Nasal Mucosal Tissue Research Articles

CircMIRLET7BHG, upregulated in an m6A-dependent manner, induces the nasal epithelial barrier dysfunction in allergic rhinitis pathogenesis

ObjectiveAllergic rhinitis (AR) remains a frequent aspiratory allergic inflammatory disorder with a high incidence. Circular RNAs (circRNAs) have been revealed to participate in the pathogenesis of AR. This study investigated the biological function of circMIRLET7BHG (hsa_circ_0008668) in AR progression. MethodsOvalbumin (OVA)-exposed human nasal epithelial cell line (HNEpC) and mice were adopted as the in vitro and in vivo models of AR. Immunofluorescence staining was used to determine epithelial tight junction protein expression. Target molecule levels were assessed by RT-qPCR and Western blotting. Localization of circMIRLET7BHG and IGF2BP1 was observed by RNA-FISH and immunofluorescence. Epithelial barrier damage was determined by transepithelial electrical resistance and fluorescein isothiocyanate-dextran (FD4) permeability. Serum concentrations of IgE, sIgE, IFN-γ, IL-4, and IL-5 were detected by ELISA. Apoptosis, pathological changes, and eosinophil infiltration in nasal mucosa tissues were evaluated by TUNEL, H&E, and Sirius red staining, respectively. Molecular mechanism was analyzed by RNA pull-down, RIP, and MeRIP assays. ResultsAn increased expression of circMIRLET7BHG was found in AR patients and experimental models. Down-regulation of circMIRLET7BHG attenuated OVA-induced allergic symptoms via relieving epithelial thicknesses, eosinophil infiltration, apoptosis, and inflammatory response in mice. Subsequently, circMIRLET7BHG deficiency prevented OVA-induced epithelial barrier dysfunction by reducing epithelial permeability, and inhibiting tight junction proteins. Mechanistically, methyltransferase-like 3 (METTL3) enhanced circMIRLET7BHG expression via m6A methylation, which enhanced ADAM10 mRNA stability via interaction with IGF2BP1. ConclusionMETTL3-mediated m6A modification increased circMIRLET7BHG expression that consequently raised ADAM10 mRNA stability via interplay with IGF2BP1, thereby promoting AR by inducing epithelial barrier dysfunction.

Porous silicon embedded in a thermoresponsive hydrogel for intranasal delivery of lipophilic drugs to treat rhinosinusitis

Intranasal delivery is the most preferred route of drug administration for treatment of a range of nasal conditions including chronic rhinosinusitis (CRS), caused by an infection and inflammation of the nasal mucosa. However, localised delivery of lipophilic drugs for persistent nasal inflammation is a challenge especially with traditional topical nasal sprays. In this study, a composite thermoresponsive hydrogel is developed and tuned to obtain desired rheological and physiochemical properties suitable for intranasal administration of lipophilic drugs. The composite is comprised of drug-loaded porous silicon (pSi) particles embedded in a poloxamer 407 (P407) hydrogel matrix. Mometasone Furoate (MF), a lipophilic corticosteroid (log P of 4.11), is used as the drug, which is loaded onto pSi particles at a loading capacity of 28 wt%. The MF-loaded pSi particles (MF@pSi) are incorporated into the P407-based thermoresponsive hydrogel (HG) matrix to form the composite hydrogel (MF@pSi-HG) with a final drug content ranging between 0.1 wt% to 0.5 wt%. Rheomechanical studies indicate that the MF@pSi component exerts a minimal impact on gelation temperature or strength of the hydrogel host. The in-vitro release of the MF payload from MF@pSi-HG shows a pronounced increase in the amount of drug released over 8 h (4.5 to 21-fold) in comparison to controls consisting of pure MF incorporated in hydrogel (MF@HG), indicating an improvement in kinetic solubility of MF upon loading into pSi. Ex-vivo toxicity studies conducted on human nasal mucosal tissue show no adverse effect from exposure to either pure HG or the MF@pSi-HG formulation, even at the highest drug content of 0.5 wt%. Experiments on human nasal mucosal tissue show the MF@pSi-HG formulation deposits a quantity of MF into the tissues within 8 h that is >19 times greater than the MF@HG control (194 ± 7 μg of MF/g of tissue vs. <10 μg of MF/g of tissue, respectively).

RORα overexpression reduced interleukin-33 expression and prevented mast cell degranulation and inflammation by inducing autophagy in allergic rhinitis.

