MUC5AC mRNA Levels Research Articles

Middle Ear microRNAs Drive Mucin Gene Response.

To investigate the role of microRNA-378 (miR-378) in the regulation of mucin gene expression and inflammatory response in human middle ear epithelial cells (HMEEC) during bacterial infection by non-typeable Haemophilus influenzae (NTHi). Human middle ear epithelial cells (HMEEC) were cultured and transfected with miR-378 or control miRNA. Post-transfection, cells were exposed to NTHi lysates. mRNA levels of MUC5B, MUC5AC, and IL-8 were quantified using RT-qPCR, and promoter activity was measured via luciferase assays. The effects of miR-378 on mucin and cytokine gene expression were analyzed. Transfection with miR-378 significantly increased the expression of MUC5B (3.6 fold, p < 0.01), MUC5AC (19.1 fold, p < 0.01), and IL-8 (2.01 fold, p < 0.05) in HMEEC. NTHi exposure reduced MUC5B (1.385 fold, p < 0.05) and MUC5AC (1.61 fold, p < 0.05) gene expression in miR-378 transfected cells but significantly increased IL-8 levels (1.32 fold, p < 0.05). Luciferase assays showed that miR-378 upregulated the promoter activity of MUC5B (1.4 fold, p < 0.01) and MUC5AC (1.6 fold, p < 0.01) genes, indicating its role in transcriptional regulation. miR-378 plays a crucial role in promoting mucin overproduction and an inflammatory response in the middle ear epithelium during OM. Targeting miR-378 could offer a novel therapeutic strategy for preventing the progression from AOM to COM. na Laryngoscope, 2024.

Effect of chronic stress on gel-forming mucins in the small intestine of BALB/c mice.

Intestinal homeostasis involves the collaboration of gut barrier components, such as goblet cells and IgA-microbiota complexes, that are under the control of stress that promotes inflammatory responses addressed primarily in the colon. The aim of this study was to evaluate the effect of stress on mucins, goblet cells, and proinflammatory parameters in the proximal and distal regions of the small intestine. A group (n = 6) of female 8-week-old BALB/c mice underwent board immobilization stress (2 h per day for 4 days) and were sacrificed with isoflurane. Samples from proximal and distal small segments were collected to analyze the following: 1) goblet cells stained with periodic acid-Schiff (PAS) and with alcian blue (AB) to visualize histologically neutral and acidic mucins, respectively; 2) IgA-microbiota complexes identified by flow cytometry in intestinal lavages; and 3) MUC2, MUC5AC, and IL-18 mRNA levels in whole mucosal scrapings by reverse transcription-qPCR. Regarding the unstressed group, in the proximal region of small intestine both PAS+ and AB+ goblet cells were unchanged; however, MUC5AC and IL-18 mRNA levels were increased, and the percentage of IgA-microbiota complexes was reduced. In the distal segment, the number of PAS+ goblet cells was increased, whereas the number of AB+ goblet cells was reduced and did not affect the remaining parameters. The data suggest that stress induces inflammation in the proximal small intestine; these findings may provide an experimental reference for human diseases that may affect the proximal small intestine, such as Crohn's disease, in which stress contributes to the progression of intestinal inflammation or relapse.

Bei Mu Gua Lou San facilitates mucus expectoration by increasing surface area and hydration levels of airway mucus in an air-liquid-interface cell culture model of the respiratory epithelium

BackgroundBei Mu Gua Lou San (BMGLS) is an ancient formulation known for its moisturizing and expectorant properties, but the underlying mechanisms remain unknown. We investigated concentration-dependent effects of BMGLS on its rehydrating and mucus-modulating properties using an air-liquid-interface (ALI) cell culture model of the Calu-3 human bronchial epithelial cell line and primary normal human bronchial epithelial cells (NHBE), and specifically focused on quantity and composition of the two major mucosal proteins MUC5AC and MUC5B.MethodsALI cultures were treated with BMGLS at different concentrations over three weeks and evaluated by means of histology, immunostaining and electron microscopy. MUC5AC and MUC5B mRNA levels were assessed and quantified on protein level using an automated image-based approach. Additionally, expression levels of the major mucus-stimulating enzyme 15-lipoxygenase (ALOX15) were evaluated.ResultsBMGLS induced concentration-dependent morphological changes in NHBE but not Calu-3 ALI cultures that resulted in increased surface area via the formation of herein termed intra-epithelial structures (IES). While cellular rates of proliferation, apoptosis or degeneration remained unaffected, BMGLS caused swelling of mucosal granules, increased the area of secreted mucus, decreased muco-glycoprotein density, and dispensed MUC5AC. Additionally, BMGLS reduced expression levels of MUC5AC, MUC5B and the mucus-stimulating enzyme 15-lipoxygenase (ALOX15).ConclusionsOur studies suggest that BMGLS rehydrates airway mucus while stimulating mucus secretion by increasing surface areas and regulating goblet cell differentiation through modulating major mucus-stimulating pathways.

