Differences in the health-related quality of life in patients with asthma living in urban and rural areas in the Amur Region of Russian Federation.

BACKGROUND: Asthma usually arises from an interaction between host and environmental factors. Growing attention has been paid to a place of residence as a factor shaping health-related quality of life (QoL). This study investigated the rural-urban disparity in QoL among adult asthma patients in the Amur region of Russian Federation. MATERIALS AND METHODS: This cross-sectional study included 351 randomly selected adults with asthma. We analyzed QoL (SF-36 and AQLQ scores), asthma control (ACT), and anxiety and depression (HADS) depending on the place of residence (urban vs. rural). RESULTS: The scale "Role Emotional" (RE) of SF-36 was significantly lower in patients from rural areas compared to urban residents (59.3±3.1 vs. 70.4±2.3 points; p = 0.0042). In the urban group, the correlation analysis demonstrated a clear influence of RE on patients' own assessment of their physical functioning (PF, r = 0.53; p<0.0001). Both groups demonstrated low "Social Functioning" (SF). In the group of urban residents, correlation analysis revealed the presence of positive correlations of SF-36 domains reflecting physical (PF, RP, BP) and social functioning (SF, VT) with most scales of both QoL questionnaires. The domains of the emotional sphere (RE and MH) positively correlated with all scales of both QoL questionnaires among urban residents. In the group of rural residents, a comparative analysis showed the absence of significant correlations between more of the QoL scales. Although Asthma Control Test did not differ between groups, we noted a significantly higher need for ß2-agonists in rural areas compared to urban areas (4.2±0.6 vs. 2.7±0.3 inh/day, respectively; p = 0.0221). The frequency of urban residents with a clinically significant level of anxiety (56 persons, or 25.2%) turned out to be lower compared to rural residents (45 persons, or 34.8%; χ2 = 34.08; p<0.001). CONCLUSION: The burden of asthma introduces a greater imbalance in the health-related QoL of rural residents compared to urban residents in the Amur region of the Russian Federation. The absence of interrelationships of some QoL domains in rural residents suggested a disunity of the physical, psychological and social aspects of life. The rural residents suppress physical discomfort by the more frequent use of short bronchodilators. They often showed emotional instability with a predominance of anxiety, which affected the decrease in QoL in the psycho-emotional sphere.

The Degradation of Airway Epithelial Tight Junctions in Asthma Under High Airway Pressure Is Probably Mediated by Piezo-1.

Full functioning of the airway physical barrier depends on cellular integrity, which is coordinated by a series of tight junction (TJ) proteins. Due to airway spasm, edema, and mucus obstruction, positive end-expiratory alveolar pressure (also termed auto-PEEP) is a common pathophysiological phenomenon, especially in acute asthma attack. However, the influence of auto-PEEP on small airway epithelial TJs is currently unclear. We performed studies to investigate the effect of extra pressure on small airway epithelial TJs and its mechanism. The results first confirmed that a novel mechanosensitive receptor, piezo-1, was highly expressed in the airway epithelium of asthmatic mice. Extra pressure induced the degradation of occludin, ZO-1 and claudin-18 in primary human small airway epithelial cells (HSAECs), resulting in a decrease in transepithelial electrical resistance (TER) and an increase in cell layer permeability. Through in vitro investigations, we observed that exogenous pressure stimulation could elevate the intracellular calcium concentration ([Ca2+] i ) in HSAECs. Downregulation of piezo-1 with siRNA and pretreatment with BAPTA-AM or ALLN reduced the degradation of TJs and attenuated the impairment of TJ function induced by exogenous pressure. These findings indicate the critical role of piezo-1/[Ca2+] i /calpain signaling in the regulation of small airway TJs under extra pressure stimulation.

The Cosmc-mediated effects of neutrophil elastase on T antigen expression in BEAS-2B cells.