Retinoid acid receptor related orphan receptor α (RORα) is a nuclear receptor that along with other bioactive factors regulates cell proliferation, differentiation, and immunomodulation in vivo. The objective of this study was to explore the function and mechanism of RORα in allergic rhinitis (AR). Derp1 was used to construct an AR cell model in HNEpC cells, and RORα was overexpressed or silenced in the AR HNEpC cells. Next, LAD2 cells were co-cultured with the Derp1-treated HNEpC cells. Additionally, an AR mouse model was established using by OVA, and a RORα Adenovirus was delivered by nebulizing. Pathological tissue structures were evaluated by hematoxylin-eosin staining, and the levels of RORα, interleukin-33 (IL-33), and other proteins were analyzed immunohistochemistry, western blotting, and immunofluorescence staining. IL-33, IL-4, IL-5, and IL-13 levels were detected using enzyme-linked immunosorbent assay kits and cell migration was assessed by Transwell assays. Our data showed that RORα was downregulated in the nasal mucosa tissues of AR patients. Derp1 treatment could cause a downregulation of RORα, upregulation of IL-33, the induction of NLRP3 inflammasomes, and cell migration in HNEpC cells. Furthermore, RORα overexpression dramatically attenuated IL-33 levels, NLRP3 inflammasome activity, and the migration of AR HNEpC cells induced with Derp1. Moreover, RORα in AR HNEpC cells could prevent mast cell (MC) degranulation and inflammation by accelerating autophagy, RORα overexpression inhibited MC degranulation and NLRP3-induced inflammation in the AR model mice. RORα overexpression reduced IL-33 expression in nasal epithelial cells, and also suppressed MC degranulation and inflammation by promoting autophagy. RORα inhibits NLRP3 inflammasome in HNEpC, and attenuated mast cells degranulation and inflammation through autophagy in AR.

Melatonin as a Potential Therapeutic Tool in Allergic Rhinitis Induced by House-Dust Mite

Melatonin is known as an important regulator of circadian rhythm in humans. In the literature, there are no studies evaluating the efficacy of melatonin in the management of allergic rhinitis (AR) or nasal polyps (Np). Np tissue was taken from nasal cavity and mucosal tissue (Mu) was taken from the nasal septal area. Melatonin (25-200nM) and Mite Allergen (2.5-12.5%) were prepared in complete media. Cell viability, apoptosis, intracellular reactive oxygen species production and gene expression levels were determined. Our results showed that there is no toxic effect of Melatonin, Mite and their combination which was given to Np-MSCs and Mu-MSCs. Melatonin significantly reduced reactive oxygen species levels in both mite-treated Np-MSCs and Mu-MSCs. Indoleamine 2,3-dioxygenase level was significantly decreased in melatonin-treated cells. Cyclooxygenase-1 level was significantly decreased in melatonin-treated healthy and allergic Np-MSCs while there was no significant difference in 100 and 150nM Melatonin-treated Mu-MSCs. Interestingly, 50nM Melatonin significantly increased Cyclooxygenase-1 level in Mu-MSCs. 50, 100 and 150nm Melatonin significantly decreased Interleukin-6 level in Mite-treated Np-MSCs. In addition, 100 and 150nM Melatonin significantly decreased Interleukin-6 level in Mite-treated Mu-MSCs. Melatonin has well-established anti-oxidant and anti-neoplastic activity, could be a promising therapeutic agent in the treatment of AR and nasal polyposis.