Qufeng Xuanbi Formula inhibited benzo[a]pyrene-induced aggravated asthma airway mucus secretion by AhR/ROS/ERK pathway

Ethnopharmacological relevanceExcessive secretion of airway mucus may be an important pathological factor of air pollution-induced acute asthma attacks. Treatment of airway mucus hypersecretion improves asthma aggravated by air pollutants. Qufeng Xuanbi Formula (QFXBF) has been used to treat asthma for more than 30 years. However, whether QFXBF inhibits asthmatic mucus secretion exacerbated by air pollutants has not yet been established. Aim of the studyThis study aimed to evaluate the effect of QFXBF on airway mucus secretion and the mechanism of action in an air pollutant benzo[a]pyrene (BaP)-induced mouse model of aggravated asthma. Materials and methodsOvalbumin (OVA) and BaP co-exposure were used to establish the aggravated asthma model. The average enhanced pause (Penh), serum OVA-specific IgE, and changes in lung histopathology were determined. 16HBE cells exposed to BaP, treatment with QFXBF, arylhydrocarbon receptor (AhR) signal antagonist SR1, reactive oxygen species (ROS) antagonist NAC, or extracellular signal-regulated kinase (ERK1/2) signal antagonist U0126 were established to investigate the effect of QFXBF on BaP-induced mucus secretion and its target. The mRNA and protein expression levels of MUC5AC in the lung tissue and 16HBE cells were examined. We also studied the effect of QFXBF on ROS production. Finally, the protein expression of AhR, phospho-extracellular signal-regulated kinases (p-ERK1/2), and ERK1/2 in 16HBE cells and lung tissues was determined by western blotting. ResultsAdministration of QFXBF significantly alleviated the pathological symptoms, including Penh, serum OVA-specific IgE, and changes in lung histopathology in a BaP-induced mouse model of aggravated asthma. QFXBF inhibited MUC5AC expression in asthmatic mice and 16HBE cells exposed to BaP. ROS production, AhR expression, and ERK1/2 phosphorylation were significantly increased in BaP-induced asthmatic mice and 16HBE cells. Signaling pathway inhibitors StemRegenin 1 (SR1), NAC, and U0126 significantly inhibitedBaP-induced MUC5AC expression in 16HBE cells. SR1 reversed Bap-induced ROS production and ERK activation, and NAC inhibited Bap-induced ERK activation. In addition, QFXBF regulated AhR signaling, inhibited ROS production, reversed ERK activation, and downregulated mucus secretion to improve asthma aggravated by air pollutant BaP. ConclusionsQFXBF can ameliorate mucus secretion in BaP-induced aggravated asthmatic mice and 16HBE cells, and the specific mechanism may be related to the inhibition of the AhR/ROS/ERK signaling pathway.

MiR-193b-3p Regulates TLR4 Expression to Inhibit Inflammation by Targeting ETS1 in Allergic Rhinitis

Introduction: Allergic rhinitis (AR) is identified as a multifactorial disease caused by the interaction of genes and surroundings, which is difficult to cure. MicroRNAs were reported to be engaged in AR development. Here, we aimed to seek the anti-inflammatory effects and regulatory mechanism of miR-193b-3p in AR. Methods: Mucosal tissues from AR patients and healthy volunteers were collected, and human nasal epithelial cells (HNECs) were treated with IL-13 to establish a cell model of AR. The gene expression of miR-193b-3p, ETS1, TLR4, GM-CSF, eotaxin, and MUC5AC was determined by RT-qPCR. The protein levels of ETS1 and TLR4 were examined using Western blot. Enzyme-linked immunosorbent assay was performed to measure protein concentration of GM-CSF, eotaxin, and MUC5AC in cell supernatant. Dual luciferase assay was applied to verify the interaction among miR-193b-3p, ETS1, and TLR4. Results: The expression of miR-193b-3p was declined in clinical samples from AR patients and in IL-13-induced HNECs, while the mRNA and protein levels of ETS1 and TLR4 were elevated. MiR-193b-3p overexpression or ETS1 silencing notably decreased the mRNA and protein levels of GM-CSF, eotaxin, and MUC5AC in IL-13-treated HNECs. Mechanistically, miR-193b-3p directly combined with ETS1 to silence ETS1 expression. ETS1 promoted the transcriptional activity of TLR4 through interacting with TLR4 promoter. Furthermore, rescue experiments revealed that ETS1 overexpression abolished miR-193b-3p sufficiency-mediated suppression of the mRNA and protein levels of GM-CSF, eotaxin, and MUC5AC in IL-13-treated HNECs. Similarly, TLR4 overexpression compromised the inhibitory impacts of ETS1 downregulation on the mRNA and protein levels of GM-CSF, eotaxin, and MUC5AC in IL-13-induced HNECs. Discussion: MiR-193b-3p repressed IL-13-induced inflammatory response in HNECs by suppressing ETS1/TLR4 axis, which indicated that miR-193b-3p might be a therapeutic target for AR treatment.