Mucin 5AC (MUC5AC) is a highly O-glycosylated mucin secreted by human bronchial epithelial cells during pulmonary inflammatory diseases. T antigen, a component of the MUC5AC glycans, is the product of the O-glycosylation transferase T-synthase and its chaperone Cosmc. Since the expression of Cosmc is mediated by signaling pathways and inflammatory factors affecting mucin O-glycosylation, we analyzed the impact of neutrophil elastase (NE)-mediated Cosmc and T antigen expression in BEAS-2B cells derived from human bronchial epithelial cells. The expression of Cosmc and T antigen in human lung tissue was analyzed by immunohistochemistry. Cellular immunohistochemistry and western blot analysis demonstrated elevated expression of T antigen in BEAS-2B cells after NE stimulation. Altered Cosmc expression in BEAS-2B cells after NE stimulation was analyzed by confocal microscopy, western blot analysis and quantitative RT-PCR. To assess the biological implications of Cosmc function for T-synthase activity and T antigen synthesis after NE stimulation, BEAS-2B cells were transfected with shRNA to silence the expression of Cosmc. The changes in signaling pathways were analyzed by western blotting. The expression of Cosmc and T antigen increased in lung tissue exposed to chronic inflammation. The expression of Cosmc and T antigen increased in NE-stimulated BEAS-2B cells. NE induced increases in T antigen expression and T-synthase transferase activity in BEAS-2B cells expressing Cosmc, highlighting the importance of Cosmc in the relationship between NE and T antigen. Cosmc and phosphatidylinositol-3-kinase (PI3K) played important roles in the signaling pathway that stimulated hyperexpression of T antigen.

The role of the XBP-1/AGR2 signaling pathway in the regulation of airway Mucin5ac hypersecretion under hypoxia.

Oversecretion of Mucin5ac (MUC5AC), which is primarily synthesized by goblet cells and is the major gel-forming mucin, is a hallmark of various pulmonary inflammatory diseases. Hypoxia is considered a common pathophysiologic feature in various pulmonary inflammatory diseases. It has been suggested that hypoxia-inducible factor 1α (HIF-1α) acts as a key factor in hypoxia-induced MUC5AC hypersecretion; however, the exact mechanisms that maintain the stability of HIF-1α and support oversecretion by airway epithelial cells under hypoxia are still unclear. With immunohistochemistry, we found overexpression of anterior gradient 2 (AGR2) in the bronchial epithelial cells of hypoxia-treated mice. With specific shRNA transduction, AGR2 was demonstrated to be a key factor in MUC5AC hypersecretion in vitro. Additionally, co-immunoprecipitation, cell immunochemistry and confocal microscopy experiments were performed to explore the interaction between HIF-1α and AGR2 during hypoxia-induced MUC5AC hypersecretion in vitro. The results indicated increased binding and intracytoplasmic colocation of HIF-1α and AGR2. Our findings suggest that AGR2 acts as a key regulator in hypoxia-induced airway MUC5AC hypersecretion by increasing the stability of HIF-1α. Additionally, the elevated expression of AGR2 induced by hypoxia in bronchial epithelial cells likely depends on an XBP-1-associated pathway.

MicroRNA-155 regulates lipopolysaccharide-induced mucin 5AC overproduction via a suppressor of cytokine signaling 1-mediated mechanism in human bronchial epithelial cells.

Chronic inflammatory lung diseases accompanied by Gram-negative bacteria infection are characterized by excessive mucin production. Lipopolysaccharide (LPS), the major endotoxin released from Gram-negative bacteria, is a potent inflammatory agonist for mucin overproduction. In this study, we sought to examine whether the toll-like receptor (TLR)-responsive microRNA miR-155 plays a role in LPS-provoked induction of mucin 5AC (MUC5AC) and the potential role of suppressor of cytokine signaling 1 (SOCS1) involved in this process. We found that LPS increased the expression of MUC5AC in association with TLR4-dependent miR-155 induction. The suppression of miR-155 by antagomir led to an excessive production of SOCS1, thereby downregulation of MUC5AC production. Collectively, these data imply that miR-155 is involved in LPS-induced MUC5AC overproduction through a TLR4-dependent manner and thereby the downregulation of SOCS1.

Vaccine Prophylaxis of Pneumococcal Infections in Children under Conditions of Severe Flood in the Amur River Basin.

