The Expression and Regulation of Na+-K+-ATPase in Nasal Epithelial Cells of Chronic Rhinosinusitis with Nasal Polyps.

OBJECTIVE: Na+-K+-ATPase (NKA) is essential in maintaining cell permeability, reserving potential energy, and preventing cellular edema. Nevertheless, how NKA expression is altered and regulated in chronic rhinosinusitis with nasal polyps (CRSwNPs) remain uncertain. Therefore, the present study aimed to explore the expression and regulation of NKA in CRSwNP. METHODS: NKA immunolabeling was assessed by the immunohistochemistry method, NKA protein levels were detected with the Western blotting method, and mRNA levels of NKA and aquaporin-5 (AQP5) were assayed by real-time PCR in nasal tissues from CRSwNP and control subjects. The co-localization of NKA with inflammatory cells was evaluated by immunofluorescence staining. In addition, human nasal epithelial cells (HNECs) were cultured and stimulated using various stimulators to evaluate the regulation of NKA. RESULTS: We found significantly decreased NKA positive cells, NKA protein levels, and mRNA levels of NKA and AQP5 in nasal tissues from CRSwNP patients compared to control subjects, especially in eosinophilic CRSwNP. Furthermore, NKA mRNA levels in HNECs were downregulated by staphylococcal enterotoxin B (SEB), lipopolysaccharides (LPSs), inflammatory cytokine (IFN)-γ, IL-4, IL-13, and IL-1ß. CONCLUSION: NKA and AQP5 expressions were decreased in CRSwNP. NKA in HNECs could be suppressed by SEB, LPS, IFN-γ, IL-4, IL-13, and IL-1ß. Impairment of NKA may contribute to the genesis and development of CRSwNP via inducing AQP5 downregulation and edema.

Therapeutic effects of SKF-96365 on murine allergic rhinitis induced by OVA.

INTRODUCTION: SKF-96365 is regarded as an inhibitor of receptor-mediated calcium ion (Ca2+) entry. The current study aimed to explore the effects of SKF-96365 on murine allergic rhinitis (AR). METHODS: Intranasal SKF-96365 administration was performed on OVA induced murine AR. Serum and nasal lavage fluid (NLF) from mice were harvested to assay IgE and inflammatory cytokines using ELISA method. Inflammatory cells were counted and analyzed in NLF. Nasal mucosa tissues were collected from mice and used for HE staining, immunohistochemistry (IHC) staining, and real-time PCR detection. RESULTS: SKF-96365 had therapeutic effects on murine AR manifesting attenuation of sneezing, nasal rubbing, IgE, inflammatory cytokines, inflammatory cells, TRPC6 immunolabeling, and TRPC6, STIM1 and Orai1 mRNA levels in AR mice. CONCLUSION: SKF-96365 could effectively alleviate the symptoms of murine AR. SKF-96365 could suppress TRPC6, STIM1, and Orai1 activities, leading to the downregulation of inflammatory cytokines and inflammatory cells in murine AR.

RGFP966, a selective HDAC3 inhibitor, ameliorates allergic and inflammatory responses in an OVA-induced allergic rhinitis mouse model.

RGFP966 is a selective inhibitor of histone deacetylase 3 (HDAC3) playing crucial roles in triggering allergic and inflammatory responses. Whereas, its role in allergic rhinitis (AR) remains uncertain. This study sought to illustrate the role and mechanism of HDAC3 inhibitor RGFP966 on allergic and inflammatory responses in murine AR. RGFP966 administration was applied on murine AR. HE staining, PAS staining, toluidine blue staining, immunohistochemistry staining and real-time PCR methods were used to assess eosinophils, goblet cells, mast cells, HDAC3 positive cells and mRNA levels in nasal tissues of mice. HDAC3 activities in nasal tissues were quantified with HDAC3 Activity Assay Kit. We collected blood and nasal lavage fluid (NLF) of mice for assaying IgE, inflammatory cytokines and inflammatory cells. Results indicated that RGFP966 intervention attenuated sneezing, nose rubbing, IgE, inflammatory cytokines, eosinophils, goblet cells, mast cells, inflammatory cells, HDAC3 levles and activities in RGFP966 treated mice. In conclusion, RGFP966 might reduce HDAC3 expression and HDAC3 activities, and then eosinophils and mast cells recruitment, goblet cells proliferation and inflammatory cytokines levels are decreased, resulting in the alleviation of allergic and inflammatory responses in AR mice.

Evidence for role of acid-sensing ion channel 1a in chronic rhinosinusitis with nasal polyps.