Mollugin ameliorates murine allergic airway inflammation by inhibiting Th2 response and M2 macrophage activation

Mollugin, isolated from Rubia cordifolia L, is a pharmacological compound with anti-inflammatory activity. This study aimed to investigate whether mollugin protects mice against shrimp tropomyosin (ST)-induced allergic airway inflammation. Mice were sensitized with ST combined with Al(OH)3 administered intraperitoneally (i.p.) once weekly for 3 wk followed by ST challenge for 5 d. Mice were i.p.-administered daily with mollugin for 7 d. Results showed that mollugin attenuated ST-induced infiltration of eosinophils and epithelial mucus secretion in the lung tissues and suppressed lung eosinophil peroxidase activity. Additionally, mollugin lowered the Th2 cytokine, IL-4 and IL-5, production and downregulated the mRNA levels of Il-4, Il-5, Il-13, eotaxin, Ccl-17, Muc5ac, arginase-1, Ym-1, and Fizz-1 in the lung tissues. Network pharmacology was employed to predict core targets, and the molecular docking approach was used to verify the compound targets. The results of the molecular docking study of mollugin into p38 MAPK or poly(ADP-ribose) polymerase 1 (PARP1) binding sites revealed that its mechanism was possibly similar to that of SB203580 (a p38 MAPK inhibitor) or olaparib (a PARP1 inhibitor). Immunohistochemistry analysis revealed that mollugin mitigated ST-induced elevation of arginase-1 expression and macrophage levels in the lungs and bronchoalveolar lavage fluid, respectively. Furthermore, arginase-1 mRNA level and phosphorylation of p38 MAPK were inhibited in IL-4-stimulated peritoneal macrophages. In ST-stimulated mouse primary splenocytes, mollugin notably inhibited IL-4 and IL-5 production and downregulated PARP1 and PAR protein expression. According to our findings, mollugin ameliorated allergic airway inflammation by inhibiting Th2 response and macrophage polarization.

A systematic exploration of ginsenoside Rg5 reveals anti-inflammatory functions in airway mucosa cells

BackgroundHyperactivated airway mucosa cells overproduce mucin and cause severe breathing complications. Here, we aimed to identify the effects of saponins derived from Panax ginseng on inflammation and mucin overproduction. MethodsNCI–H292 cells were pre-incubated with 16 saponins derived from P. ginseng, and mucin overproduction was induced by treatment with phorbol 12-myristate 13-acetate (PMA). Mucin protein MUC5AC was quantified by enzyme-linked immunosorbent assay, and mRNA levels were analyzed using quantitative polymerase chain reaction (qPCR). Moreover, we performed a transcriptome analysis of PMA-treated NCI–H292 cells in the absence or presence of Rg5, and differential gene expression was confirmed using qPCR. Phosphorylation levels of signaling molecules, and the abundance of lipid droplets, were measured by western blotting, flow cytometry, and confocal microscopy. ResultsGinsenoside Rg5 effectively reduced MUC5AC secretion and decreased MUC5AC mRNA levels. A systematic functional network analysis revealed that Rg5 upregulated cholesterol and glycerolipid metabolism, resulting in the production of lipid droplets to clear reactive oxygen species (ROS), and modulated the mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathways to regulate inflammatory responses. Rg5 induced the accumulation of lipid droplets and decreased cellular ROS levels, and N-acetyl-l-cysteine, a ROS inhibitor, reduced MUC5AC secretion via Rg5. Furthermore, Rg5 hampered the phosphorylation of extracellular signal-regulated kinase and p38 proteins, affecting the NF-κB signaling pathway and pro-inflammatory responses. ConclusionRg5 alleviated inflammatory responses by reducing mucin secretion and promoting lipid droplet-mediated ROS clearance. Therefore, Rg5 may have potential as a therapeutic agent to alleviate respiratory disorders caused by hyperactivation of mucosa cells.