BACKGROUND: Pneumococcal infection being one of the dominant causes of acute respiratory diseases and exacerbation of chronic ones is a serious problem for human health and society. The flood in the Amur river basin in the summer of 2013 created a special zone and risk conditions for the formation of respiratory pathology in the Far-Eastern region of Russia. We aimed to give clinical and epidemiological assessment of the effectiveness of vaccination programs of respiratory viral and pneumococcal infections and generalization of regional experience in the organization of a set of measures aimed at their prevention in the postflood period in the Far-Eastern region. METHODS: The monitoring program includes children aged 2 to 5 years in the amount of 4988 with risk factors for pneumococcal infection. The pneumococcal conjugate vaccine Prevenar-13 was used for immunization. Data on the incidence of ARVI and pneumonia in children in pre- and postvaccination periods were to be recorded. The indicators and special criteria were used to assess the effectiveness of vaccination. To study the circulation of serovariants of pneumococcus in inflammatory diseases of the respiratory tract and nasopharyngeal carrier, bacteriological and molecular genetic methods (RT-PCR in the mode of multiprime detection) were used. RESULTS: Differences in the frequency and range of serovariants of circulating isolates of pneumococcus in the postvaccinal period and in unvaccinated children, elimination of a number of serotypes, and appearance of circulation of nonvaccinated strains were revealed. The incidence of acute respiratory diseases and pneumonia among the vaccinated population for 2 years in the region decreased by 2.5 times. The coefficient of effectiveness of vaccination according to the indicator of morbidity of children with pneumonia reaches 75-100% with direct dependence on the age of children (r=0.98). CONCLUSION: Comparative statistical analysis revealed a high degree of effectiveness of regional programs with the methods of immunoprophylaxis of pneumococcal infections.

Munc13­4 mediates human neutrophil elastase­induced airway mucin5AC hypersecretion by interacting with syntaxin2.

The overexpression and hypersecretion of mucus is a hallmark of chronic pulmonary inflammatory disease. Mucin5AC (MUC5AC) is a major component of airway gel­forming mucin. Members of the Unc13 (Munc13) protein family act as important activators of granule exocytosis from various types of mammalian cells. The present study aimed to determine the role of Munc13 family proteins in MUC5AC secretion via an in vitro study with BEAS­2B and Calu­3 cell lines. Reverse transcription­quantitative polymerase chain reaction and western blotting indicated that stimulation of the cells with 100 nM human neutrophil elastase (hNE) for 1 h did not affect the expression of either unc13 homolog B (Munc13­2) or unc13 homolog D (Munc13­4), but immunofluorescence analysis demonstrated that hNE treatment was associated with the recruitment of Munc13­4 to the plasma membrane. Co­immunoprecipitation analysis indicated increased binding between Munc13­4 and syntaxin2 followingh NE stimulation; however, Munc13­2 formed a stable interaction with syntaxin2 with or without hNE stimulation. Subsequently, Munc13­2 and Munc13­4 expression levels were downregulated in BEAS­2B and Calu­3 cells using small interfering RNA (siRNA). ELISAs and immunofluorescence analysis were performed to assess MUC5AC secretion and intracellular retention, respectively. Munc13­2 siRNA transfection did not alter the expression levels of intracellular or secreted MUC5AC following hNE stimulation in either cell line; however, it increased the baseline intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC. Conversely, Munc13­4 siRNA transfection increased the intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC following hNE stimulation, but did not affect their baseline quantities. The results of the present study indicate that Munc13­2 may be an essential regulator of basal MUC5AC exocytosis, while Munc13­4 appears to be a Munc13 protein subtype that may to be sensitive to hNE stimulation during airway MUC5AC hypersecretion.

Cold-inducible RNA-binding protein mediates airway inflammation and mucus hypersecretion through a post-transcriptional regulatory mechanism under cold stress.

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.

Transient receptor potential melastatin 8 gene polymorphism is associated with cold-induced airway hyperresponsiveness in bronchial asthma.

