. BAPTA-AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+ -dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2+. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the influence of scutellarin on mucus production induced by human neutrophil elastase (HNE) and the possible in vitro and in vivo mechanisms. To this purpose, cells were incubated with saline, scutellarin or gefitinib for 60 min and exposed to 0.1 μM HNE for 24 h. After being pretreated respectively with saline, scutellarin or gefitinib, rats were challenged intratracheally with HNE by means of nebulization for 30 days. The expression of mucin (MUC) 5AC, protein kinase C (PKC), and extracellular signal-regulated kinase 1/2 (ERK1/2) was assessed by ELISA, RT-PCR or Western blotting. The results showed that scutellarin inhibited MUC5AC mRNA and protein expressions induced by HNE in a concentration-dependent manner in vitro. In the in vivo model, scutellarin significantly attenuated MUC5AC mRNA expression and goblet cell hyperplasia in rats treated with HNE for 30 days, as well as decreased the phosporylation of PKC and ERK1/2 compared to the HNE control group. Therefore, our study showed that scutellarin could prevent mucus hypersecretion by inhibiting the PKC-ERK signaling pathway. Inhalation scutellarin may be valuable in the treatment of chronic inflammatory lung disease. [ABSTRACT FROM AUTHOR]

Abstract: Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. [Copyright &y& Elsevier]

Abstract: Mucus hypersecretion is a common feature in chronic airway diseases, and serine proteases play a critical role in this process. However, the mechanisms by which serine proteases induce mucin5AC hypersecretion have not been fully explored. In this study, we characterized human airway trypsin-like protease (HAT), a serine protease that is found in the mucoid sputum of patients with chronic airway diseases and is an agonist of protease-activated receptor 2 (PAR2)-induced cellular responses in human bronchial epithelial cells (16HBE). We also investigated the potential involvement of PAR2 in this process. We found that both HAT and PAR2-AP enhance the exocytosis of mucin5AC protein, whereas HAT, but not PAR2-AP, enhances the expression of mucin5AC mRNA. PAR2 is expressed at a much higher level in the cells than the other three PARs. Transfection with an siRNA against the PAR2 receptor or Gαq/11 protein or pretreatment with the Gαq/11 protein inhibitor YM-254890, the PLC inhibitor U73122 or the intracellular Ca2+ chelator BAPTA-AM all effectively attenuated the HAT-induced cellular responses. Taken together, these results indicate that HAT can stimulate mucin5AC hypersecretion through a PAR2-mediated signaling pathway in 16HBE cells. Thus, PAR2 could represent a novel therapeutic target for chronic airway diseases with mucus hypersecretion. [Copyright &y& Elsevier]