Structure, immunostimulatory activity, and the effect of ameliorating airway inflammation of polysaccharides from Pyrus sinkiangensis Yu

Purified acid polysaccharides PSAP-1 and PSAP-2 with apparent molecular weights of 64.6 and 38.9 kDa, respectively, were isolated from Pyrus sinkiangensis Yu. through combined techniques of ion-exchange and gel permeation chromatography. Both polysaccharides were composed of predominant amounts of GalA and small amounts of Ara, Rha, and Gal. They are deduced to be native pectin-type polysaccharides containing the HG backbone consisting of α-1,4-GalAp and methyl-esterified α-1,4-GalAp residues by IR, GC–MS and NMR spectra analyses. The immunoregulatory activity test showed that PSAP-1 and PSAP-2 could increase the cell viability and the release of NO, IL-6, and TNF-α on the RAW264.7 macrophage. It indicated that PSAP-1 and PSAP-2 could increase macrophage-mediated immunostimulatory activity. The airway inflammation test of antiasthmatic mice showed that PSAP-1 could decrease the contents of IL-4, IL-5, and IL-13 and the number of inflammatory cells in BALF and improve the pathological changes in lung tissue. PSAP-1 could also decrease the amount of mucus secreted by goblet cells and the expression levels of NF-κB p65, IκBα, IKK, ERK, JNK, P38, and Muc5ac mRNA and increase the expression levels of TLR2 and TLR4 mRNA in lung tissues. This suggested that PSAP-1 may resist airway inflammation in mice. PSAP-1 and PSAP-2 had potential clinical application value.

Guifu Dihuang Pills Ameliorated Mucus Hypersecretion by Suppressing Muc5ac Expression and Inactivating the ERK-SP1 Pathway in Lipopolysaccharide/Cigarette Smoke-Induced Mice.

Mucus hypersecretion is a hallmark of chronic obstructive pulmonary disease (COPD) and is associated with increasing sputum production and declining pulmonary function. Therefore, reducing mucus secretion can be a new therapeutic opportunity for preventing COPD. The Guifu Dihuang pill (GFDHP) is a classical Chinese medicine and has been used as an immunoregulator for treatment of kidney yang deficiency syndrome, including hypothyroidism, adrenocortical hypofunction, chronic bronchitis, and COPD, for more than 2000 years. However, the protective effects and mechanisms of GFDHP against mucus hypersecretion in COPD remain obscure. The aim of the present study was to explore the inhibitory effects of GFDHP on lipopolysaccharide/cigarette smoke- (LPS/CS-) induced Mucin5ac (Muc5ac) overproduction and airway goblet cell hyperplasia in mice. The mice were randomly assigned into 6 groups: control, model, GFDHP-L, GFDHP-M, GFDHP-H, and dexamethasone. The mice were given LPS twice through intranasal inhalation and then exposed to CS daily for 6 weeks. Three doses of GFDHP were orally administered daily during the last 3 weeks of the experiment. Pulmonary function was examined with an EMKA pulmonary system, and pulmonary hyperpermeability and lung damage were evaluated with an in vivo imaging system. Inflammatory cells and cytokines in bronchoalveolar lavage fluid (BALF) were detected with a cell count analyzer and though ELISA analysis, respectively. Lung pathological changes and airway goblet cell hyperplasia were analyzed with hematoxylin and eosin and Alcian blue periodic acid Schiff staining. The protein expression levels of Muc5ac and extracellular signal-regulated kinase (ERK)-specificity protein1 (SP1) signaling pathway were measured with Western blot and immunohistochemistry. The results demonstrated that GFDHP improved pulmonary function and suppressed mouse pulmonary hyperpermeability and edema. GFDHP suppressed inflammatory cell infiltration and cytokine release in BALF, thereby elevating pulmonary function. It ameliorated lung pathological changes and airway goblet cell hyperplasia, and suppressed expression levels of Muc5ac mRNA and protein and phospho-ERK and SP1 levels in the lung tissues of the COPD mice. In conclusion, GFDHP inhibited mucus hypersecretion induced by LPS/CS by suppressing the activation of the ERK-SP1 pathway.