BACKGROUND AND OBJECTIVE: Cold-induced airway hyperresponsiveness (CAH) is common in bronchial asthma (BA) patients and represents a problem for those living in cold climate. Transient receptor potential melastatin 8 (TRPM8) channel is the main cold temperature sensor in humans that could mediate cold response in asthmatics with CAH. No associations between TRPM8 gene polymorphisms and CAH have been reported. METHODS: The present study involved 123 BA patients. CAH was assessed by 3-min isocapnic (5% CO2 ) cold air (-20°C) hyperventilation challenge. The c.750G > C (rs11562975), c.1256G > A (rs7593557), c.3048C > T (rs11563208) and c.3174C > G (rs11563071) polymorphisms of TRPM8 gene were genotyped by allele-specific polymerase chain reaction (PCR) and PCR with subsequent restriction fragment length polymorphism analysis. RESULTS: GC genotype and C allele carriers of the c.750G > C (rs11562975) polymorphism were more frequently observed to exhibit CAH. The estimated odds ratio for the GC genotype was 3.73 95%CI (1.48; 9.37), P = 0.005. Furthermore, GC heterozygotes had a prominent decrease in forced expiratory volume in 1 s after the challenge as compared to GG homozygotes (-12% (-16; -8.1) vs -6.45% (-11; -2.1), P < 0.001). GC carriers also had a marked reduction in other spirometric parameters. CONCLUSIONS: The GC variant of the TRPM8:c.750G > C (rs11562975) polymorphism is associated with CAH in patients with BA, which suggests a potential role of TRPM8 in CAH development.

Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells.

BACKGROUND/AIM: Increased mucin secretion is a characteristic feature of many chronic airway diseases, particularly during periods of exacerbation; however, the exact mechanism of mucin secretion remains unclear. Ezrin, which is a specific marker of apical membranes, is predominantly concentrated in exocyst-rich cell surface structures, crosslinking the actin cytoskeleton with the plasma membrane. In the present study, we examined whether Ezrin is involved in mucin 5AC (MUC5AC) secretion after neutrophil elastase (NE) attack, and we investigated the role of the exocyst complex docking protein Sec3 in this process. METHODS: NE was used as a stimulator in a 16HBE14o- cell culture model. The expression and location of Ezrin and Sec3 were investigated, and the interaction between Ezrin and Sec3 in 16HBE14o-cells was assayed after treatment with NE, Ezrin siRNA, Sec3 siRNA, neomycin or PIP2-Ab. RESULTS: We found that Ezrin was highly expressed in the bronchi of humans with chronic airway diseases. NE induced robust MUC5AC protein secretion. The Ezrin siRNA, Sec3 siRNA, and neomycin treatments led to impaired MUC5AC secretion in cells. Both Ezrin and Sec3 were recruited primarily to the cytoplasmic membrane after NE stimulation, and the neomycin and PIP2-Ab treatments abrogated this effect. Immunoprecipitation analysis revealed that Ezrin and Sec3 combined to form complexes; however, these complexes could not be detected in Ezrin∆1-333 mutant-transfected cells, even when PIP2 was added. CONCLUSIONS: These results demonstrate that Ezrin/Sec3 complexes are essential for MUC5AC secretion in NE-stimulated airway epithelial cells and that PIP2 is of critical importance in the formation of these complexes.

Effects of ATP release on mucin5AC secretion in airway epithelial cells by mechanical stretching.

This study is to determine the effects of ATP and Ca(2+) on mucin5AC (MUC5AC) overexpression in airway epithelial cells in mechanical ventilation. Oxygen was injected into the closed box used in this study to increase the pressure. Gravity-driven draining flow led to formation of a thin liquid film on the upper portion of cell monolayer, exposing cells to the tension forces at the air-liquid interface. The levels of MUC5AC protein and ATP in culture medium were detected by ELISA and high performance liquid chromatography, respectively. Ca(2+) and MUC5AC mRNA in culture cells were detected by flow cytometry and RT-PCR, respectively. Mechanical stretching increased the expression of MUC5AC in cells and the concentration of MUC5AC and ATP in supernatant. BAPTA-AM and EGTA partially reduced the increases in the concentrations of MUC5AC and ATP in supernatant with mechanical ventilation. BAPTA-AM completely inhibited ATP in supernatant with normal breathing conditions. Our results showed that mechanical ventilation increases the secretion of MUC5AC in airway epithelial cells. This is possibly related to Ca(2+)-dependent ATP release and intracellular and external Ca(2+).