Role of NLRP3 in the exacerbation of ozone-induced allergic rhinitis

ObjectiveAllergic rhinitis (AR) is an immunoglobulin E (IgE)-mediated inflammatory disorder of the nasal mucosa, and the impact of ozone on AR is gaining increasing attention. Although NOD-like receptor thermal protein domain associated protein 3 (NLRP3) plays a crucial role in the pathogenesis of AR, its regulatory mechanisms in ozone-induced exacerbation remain unclear. Therefore, we explored the impact of ozone inhalation on inflammation in AR and investigated the regulatory mechanisms involving NLRP3. MethodsFifty female Sprague–Dawley rats were selected and divided into five groups: normal control (NC), normal with ozone exposure (NE), AR model, AR with ozone exposure (ARE), and ARE treated with the NLRP3 inhibitor MCC950 (ARE+MCC950). Behavioral changes were observed in the rats, and the expression of NLRP3, active-caspase 1, and GSDMD-N was detected by western blotting. The expression levels of interleukin (IL)− 4, IL-5, IL-13, IL-1β, and ovalbumin-specific IgE (OVA-sIgE) in nasal lavage fluid as well as IL-6 in the serum were measured by ELISA. The expression and distribution of NLRP3 and IL-1β in nasal mucosal tissue were detected by immunohistochemistry, and pathological changes and eosinophilic infiltration in nasal mucosal tissue were observed by hematoxylin and eosin (HE) staining. The effects of ozone exposure on inflammation in the nasal mucosal tissue of rats with AR and the relationship between NLRP3 and inflammation were analyzed. ResultsUpregulation of NLRP3 was observed in the AR rat model, and ozone further aggravated the expression of NLRP3 in the nasal mucosal tissue. Compared to the AR, NC, and NE groups, NLRP3 inflammasomes were activated in the ARE group, and the expression levels of related indexes active-caspase 1 and GSDMD-N were significantly increased; the expression levels of Th2 inflammatory factors IL-4, IL-5, IL-13, and OVA-sIgE were increased, and inflammatory factors such as IL-1β and IL-6 expression was also significantly increased. HE staining revealed that ozone aggravated damage to the nasal mucosal tissue in AR. Compared with the ARE group, the expression of NLRP3 inflammasomes was downregulated, sneezing and scratching symptoms were reduced, inflammatory indicators in nasal lavage fluid were decreased, and nasal mucosal tissue damage was alleviated in rats in the ARE+MCC950 group. ConclusionOzone exposure significantly increased the inflammatory response in an animal model of AR. MCC950 can selectively inhibit the expression of NLRP3, inhibit the activity of inflammasomes, and reduce nasal mucosal inflammation by regulating the NLRP3-caspase-1-IL-1β pathway.

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues. This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring ≥5 points. Hematoxylin and eosin (H&E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of RORγt and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 1-Establishing a Nasal In Vitro Model for Drug Delivery Testing Based on a Novel Cell Line.

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air-liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems.

Effect of TFF3 on tight junction protein in eosinophilic chronic sinusitis and its related mechanism

Objective: To study the effect of trefoil factor family (TFF) 3 on the expression of tight junctions (TJs) in the nasal mucosa epithelium of eosinophilic chronic rhinosinusitis (eCRS) and its mechanism. Methods: From September to December 2020, eligible patients from the Department of Otorhinolaryngology of the First Affiliated Hospital of Nanchang University were recruited, including 11 control patients and 37 patients with chronic rhinosinusitis with nasal polyps (CRSwNP), from whom nasal mucosa and nasal polyp tissue samples were collected. Immunohistochemistry (IHC) was used to detect the localization and expression intensity of TFFs (TFF1, TFF2 and TFF3) and TJs (occudin, claudin-1 and ZO-1) in nasal mucosa. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the mRNA and protein expression. A cell model of tight junction injury in human nasal epithelial cells (HNECs) through stimulation with interleukin (IL)-13 was also established. The optimal modeling concentration and time for HNECs were determined, which were subsequently treated with TFF3 and/or a phosphoinositide 3-kinase (PI3K)-specific inhibitor (LY294002). Finally, RT-qPCR and WB were used to assess the effects of TFF3 on tight junctions and the PI3K/serine/threonine kinase (Akt) signaling pathway. Data were analyzed statistically using GraphPad Prism 7 software. Results: IHC results showed that the expression of TFF1 and TFF3 in nasal mucosa of eCRS group was significantly higher than that of control group (t=4.62, P=0.002; t=5.89, P<0.001), respectively, mainly expressed in goblet cell. The expression of occludin, claudin-1 and ZO-1 in the nasal mucosa of the eCRS group was lower than that of the control group (occludin t=3.98, P=0.019; claudin-1 t=5.15, P=0.002; ZO-1 t=5.42, P=0.001), respectively. WB results showed that the expression of TFF3 in non-eosinophilic chronic sinusitis (Non-eCRS) group and eCRS group was higher than that in the control group (t=3.62, P=0.036; t=5.93, P<0.001). The expression of occludin, claudin-1 and ZO-1 in eCRS group was lower than that in the control group (occludin t=5.14, P=0.002; claudin-1 t=6.35, P<0.001; ZO-1 t=6.64, P<0.001), respectively. The RT-qPCR results showed that compared with the control group, the levels of TFF1 and TFF3 mRNA were increased in the nasal mucosal epithelium of the Non-eCRS and eCRS groups (TFF1 t=3.98, P=0.046, t=4.89, P=0.002; TFF3 t=3.50, P=0.044, t=6.78, P<0.001). There was no statistically significant difference in TFF2 mRNA levels between the Non-eCRS and eCRS groups (t=1.34, P=0.061; t=3.37, P=0.055). Compared with the control group, Non-eCRS and eCRS groups showed a decrease in the mRNA levels of occludin, claudin-1 and ZO-1 (occludin t=4.27, P=0.011, t=5.61, P=0.007; claudin-1 t=3.62, P=0.036, t=6.80, P<0.001; ZO-1 t=3.47, P=0.047, t=7.86, P<0.001). The mRNA levels of TFF3 and TJs in eCRS nasal mucosa tissue showed a moderate positive correlation (occludin r=0.661, claudin-1 r=0.614, ZO-1 r=0.548, all P<0.001); TFF1 showed a low degree of positive correlation with the expression of occludin, claudin-1 and ZO-1 (occludin r=0.467, P=0.040; claudin-1 r=0.362, P=0.012; ZO-1 r=0.425, P=0.025). The establishment of cell models showed that compared with normal HNECs, the mRNA expression of TFF3 was most significantly increased at a concentration of 50 ng/ml stimulated by IL-13 (t=3.72, P=0.013); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.18, P=0.031; claudin-1 t=3.86, P=0.010; ZO-1 t=5.16, P=0.002). The expression of TFF3 mRNA increased most significantly after 15 hours of IL-13 stimulation (t=3.14, P=0.034); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.97, P=0.010; claudin-1 t=4.78, P=0.004; ZO-1 t=5.16, P=0.004). TJs damage model could be established by treating HNECs with 50 ng/ml IL-13 for 15 hours. Intervention experiments showed that compared with the IL-13 group, the IL-13+TFF3 group showed an increase in TJs mRNA expression (occludin t=6.10, P=0.009; claudin-1 t=5.90, P=0.013; ZO-1 t=9.44, P=0.007). Compared with the IL-13 group, the expression of TJs protein in the IL-13+TFF3 group increased (occludin t=3.23, P=0.013; claudin-1 t=9.40, P=0.017; ZO-1 t=2.23, P=0.032); The expression of TJs protein decreased in the IL-13+TFF3+LY294002 group (occludin t=4.73, claudin-1 t=8.77, ZO-1 t=3.51, all P<0.001). Compared with the IL-13+TFF3 group, the IL-3+TFF3+LY294002 group showed a decrease in PI3K and p-Akt/Akt protein expression (PI3K t=13.29, p-Akt/Akt t=10.30, all P<0.001). The increased mRNA and protein expression of occludin, claudin-1 and ZO-1 induced by TFF3 were also inhibited by LY294002. Conclusion: TFF3 can up-regulate the expression of occludin, claudin-1, and ZO-1 through PI3K/Akt pathway, and has a certain protective effect on the nasal mucosal epithelial barrier, providing a new idea for treating eCRS.