Human Neutrophil Elastase Mediates MUC5AC Hypersecretion via the Tumour Necrosis Factor-α Converting Enzyme-Epidermal Growth Factor Receptor Signalling Pathway in vivo

Objectives: The objective of this study is to examine the role of the tumour necrosis factor-α converting enzyme-epidermal growth factor receptor (TACE-EGFR) pathway in human neutrophil elastase (HNE)-induced MUC5AC mucin expression in mice. Method: Four groups of mice, treated with HNE alone (HNE group), HNE plus TACE inhibitor (HNE + TAPI-2 group), HNE plus EGFR inhibitor (HNE + AG1478 group), and untreated (control group), were used in the experiment. Histopathological changes were monitored by haematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. TACE, EGFR, and MUC5AC expression in the nasal mucosa were determined using immunohistochemistry. The expression of p-EGFR, EGFR, and TACE protein was analysed on Western blots, and MUC5AC protein levels were assessed via ELISA. TACE, EGFR, and MUC5AC expression in the nasal mucosa were determined using real-time quantitative PCR. Results: Compared to the control group, HE-stained tissues from the HNE group showed an irregular epithelium as well as goblet cell and submucosal glandular hyperplasia. In the nasal mucosa, strongly positive fuchsia granules were seen in PAS staining and significant increases in TACE, EGFR, MUC5AC mRNA, and protein expression were detected (p < 0.01). The HNE + TAPI-2 and HNE + AG1478 groups had significantly less goblet cell and submucosal gland hyperplasia as well as weaker PAS staining. Compared to mice treated with HNE alone, in HNE + TAPI-2-treated mice, the levels of TACE, EGFR, and MUC5AC mRNA and protein as well as p-EGFR protein were significantly reduced (p < 0.01). In HNE + AG1478-treated mice, EGFR and MUC5AC mRNA and protein levels and p-EGFR protein expression were reduced significantly (p < 0.01), but the difference in TACE mRNA and protein expression between the HNE + AG1478 and HNE groups was not significant (p > 0.05). Conclusion: Using a newly developed, stable experimental model of nasal hypersecretion in mice, we showed that TAPI-2 or AG1478 inhibited HNE-induced MUC5AC production. This suggests that MUC5AC mucin expression in vivo is mediated by a cascade involving the HNE-TACE-EGFR signalling pathway.

Alterations in Mucin-Associated Gene Expression on the Ocular Surface in Active and Stable Stages of Atopic and Vernal Keratoconjunctivitis.

Purpose To evaluate the presence of ocular surface mucin in patients with atopic and vernal keratoconjunctivitis (AKC/VKC), we investigated the mRNA expression levels of SAM-pointed domain-containing ETS-like factor (SPDEF) and mucin-related genes on the ocular surface. Methods Nineteen patients with AKC or VKC were divided into two groups based on the severity of the disease as determined by their clinical scores for AKC/VKC: the stable group and the active group. Impression cytology was performed in all patients using filter paper, and the expression levels of SPDEF, MUC1, MUC4, MUC5AC, MUC16, and eotaxin-2 mRNA were determined by real-time reverse-transcription polymerase chain reaction. Results The results showed that the expression levels of SPDEF and MUC5AC mRNA in the active group were significantly decreased compared with those in the stable group. Furthermore, clinical scores were significantly negatively correlated with the expression levels of SPDEF mRNA and significantly positively correlated with the expression levels of eotaxin-2, which is a biomarker for eosinophilic inflammation on the ocular surface. Cluster analysis classified the patients with AKC/VKC into three clusters, and the stable group was divided into two clusters according to the condition of ocular surface mucin. Conclusions Ocular surface mucin in patients with AKC/VKC is altered in accordance with the clinical severity of the disease.

Antiasthmatic effect of atorvastatin via modulation of macrophage activation

Purpose: Asthma is a chronic airway inflammatory disorder and is associated with macrophages. Statin, a well-known lipid-lowering agent, has recently been noted for its anti-inflammatory effect on macrophage. This study was designed to evaluate the antiasth matic effect of atorvastatin via modulation of macrophage activation by using an animal model of allergic asthma. Methods: Atorvastatin 40 mg/kg was given by gavage once a day for 3 days before challenge of ovalbumin (OVA); airway hyperre -sponsiveness (AHR), airway inflammatory cells, and cytokines were evaluated in the murine asthma model. The direct effect of ator vastatin on the activation of macrophages in vitro was determined using the alveolar macrophage cell line CRL-2456. Results: Administration of atorvastatin reduced the numbers of total inflammatory cells, macrophages, and eosinophils as well as lung histology enhanced in the murine asthma model. AHR measured by enhanced pause was significantly reduced after atorvas tatin administration in the murine asthma model (P<0.05). Atorvastatin administration resulted in the reduction in serum OVA-spe cific IgE levels and the increase in serum OVA-specific IgG2a levels (P<0.05). The mRNA levels of Ccr3, Il-17, and Muc5ac enhanced by OVA challenge were decreased by treatment with atorvastatin (P<0.05). Along with these improvement in allergic inflammatory changes, the population of CD11c-CD206+ macrophages as well as the expression of Ym-1 and Relm-α in the lungs were reduced with atorvastatin (P<0.05). In vitro test with CRL-2456 showed that atorvastatin reduced the expression of Cd206, Arg-1, and Fgf-2 in duced by IL-4 stimulation (P<0.05). Conclusion: This study highlighted the antiasthmatic effect of atorvastatin on the suppression of M2 macrophage activation in al lergic asthma. (Allergy Asthma Respir Dis 2021;9:27-35)

