Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

Panax notoginseng saponin R1 modulates TNF-α/NF-κB signaling and attenuates allergic airway inflammation in asthma.

BACKGROUD: Panax notoginseng saponin R1 (PNS-R1) is one of the most important chemical monomers derived from the panax notoginseng, and our previous study found that PNS-R1 reduced glucocorticoid-induced apoptosis in asthmatic airway epithelial cells. Thus, in this study, we explored the effects of the PNS-R1 on inflammation of allergic asthma. METHODS: The asthmatic mice were administered 15 mg/kg PNS-R1 by intraperitoneal injection three days before sensitized to OVA. The effects of PNS-R1 on asthmatic mice were detected by airway hyperresponsiveness, inflammation, mucus hypersecretion and inflammatory cytokines such as interleukin (IL)-13, IL-4, IL-5, IL-8 and tumor necrosis factor (TNF)-α were studied. We also treated human bronchial epithelial cells (16HBE) with house dust mites (HDM) and then detected the secretion of cellular inflammatory factors (IL-13 and TNF-α). Western blot and immunofluorescence were used to examine the effect of PNS-R1 on TNF-α/NF-κB pathway. TNF-α/NF-κB/IKK signal pathway activator was used in PNS-R1-treated asthmatic mice. RESULTS: PNS-R1 significantly reduced the airway inflammatory, mucus secretion and hyperresponsiveness in asthma model. It also reduced the levels of IL-13, IL-4, IL-5 and IL-8 in bronchoalveolar lavage fluid (BALF) and IgE and OVA-specific IgE in serum for asthma mice. PNS-R1 reduced IL-13 and TNF-α secretion in HDM-treated 16HBE cells. In addition, PNS-R1 suppressed TNF-α/NF-κB pathway in both asthmatic mice and 16HBE. Activation of NF-kB pathway reversed the therapeutic effect of PNS-R1 on asthmatic mice. CONCLUSION: The results indicated that PNS-R1 effectively suppresses allergic airway inflammation of asthma partly through TNF-α/NF-κB pathway. PNS-R1 may play a potential role in allergic asthma treatment in the future.

Lipopolysaccharide induces steroid-resistant exacerbations in a mouse model of allergic airway disease collectively through IL-13 and pulmonary macrophage activation.

BACKGROUND: Acute exacerbations of asthma represent a major burden of disease and are often caused by respiratory infections. Viral infections are recognized as significant triggers of exacerbations; however, less is understood about the how microbial bioproducts such as the endotoxin (lipopolysaccharide (LPS)) trigger episodes. Indeed, increased levels of LPS have been linked to asthma onset, severity and steroid resistance. OBJECTIVE: The goal of this study was to identify mechanisms underlying bacterial-induced exacerbations by employing LPS as a surrogate for infection. METHODS: We developed a mouse model of LPS-induced exacerbation on the background of pre-existing type-2 allergic airway disease (AAD). RESULTS: LPS-induced exacerbation was characterized by steroid-resistant airway hyperresponsiveness (AHR) and an exaggerated inflammatory response distinguished by increased numbers of infiltrating neutrophils/macrophages and elevated production of lung inflammatory cytokines, including TNFα, IFNγ, IL-27 and MCP-1. Expression of the type-2 associated inflammatory factors such as IL-5 and IL-13 were elevated in AAD but not altered by LPS exposure. Furthermore, AHR and airway inflammation were no longer suppressed by corticosteroid (dexamethasone) treatment after LPS exposure. Depletion of pulmonary macrophages by administration of 2-chloroadenosine into the lungs suppressed AHR and reduced IL-13, TNFα and IFNγ expression. Blocking IL-13 function, through either IL-13-deficiency or administration of specific blocking antibodies, also suppressed AHR and airway inflammation. CONCLUSIONS & CLINICAL RELEVANCE: We present evidence that IL-13 and innate immune pathways (in particular pulmonary macrophages) contribute to LPS-induced exacerbation of pre-existing AAD and provide insight into the complex molecular processes potentially underlying microbial-induced exacerbations.

[Effect of methyleugenol on expression of MUC5AC in nasal mucosa of rats with allergic rhinitis].