Secretoneurin induces airway mucus hypersecretion by enhancing the binding of EGF to NRP1.

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.

Annexin II mediates the neutrophil elastase-stimulated exocytosis of mucin 5ac.

The overexpression and hypersecretion of mucus is a hallmark of several chronic pulmonary inflammatory diseases, including chronic obstructive pulmonary disease (COPD), asthma and cystic fibrosis. Mucin 5ac (MUC5AC) is a major component of airway mucus. Annexin II (ANXII) has been reported to be expressed in various cells and is associated with the fusion of secretory vesicles. Neutrophil elastase (NE) is present at high concentrations in the airway surface fluid in patients with cystic fibrosis and various other severe diseases. However, the role of ANXII in NE-induced secretion of MUC5AC granules remains unclear. It was determined that NE upregulates the transcription and protein synthesis of ANXII in 16HBE human bronchial epithelial cells. Following stimulation with NE, ANXII is recruited to the cell membrane, as visualised by cell immunochemistry and laser confocal microscopy, and the redistribution of ANXII is inhibited by the protein kinase-C (PKC) inhibitor bisindolylmaleimide I. Conversely, depleting endogenous ANXII decreases MUC5AC secretion into the cell culture supernatant and increases the levels of intracellular MUC5AC protein. The data indicated that ANXII is associated with the secretion of MUC5AC granules.

Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion.

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-(o-aminophenoxy)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.

[Main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion].

OBJECTIVE: To explore the main mediated molecules of mucin (MUC) 5AC extracellular secretion stimulated by airway shear stress (SS). METHODS: The 16 human bronchial epithelial (HBE) cells were cultured and randomized divided by Stata software into 5 groups: A. control group; B. SS stimulated group; C. SS stimulated & NSC23766 (a specific inhibitor of Rac-1) incubated group; D. SS stimulated & Cytochalasin D incubated group; E. Cortactin-siRNA (a small interfering RNA of Cortactin) transfected & SS stimulated group. Each group consisted of 6 parallel wells. Triplicate experiments were performed for statistical analysis. Rhythmic rotating device was used to simulate the breathing air flow mediated shear stress. The function of Cortactin was inhibited by Cortactin-siRNA. The relative content of MUC5AC in supernatant was measured by enzyme linked immunosorbent assay (ELISA). The p-Cortactin (phosphorylation Cortactin) relative level, Cortactin relative level and the effect of transfection were measured with Western blotting. And laser confocal microscope was used to observe the polymerization of F-actin. RESULTS: The transfection of Cortactin-siRNA successfully inhibited the function of Cortactin. The relative content of MUC5AC was (0.210 ± 0.013), (0.631 ± 0.025), (0.473 ± 0.112), (0.330 ± 0.067), (0.272 ± 0.019) in groups A, B, C, D and E, the group B was significantly higher than any other group (P = 0.000, 0.043, 0.000, 0.000). The Cortactin relative level in group B (0.670 ± 0.048) was significantly higher than that in group E (0.132 ± 0.014) (P < 0.01). But as compared with groups A, C, D (0.641 ± 0.016, 0.622 ± 0.012, 0.653 ± 0.027), there was no significance (all P > 0.05). The p-Cortactin relative level in group B (0.582 ± 0.067) was significantly higher than that in groups A, C, E (0.131 ± 0.011, 0.393 ± 0.045, 0.170 ± 0.016) (P = 0.000, 0.021, 0.000). But as compared with group D (0.511 ± 0.029), there was no significance (P = 0.246). CONCLUSION: Rac-1, Cortactin and F-actin are the main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion.

The degradation of airway tight junction protein under acidic conditions is probably mediated by transient receptor potential vanilloid 1 receptor.