Effect of Tobacco-specific Nitrosamines on MUC5AC Expression in Human Airway Epithelial Cells

Background and Objectives: Nicotine is oxidized into tobacco-specific nitrosamines (TSNAs; NAB, NAT, NNN, NNAL, NNK) at high temperature and high pressure. TSNAs are associated with airway diseases characterized by mucus hypersecretion as a major pathophysiologic phenomenon. The aim of study is to investigate the effect of TSNAs on mucin overexpression and its molecular mechanism in human airway epithelial cells.Materials and Method: The cytotoxicity of TSNAs was evaluated using EX-Cytox and inverted microscopy. The mRNA and protein levels of MUC5AC and MUC5B were measured using real-time PCR and ELISA.Results: NAB, NNN, NNAL, and NNK did not affect cell viability. NAT did not affect cell viability up to a concentration of 100 μM in human airway epithelial cells. NAT, NNN, NNAL, and NNK significantly induced MUC5AC expression, but not MUC5B expression. NAB did not affect the expression of MUC5AC and MUC5B. Propranolol (a β-adrenergic receptor antagonist) inhibited NAT, NNN, NNAL, and NNK-induced MUC5AC expression, whereas α-bungarotoxin (an α7-nicotinic acetylcholine receptor antagonist) only inhibited NNN- and NNK-induced MUC5AC expression.Conclusion: These results suggested that NAT, NNN, NNAL, and NNK induce MUC5AC expression through β-adrenergic receptor and/or α7-nicotinic acetylcholine receptor in human airway epithelial cells, which may be involved in mucus hypersecretion in inflammatory airway diseases.

Effects of dexamethasone on VEGF-induced MUC5AC expression in human primary bronchial epithelial cells: Implications for asthma

Mucins are major macromolecular components of lung mucus that are mainly responsible for the viscoelastic property of mucus. MUC5AC is a major mucin glycoprotein that is hypersecreted in asthmatic individuals. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Our previous studies indicate that VEGF upregulates MUC5AC expression by interacting with VEGF receptor 2 (VEGFR2). It has been shown that dexamethasone (Dex) downregulates MUC5AC expression; however, the underlying mechanisms have not been completely elucidated. Therefore, we sought to investigate the effect of Dex on MUC5AC expression induced by VEGF and study the underlying mechanisms. We tested the effects of Dex on VEGFR2 and RhoA activation, caveolin-1 expression, and the association of caveolin-1 and VEGFR2 in primary bronchial epithelial cells. Dex downregulated MUC5AC mRNA and protein levels in a dose- and time-dependent manner, and suppressed the activation of VEGFR2 and RhoA induced by VEGF. Additionally, Dex upregulated caveolin-1 protein levels in a dose- and time-dependent manner. Furthermore, phospho-VEGFR2 expression was decreased through overexpression of caveolin-1 and increased after caveolin-1 knockdown. Dex treatment attenuated the VEGF-decreased association of caveolin-1 and VEGFR2. Collectively, our findings suggest that Dex downregulates VEGF-induced MUC5AC expression by inactivating VEGFR2 and RhoA. Furthermore, decreased MUC5AC expression by Dex was related to the increased association of caveolin-1 with VEGFR2. Further studies characterizing these mechanisms are required to facilitate the development of improved treatment strategies for asthma.

Occupational effect of sugarcane biomass burning on the conjunctival mucin profile of harvest workers and residents of an adjacent town - A Brazilian panel study