Objective: To investigate the effect of methyleugenol on expression of MUC5AC in nasal mucosa of rats with allergic rhinitis (AR). Methods: Seventy-two Wistar rats were randomly divided into 6 groups:normal control group, AR group, loratadine group, low-dose methyleugenol group, middle-dose methyleugenol group and high-dose methyleugenol group with 12 rats in each group. AR was induced by intraperitoneal injection of ovalbumin in latter 5 groups. 10 mg loratadine q.d was given to rats in loratadine group by gavage; and 10 mg/kg, 20 mg/kg and 40 mg/kg methyleugenol were given by gavege q.d to rats in low-, middle-and high-dose methyleugenol groups, respectively. Nasal mucosa samples were obtained from rats at 1, 2, 4 and 6 weeks after drug intervention. The expression of MUC5AC protein and mRNA in nasal mucosa was detected by immunohistochemistry and real-time fluorescence quota PCR (RT-PCR), respectively. Results: Compared with AR, the percentage of cells staining positively for MUC5AC protein and the relative quantity of MUC5AC mRNA in middle-and high-dose methyleugenol groups were significantly decreased after 2 and 4 weeks of drug intervention (P<0.05), but no such decrease was observed in low-dose methyleugenol group at all time points (P>0.05). The percentage of cells with positive expression of MUC5AC protein and mRNA in loratadine group were significantly decreased after 1 week of administration (P<0.05). The percentage of cells with positive MUC5AC protein in middle-dose methyleugenol group was higher than that in loratadine group (P<0.05) after 6 week of drug intervention, but the difference was not seen in high-dose group (P>0.05). There was no significant difference in relative quantities of MUC5AC mRNA after 4 weeks of administration between high-and middle-dose methyeugenol groups and loratadine group (P>0.05). Conclusion: Methyleugenol can attenuate AR through inhibiting the expression of MUC5AC mRNA and protein in nasal mucosa of AR rats.

Anti-inflammatory effects of a novel non-antibiotic macrolide, EM900, on mucus secretion of airway epithelium.

OBJECTIVE: Low-dose, long-term use of 14-membered macrolides is effective for treatment of patients with chronic airway inflammation such as diffuse panbronchiolitis or chronic rhinosinusitis. However, long-term use of macrolides can promote the growth of drug-resistant bacteria, and the development of anti-inflammatory macrolides that lack antibiotic effects is desirable. Previously, we developed EM900, a novel 12-membered erythromycin A derivative, which has potent anti-inflammatory and immunomodulatory activities and lacks any antibacterial activity. We examined the anti-inflammatory effects of EM900 on mucus secretion from airway epithelial cells. METHODS: To examine the in vivo effects of EM900 on airway inflammation, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium via intranasal instillation of lipopolysaccharides. In vitro effects of EM900 on airway epithelial cells were examined using cultured human airway epithelial (NCI-H292) cells. Mucus secretion was evaluated via enzyme-linked immunosorbent assays with an anti-MUC5AC monoclonal antibody. RESULTS: Oral administration of EM900 or clarithromycin (CAM) significantly inhibited LPS-induced mucus production from rat nasal epithelium. EM900, CAM, or erythromycin significantly inhibited MUC5AC secretion induced by tumor necrosis factor-α from NCI-H292 cells. MUC5AC mRNA expression was also significantly lower in EM900-treated cells. CONCLUSION: These results indicated that a novel non-antibiotic macrolide, EM900 exerted direct inhibitory effects on mucus secretion from airway epithelial cells, and that it may have the potential to become a new anti-inflammatory drug for the treatment of chronic rhinosinusitis.

Crude ethanolic extracts of Zingiber cassumunar ROXB. inhibit PMA-induced MUC2 and MUC5AC expression via ERK inhibition in human airway epithelial cells.

BACKGROUND: Over expression of mucin often leads to serious airway pathologies. The rhizome of Zingiber cassumunar Roxb. ("Phlai" in Thai) has been used as an anti-asthmatic drug in Thai traditional medicine. However, the effect of this plant on mucin production has not been reported. OBJECTIVE: The aim of the present study was to investigate whether crude ethanolic extracts of Zingiber cassumunar (CEZE) suppress phorbol12-myristate 13-acetate (PMA)-induced mucin production and gene expression in human airway epithelial cells and if so, to examine whether the suppression of mucin gene expression is mediated via the mitogen-activated protein kinase (MAPK) signal transduction pathways. METHODS: Confluent NCI-H292 cells were pretreated with CEZE for 2 hours and then stimulated with 100 or 200 nmol/l PMA for 8 h. The levels of MUC2 and MUC5AC mRNA were determined by RT-PCR and real-time PCR. Levels of total mucin; MUC2 and MUC5AC inculture supernatants were measured using ELLA and ELISA assays, respectively. Extracellular signal-regulated kinase (ERK), JNK, p38 MAPK protein levels were analyzed by Western blotting. RESULTS: CEZE (5-100?g/ml) significantly inhibited total mucin production, including MUC2 and MUC5AC mRNA and proteins induced by PMA in NCI-H292 cells. The extracts obviously inhibited the phosphorylation of ERK, but not JNK and p38 in PMA-stimulated NCI-H292 cells. Our results suggest that Z. cassumunar-mediated suppression of PMA-induced MUC2 and MUC5AC mRNA operates via ERK inhibition. CONCLUSION: Z. cassumunar suppresses PMA-induced MUC2 and MUC5AC gene expression in human airway epithelial cells via inhibition of ERK MAPK-dependent pathway.

Phase IIA trial testing erlotinib as an intervention against intraductal pancreatic mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMN) are a distinct type of precursor lesions that cause about 5% of pancreatic adenocarcinoma (1, 2). Four-year survival rates of 40% to 75% are reported (3-7). There is currently no chemotherapy specifically approved for treating IPMNs. The number of IPMN cases has significantly increased over the last decade (8).