PURPOSE: A variety of inflammatory cells are infiltrated histologically in sinonasal mucosa of chronic rhinosinusitis with nasal polyps (CRSwNP), especially CRSwNP with asthma. Acid-sensing ion channel 1a (ASIC1a) is essential in the process of sensing acidification and triggering inflammation. Whereas, its role and mechanism in CRSwNP remain uncertain. The present study aimed to explore the roles and mechanism of ASIC1a in the pathogenesis of CRSwNP. METHODS: Nasal secretions from control subjects, patients with CRSwNP with or without asthma were collected for measuring pH values. Western blotting, real-time PCR and immunohistochemistry (IHC) were employed to assess ASIC1a expression in nasal tissue samples from included subjects. The co-localization of ASIC1a with inflammatory cells was evaluated by immunofluorescence staining. Then, dispersed nasal polyp cells (DNPCs) were cultured under acidified condition (pH 6.0), with or without ASIC1a inhibitor amiloride. Western blotting, real-time PCR, LDH activity kit, and ELISA were performed to assess the effects and mechanisms of stimulators on the cells. RESULTS: The pH values were significantly lower in the nasal secretions from patients with CRSwNP with asthma. Significant upregulation of ASIC1a protein, mRNA levels, and positive cells was found in CRSwNP with asthma. ASIC1a was detected in a variety of inflammatory cells. In cultured DNPCs, significant alterations of ASIC1a levels, LDH activity, HIF-1α levels, and inflammatory cytokines were found under acidified condition (pH 6.0), but were prevented by amiloride. CONCLUSION: Upregulation of ASIC1a might be essential in the process of sensing acidification and triggering inflammatory response via enhancing HIF-1α expression and LDH activity to activate inflammatory cells in the pathogenesis of CRSwNP, especially in CRSwNP with asthma.

Increased Expression of TXNIP Facilitates Oxidative Stress in Nasal Epithelial Cells of Patients With Chronic Rhinosinusitis With Nasal Polyps.

BACKGROUND: Oxidative stress plays crucial roles in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Thioredoxin-interacting protein (TXNIP) is essential in the process of triggering oxidative stress. However, its role and mechanism in CRSwNP remain unclear. The present study sought to explore the role and mechanism of TXNIP in the pathogenesis of CRSwNP. METHODS: Western blotting, real-time PCR and immunohistochemistry (IHC) were employed to assess TXNIP, thioredoxin (TRX) expression in nasal tissue samples from patients with CRSwNP and control subjects. MDA level and SOD activity in nasal tissue homogenates were measured using MDA and SOD Assay Kit. To evaluate the role and mechanism of TXNIP in CRSwNP, human nasal epithelial cells (HNECs) were cultured and stimulated using TXNIP siRNA, with or without N-acetylcysteine (NAC, an ROS scavenger). Western blotting, real-time PCR, ROS detecting dye DCFH-DA, MDA and SOD Assay Kit were performed to assess the effects and mechanisms of stimulators on the cells. RESULTS: We found significantly increased levels of TXNIP and decreased levels of TRX protein, mRNA, positive cells, increased MDA level and decreased SOD activity in CRSwNP patients compared with control subjects. In vitro study, significantly altered levels of TXNIP, TRX, MDA, SOD and ROS in HNECs were found following treatment of TXNIP siRNA with or without NAC on HNECs. CONCLUSION: TXNIP expression was increased and TRX expression was decreased in CRSwNP at both protein and mRNA levels. MDA levels were increased and SOD activities were decreased in CRSwNP. TXNIP may have negative association with TRX, and then decrease SOD activities and increase MDA levels, resulting in the upregulation of ROS and oxidative stress in HNECs, which may play a pivotal role in the pathogenesis of CRSwNP. Future studies are expected to further explore the role and mechanism of TXNIP in CRSwNP.