Pre-harvest burning of sugarcane fields produces large amounts of air pollutants which are known to cause health problems, including ocular surface abnormalities. In this study, we evaluated the effect of biomass burning on mucus quality and mucin gene expression (MUC1, MUC5AC, MUC16) in the conjunctiva of sugarcane workers (SWs) and residents of an adjacent town (RTs). Impression cytology samples of the inferior tarsal and bulbar conjunctiva of 78 SWs and 32 RTs were collected before (T1) and immediately after (T2) a 6-month harvest period. The neutral, acid and total mucus content of goblet cells was determined by PAS and AB staining. The levels of MUC5AC, MUC1 and MUC16 mRNA in the conjunctiva were measured by real-time PCR. Compared to RTs, SWs had higher levels of bulbar acid mucus and MUC16 mRNA and tarsal MUC5AC mRNA at T2 and lower levels of neutral mucus at T1 and T2. In the SW group, MUC1 mRNA levels were higher at T2 than at T1, but the levels of neutral and acid mucus were similar. In the RT group, acid mucus decreased and neutral mucus increased in the bulbar and tarsal conjunctiva at T2. In conclusion, our findings show that sugarcane harvesting is associated with abnormalities in mucus quality and content and changes in mucin mRNA levels on the ocular surface. This may help explain the ocular inflammatory signs and symptoms observed in subjects exposed to air pollutants and high temperatures from sugarcane biomass burning.

Upregulation of MUC5AC by VEGF in human primary bronchial epithelial cells: implications for asthma

BackgroundAirway mucus hypersecretion is an important pathophysiological feature in asthma. Mucins are glycoproteins that are mainly responsible for the viscoelastic property of mucus, and MUC5AC is a major mucin glycoprotein that is overproduced in asthma. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Therefore, we sought to investigate the effect of VEGF on MUC5AC expression and study the underlying mechanisms.MethodsIn order to elucidate the precise mechanism underlying the effect of VEGF on MUC5AC expression, we tested the effects of VEGF on RhoA activation and the association of caveolin-1 and VEGFR2 in Primary Bronchial Epithelial Cells.ResultsVEGF up-regulated MUC5AC mRNA and protein levels in a dose- and time-dependent manner, and activated RhoA. Additionally, VEGF-induced MUC5AC expression and RhoA activation were enhanced by disrupting caveolae with cholesterol depletion and reversed by cholesterol repletion, and inhibited by a selective VEGF receptor 2 (VEGFR2) inhibitor SU1498. Furthermore, phospho-VEGFR2 expression was decreased via overexpression of caveolin-1. VEGF treatment reduced the association of caveolin-1 and VEGFR2.ConclusionCollectively, our findings suggest that VEGF up-regulates MUC5AC expression and RhoA activation by interaction with VEGFR2, and this phenomenon was related with the association of caveolin-1 and VEGFR2. Further studies on these mechanisms are needed to facilitate the development of treatments for asthma.

Human albumin augmented airway inflammation induced by PM2.5 in NC/Nga mice.

The synergic allergic inflammatory effects of particulate matter (PM) 2.5 and human albumin were investigated in NC/Nga mice, which are hypersensitive to mite allergens. PM2.5 or PM2.5 plus human albumin with aluminum oxide was injected twice intraperitoneally for sensitization. After 7 days, PM2.5 or PM2.5 plus human albumin was administered five times intranasally to mice for further sensitization. Subsequently, PM2.5 was administered as a challenge on the 11th day. On the 12th day, mice were examined for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, mRNA expression of Th1 , Th2 cytokines, chemokines, and mucus proteins (MUC5AC and MUC5B) in the lung tissue and histopathology. Although PM2.5 or human albumin alone did not induce allergic airway inflammation, simultaneous inoculation of PM2.5 and human albumin-induced airway inflammation showing increase in AHR, total BALF cell numbers, mRNA levels of IL-13, eotaxin 1, eotaxin 2, and MUC5AC, and anti-IG against human serum albumin. Inflammation was observed around the bronchus in PM2.5 plus human albumin-induced lungs. These results demonstrate that PM2.5 can induce allergic airway inflammation through the synergistic action with human albumin in NC/Nga mice.

Polyhexamethylene guanidine phosphate-induced upregulation of MUC5AC via activation of the TLR-p38 MAPK and JNK axis

Epidemiological and toxicological studies indicate that polyhexamethylene guanidine phosphate (PHMG-p) is a guanidine-based cationic disinfectant strongly associated with interstitial lung diseases. As individuals exposed to aerosolized PHMG-p complain of respiratory problems (asthma and rhinitis), whether PHMG-p can cause respiratory diseases other than interstitial fibrosis should be investigated. MUC5AC, the predominant mucin gene expressed in airways, is associated with obstructive respiratory disease pathogenesis. Therefore, in this study, we elucidated the relationship between PHMG-p and MUC5AC overexpression. First, in immunofluorescence studies, the bronchial epithelia of mice intratracheally administrated PHMG-p appeared to be sloughing and tethered by MUC5AC. Second, Calu-3 cells exposed to PHMG-p showed concentration-dependent increases in MUC5AC mRNA and protein expression. c-Jun N-terminal kinase (JNK), p38, and c-jun were phosphorylated in cells exposed to PHMG-p. SP600125 and SB203580, JNK and p38 inhibitors, respectively, reduced the upregulation of MUC5AC by PHMG-p in Calu-3 cells. When toll-like receptor (TLR)2, 4, and 6 were silenced, PHMG-p-induced JNK and p38 phosphorylation decreased. Furthermore, TLR2-, 4-, and 6-silenced cells showed reduced levels of MUC5AC mRNA and protein induced by PHMG-p, with TLR6 knockdown showing the greatest effect. In conclusion, PHMG-p induced MUC5AC overexpression via activation of the TLR-p38 MAPK and JNK axis.