Oleanolic acid and ursolic acid derived from Cornus officinalis Sieb. et Zucc. suppress epidermal growth factor- and phorbol ester-induced MUC5AC mucin production and gene expression from human airway epithelial cells.

In this study, the effects of oleanolic acid and ursolic acid on MUC5AC mucin production and gene expression induced by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) from human airway epithelial cells were investigated. Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with EGF and PMA for 24 h, respectively. MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA. Oleanolic acid and ursolic acid were found to inhibit the production of MUC5AC mucin protein induced by EGF and PMA, and both compounds also inhibited the expression of MUC5AC mucin gene induced by EGF and PMA. These results suggest that oleanolic acid and ursolic acid can regulate mucin gene expression, and production of mucin protein, by directly acting on airway epithelial cells.

Inhibition of neutrophil elastase-induced goblet cell metaplasia by tiotropium in mice.

Airway occlusion by mucus in chronic obstructive disease (COPD) is associated with a poor prognosis. We hypothesised that tiotropium has the ability to inhibit neutrophil elastase (NE)-induced goblet cell metaplasia in mice and mucin production in vitro. On days 1, 4, and 7, tiotropium or vehicle was administered to C57BL/6 mice by inhalation and they were allowed to intranasally aspirate human NE. Bronchoalveolar lavage fluid (BALF) and lung sections were analysed on days 8, 11 and 14. The effect of late administration of tiotropium on the goblet cell metaplasia by NE aspiration was also assessed. NE-induced MUC5AC production by NCI-H292 cells was measured with ELISA. Repeated NE aspiration induced marked goblet cell metaplasia. The grading of goblet cell metaplasia, neutrophil count and eosinophil count in BALF, keratinocyte-derived chemokine level and leukotriene B(4) level in BALF, and M(3) receptor expression by immunohistochemistry, were lower in the tiotropium group than in the vehicle group. Late administration of tiotropium inhibited the established goblet cell metaplasia. Tiotropium inhibited NE-induced MUC5AC production. Tiotropium inhibited NE-induced goblet cell metaplasia and mucin production, probably mediated by suppression of inflammation and a direct action on epithelial cells. This result suggests that tiotropium may be useful for the treatment of mucus overproduction in COPD.

[6]-Gingerol suppresses interleukin-1 beta-induced MUC5AC gene expression in human airway epithelial cells.

BACKGROUND: [6]-Gingerol is a major active component of ginger and a natural polyphenol compound. The present study investigated whether [6]-gingerol suppresses interleukin (IL)-1 beta-induced MUC5AC gene expression in human airway epithelial cells and, if so, examined whether the suppression of MUC5AC gene expression is mediated via the mitogen-activated protein kinase (MAPK) signal transduction pathway. METHODS: MUC5AC mRNA and protein were measured using reverse transcription-polymerase chain reaction (PCR), real-time PCR, and Western blot analysis in cultured NCI-H292 human airway epithelial cells. Extracellular signal-regulated kinase (ERK) and p38 MAPK protein levels were analyzed by Western blot. RESULTS: Expression of MUC5AC mRNA increased in NCI-H292 cells upon treatment with 10 ng/mL of IL-1 beta for 24 hours. When the cells were pretreated with 10 microM of [6]-gingerol, expression of IL-1 beta-induced MUC5AC mRNA and protein was significantly suppressed. Suppression of IL-1 beta-induced MUC5AC mRNA was also observed in cells pretreated with ERK- or p38 MAPK-specific inhibitors, suggesting that [6]-gingerol-mediated suppression of IL-1 beta-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathways. CONCLUSIONS: [6]-Gingerol suppresses IL-1 beta -induced MUC5AC gene expression in human airway epithelial cells via the ERK- and p38 MAPK-dependent pathways; therefore, [6]-gingerol may be considered a possible anti-hypersecretory agent.

L-carbocisteine reduces neutrophil elastase-induced mucin production.

Human neutrophil elastase (HNE) exists in high concentrations in airway secretions and produces mucus hypersecretion in patients with chronic obstructive pulmonary disease (COPD). L-carbocisteine improves the quality of life and reduces exacerbation in COPD patients. However the precise mechanism is uncertain. We examined the effects of L-carbocisteine on HNE-induced mucus hypersecretion and on the production of reactive oxygen species (ROS) which is associated with mucin production induced by HNE. NCI-H292, a human lung mucoepidermoid carcinoma cell line, was treated with or without HNE and L-carbocisteine. MUC5AC mRNA expression and ROS production in the cells, and MUC5AC protein concentration in supernatants were measured. HNE increased MUC5AC mRNA expression and MUC5AC protein concentration in supernatants in the cells. L-carbocisteine reduces HNE-induced mRNA expression and protein secretion of MUC5AC. L-carbocisteine also reduced ROS production in the cells induced by HNE. Reduction of HNE-induced mucus secretion by L-carbocisteine in the pulmonary epithelial cells may partly relate to the reduction of ROS.