Acidic airway microenvironment is one of the representative pathophysiological features of chronic inflammatory respiratory diseases. Epithelial barrier function is maintained by TJs (tight junctions), which act as the first physical barrier against the inhaled substances and pathogens of airway. As previous studies described, acid stress caused impaired epithelial barriers and led the hyperpermeability of epithelium. However, the specific mechanism is still unclear. We have showed previously the existence of TRPV (transient receptor potential vanilloid) 1 channel in airway epithelium, as well as its activation by acidic stress in 16HBE cells. In this study, we explored the acidic stress on airway barrier function and TJ proteins in vitro with 16HBE cell lines. Airway epithelial barrier function was determined by measuring by TER (trans-epithelial electrical resistance). TJ-related protein [claudin-1, claudin-3, claudin-4, claudin-5, claudin-7 and ZO-1 (zonula occluden 1)] expression was examined by western blotting of insoluble fractions of cell extraction. The localization of TJ proteins were visualized by immunofluorescent staining. Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly. However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells. The decline of epithelial barrier function induced by acidic stress exhibited a TRPV1-[Ca2+]i-dependent pathway. Of the TJ proteins, claudin-3 and claudin-4 seemed to be sensitive to acidic stress. The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)].

[Effects of glycyrrhizin on airway mucus hypersecretion induced by interleukin-13 in rats].

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.

Cortactin mediates elevated shear stress-induced mucin hypersecretion via actin polymerization in human airway epithelial cells.

Mucus hypersecretion is a remarkable pathophysiological manifestation in airway obstructive diseases. These diseases are usually accompanied with elevated shear stress due to bronchoconstriction. Previous studies have reported that shear stress induces mucin5AC (MUC5AC) secretion via actin polymerization in cultured nasal epithelial cells. Furthermore, it is well known that cortactin, an actin binding protein, is a central mediator of actin polymerization. Therefore, we hypothesized that cortactin participates in MUC5AC hypersecretion induced by elevated shear stress via actin polymerization in cultured human airway epithelial cells. Compared with the relevant control groups, Src phosphorylation, cortactin phosphorylation, actin polymerization and MUC5AC secretion were significantly increased after exposure to elevated shear stress. Similar effects were found when pretreating the cells with jasplakinolide, and transfecting with wild-type cortactin. However, these effects were significantly attenuated by pretreating with Src inhibitor, cytochalasin D or transfecting cells with the specific small interfering RNA of cortactin. Collectively, these results suggest that elevated shear stress induces MUC5AC hypersecretion via tyrosine-phosphorylated cortactin-associated actin polymerization in cultured human airway epithelial cells.

Role of the JNK pathway on the expression of inflammatory factors in alveolar macrophages under mechanical ventilation.

The aim of this study was to investigate the regulatory role of the c-JUN N-terminal kinase (JNK) pathway on interleukin (IL)-8 and tumor necrosis factor (TNF)-α expression in alveolar macrophages (AMs) of injured lung. Lung injury was induced in the New Zealand white rabbit by applying continuous mechanical ventilation with or without inhibitor of JNK (SP600125), p38 (SB203580), or ERK (PD98059). Non-ventilated rabbits (controls) were compared with the different ventilation-days groups, and untreated rabbits ventilated for 3 days (controls) were compared with the different inhibitor groups. We found that mechanical ventilation caused significant decreases in partial pressures of carbon dioxide (pCO2) and oxygen (pO2) of untreated rabbits (all times, P<0.05), but the inhibitor-treated groups showed no change in either blood-gas indicator (all times, P>0.05). Mechanical ventilation caused time-dependent increases in mRNA and protein levels of TNF-α and IL-8 in AMs and in serum of untreated rabbits, with the peak levels occurring at day 3 of ventilation. The SP600125-treated group showed significantly decreased TNF-α expression, but no significant change in IL-8 expression. Neither the SB203580- nor PD98059-treated groups showed any significant change in TNF-α or IL-8 expression. MAPKs' inhibitors could reduce mechanical ventilation-induced inflammation, and SP600125 produced the most robust decrease in inflammation. Mechanical ventilation-induced lung injury stimulates IL-8 and TNF-α expression in rabbit AMs in a time-dependent manner. The JNK pathway plays an important role in mechanical ventilation-stimulated TNF-α expression in AMs, but the injury-stimulated IL-8 expression may be regulated by other signaling pathways.

The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells.

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.