Modulation of intestinal epithelial permeability and mucin mRNA (MUC2, MUC5AC, and MUC5B) expression and protein secretion in Caco-2/HT29-MTX co-cultures exposed to aflatoxin M1, ochratoxin A, and zearalenone individually or collectively

Aflatoxin M1 (AFM1), ochratoxin A (OTA), and zearalenone (ZEA) are mycotoxins commonly found in milk. Mycotoxin contamination has caused food safety concerns worldwide since most of the toxic effects in humans are serious. The combined toxic effects of these mycotoxins on intestinal epithelial cells have not been reported. Herein, we investigated the combined effects of AFM1, OTA, and ZEA on intestinal integrity and define the underlying mechanisms(s) of their effects in Caco-2/HT29-MTX co-cultures. Our results showed that the mixtures of AFM1 + OTA, AFM1 + ZEA, and AFM1 + ZEA + OTA significantly increased epithelial permeability. Immunofluorescence analysis and transmission electron microscopy revealed that mycotoxins altered TJ proteins morphology and disrupted their structures. Also, the present study showed that mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA levels and protein secretion. This study demonstrated that the effects of mixtures of mycotoxins on intestinal barrier function were more significant than AFM1 alone. More importantly, the damage of intestinal integrity caused by mycotoxins was correlated to the change of the TJ proteins location and the decrease of mucin secretion. Mixtures of AFM1, OTA, and ZEA in food might pose a health risk to consumers, particularly in children, and toxin risks should be considered.

Srolo Bzhtang, a traditional Tibetan medicine formula, inhibits cigarette smoke induced airway inflammation and muc5ac hypersecretion via suppressing IL-13/STAT6 signaling pathway in rats

Ethnopharmacological relevanceSrolo Bzhtang (SBT), a traditional Tibetan medicine formula, was composed of three herbs, Solms-Laubachia eurycarpa, Bergenia purpurascens, Glycyrrhiza uralensis, and one lac, and was first documented in the ancient Tibetan medical work Four Medical Tantras (rGyud-bzhi) in the eighth century AD. It has been widely used to treat lung “phlegm-heat” syndromes such as chronic bronchitis and chronic obstructive pulmonary disease (COPD). ObjectiveThe aim of this study was to evaluate the potential influences of aqueous extract of SBT on airway inflammation and mucus secretion and to reveal the underlying mechanism in a rat model of cigarette smoke (CS)-induced chronic bronchitis (CB). Materials and methodsSixty male Sprague-Dawley rats were randomly divided to six groups: control (room air exposure), model (CS exposure), DEX (CS exposure and 0.2 mg/kg/day dexamethasone), and three SBT (CS exposure and 1.67, 2.50, and 3.34 g/kg/day SBT) groups. DEX and the three doses of SBT were administered by oral gavage every day for eight weeks. Pathological changes and mucus expression in the lung tissue were determined by hematoxylin and eosin (H&E), Alcian blue-periodic acid-Schiff (AB-PAS) and immunohistochemical staining. The levels of cytokines in bronchoalveolar lavage fluid (BALF) were assessed by ELISA. Western blot analysis and qRT-PCR were performed to explore the effects of SBT on the expression of IL-13, STAT6 and MUC5AC. ResultsPretreatment with SBT attenuated the TNF-α, IL-8, IL-13 expression levels in BALF and the inflammatory cell infiltration in bronchial walls and peribronchial lung tissue. SBT exhibited a dose-dependent downregulation of MUC5AC expression as assessed by AB-PAS and immunohistochemical staining. The protein and mRNA levels of IL-13, STAT6/p-STAT6 and MUC5AC were also downregulated by SBT preconditioning. ConclusionThese results for the first time demonstrated that SBT exhibited protective effects on CS-induced airway inflammation and MUC5AC hypersecretion, which might be related to the downregulation of the IL-13/STAT6 signaling pathway.