Anti-allergic and anti-inflammatory effects of resveratrol via inhibiting TXNIP-oxidative stress pathway in a mouse model of allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is a type I hypersensitivity mediated by IgE in the nose. Thioredoxin-interacting protein (TXNIP) plays a pivotal role in the process of producing reactive oxygen species (ROS). Resveratrol is a TXNIP inhibitor. Nonetheless, its role and mechanism in AR are still undetermined. The present study aimed to explore the effect and mechanism of resveratrol on an ovalbumin (OVA) induced mouse model of AR. METHODS: AR murine model was established using OVA and administrated intranasally with resveratrol or N-acetylcysteine (NAC). Hematoxylin and eosin (HE) stain was used for evaluating eosinophils. Immunohistochemistry (IHC) staining and real-time PCR were employed to evaluate immunolabeling and mRNA expression of TXNIP in nasal mucosas of mice. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in nasal tissue homogenates were measured using MDA and SOD Assay Kit. Concentrations of OVA-specific IgE and histamines in serum, and OVA-specific IgE, PGD2, LTC4, ECP, IL-4, IL-5, IL-6, IL-33 and TNF-α in nasal lavage fluid (NLF) were assayed by ELISA. In vitro studies, western blotting, real-time PCR, ELISA, ROS detecting dye DCFH-DA, MDA, and SOD Assay Kit were performed to evaluate the effects and mechanisms of OVA, resveratrol or NAC on spleen mononuclear cells. RESULTS: We found significant alternations of sneezing, nasal rubbing, inflammatory cytokines, eosinophil numbers, TXNIP, MDA, and SOD levels in resveratrol or NAC treated mice compared with untreated AR mice. In cultured spleen mononuclear cells, TXNIP, MDA, SOD, ROS and inflammatory cytokines levels were altered by OVA but reversed by resveratrol or NAC. CONCLUSIONS: Resveratrol could effectively alleviate murine AR by inhibiting TXNIP-oxidative stress pathway.

Ameliorative effect of selective NLRP3 inflammasome inhibitor MCC950 in an ovalbumin-induced allergic rhinitis murine model.

Allergic rhinitis (AR) is a complex IgE-mediated nasal allergic and inflammatory disease. Nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) is essential in the process of allergic and inflammatory responses. MCC950 is a selective NLRP3 inhibitor. However, its role and mechanism in AR remains undetermined. The present study aimed to explore the effect and mechanism of MCC950 on an ovalbumin (OVA) induced mouse model of AR. The AR BALB/c mice were constructed using OVA and administrated intranasally with MCC950. Concentrations of OVA-specific IgE, histamines and leukotrienes C4 (LTC4) in serum, and OVA-specific IgE, ECP, IFN-γ, IL-4, IL-5, IL-13, IL-1ß and IL-18 in nasal lavage fluid (NLF) were assayed by enzyme-linked immunosorbent assay (ELISA). Inflammatory cells were counted in NLF. HE and PAS staing were used for evaluating eosinophils and goblet cells. Immunohistochemistry (IHC) staining were employed to evaluate immunolabeling of NLRP3, Caspase-1, ASC, IL-1ß and IL-18 in nasal mucosas of mice. Real-time PCR was conducted to assay NLRP3, Caspase-1, ASC, IL-1ß and IL-18 mRNA levels. In vitro studies, western blotting, real-time PCR and ELISA were performed to evaluate the effects and mechanisms of OVA and NLRP3 inhibitor MCC950 on spleen mononuclear cells. We found significant downregulation of sneezing, nasal rubbing, inflammatory cytokines, inflammatory cells and NLRP3, Caspase-1, ASC, IL-1ß and IL-18 expression in MCC950 treated mice compared with untreated AR mice. In spleen mononuclear cells culture and stimulation experiment, NLRP3, Caspase-1, ASC, IL-1ß and IL-18 levels were upregulated by OVA but inhibited by MCC950. In conclusion, MCC950 could effectively exert its ameliorative effect in murine AR by inhibiting NLRP3 and leads to reduction of Caspase-1, ASC, IL-1ß and IL-18, resulting in the attenuation of the allergic and inflammatory responses.

A Novel Heterozygous Mutation of the COL4A3 Gene Causes a Peculiar Phenotype without Hematuria and Renal Function Impairment in a Chinese Family.

Mutations in the COL4A3 gene are frequently reported to be associated with various types of hereditary nephropathy. COL4A3 encodes the α3 chain of type IV collagen, which is the main structural protein in the basement membrane. Mutations in this gene are always related to kidney performance, and deafness and ocular lesion have also been reported. In this study, using next-generation sequencing, we investigated the DNA of a family visiting a clinic for hearing loss. A new missense mutation was found in COL4A3 of 5 patients, c.3227C>T (p.P1076L). Based on these results, we predict that the mutation is pathogenic and leads to abnormal collagen IV. Here, we report for the first time on this autosomal dominant syndrome, characterized by hearing loss and eye abnormalities, but without renal damage, in all carriers. Since the oldest patient in the trial was less than 50 years old, however, we recommend that renal examination be reviewed regularly. Our results reveal expansion in the mutation spectrum of the COL4A3 gene and phenotypic spectrum of collagen IV disease. Our study suggests that next-generation sequencing is an economical and effective method and may help in the accurate diagnosis and treatment of these patients.