Xiao-qing-long-tang ameliorates OVA-induced allergic rhinitis by inhibiting ILC2s through the IL-33/ST2 and JAK/STAT pathways

BackgroundAllergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa that is mediated by immunoglobulin E (IgE). Xiao-qing-long-tang (XQLT) is a traditional Chinese medicine compound that is widely used to treat respiratory diseases such as AR. However, the underlying mechanism of the effect of XQLT on AR remains unclear. PurposeTo elucidate the effect of XQLT on ovalbumin (OVA)-induced AR and the mechanisms of action. MethodsThe therapeutic efficacy of XQLT was evaluated in a well-established OVA-induced AR mouse model. Nasal symptoms were analyzed, type 2 cytokines and OVA-sIgE levels were measured, nasal mucosa tissues were collected for histological analysis, and the changes of Group 2 innate lymphoid cells (ILC2s) and the IL-33/ST2 and JAK/STAT signaling pathways in the nasal mucosa were observed. ResultsXQLT significantly alleviated the nasal symptoms and histological damage to the nasal mucosa in AR mice, and reduced the levels of type 2 cytokines and OVA-sIgE. In addition, after XQLT treatment, the numbers of ILC2s in the nasal mucosa of AR mice were reduced, and the mRNA levels of the transcription factors GATA3 and ROR-α were decreased. Moreover, IL-33/ST2 signaling pathway was inhibited. The costimulatory cytokine associated JAK/STAT signaling pathway was also inhibited in ILC2s. ConclusionOur study demonstrated that XQLT regulated ILC2s through the IL-33/ST2 and JAK/STAT pathways to ameliorate type 2 inflammation in OVA-induced AR. These findings suggest that XQLT might be used to treat AR.

Effect of Neurokinin-1 Receptor Knockdown on the Expression of RANTES in Allergic Rhinitis.

Neurokinin-1 receptor (NK-1R) and normal T cell expressed and secreted (RANTES) have been shown to play important roles in allergic rhinitis (AR). However, whether the regulating effect of NK-1R in AR is achieved via RANTES remains unknown. In the present study, Sprague-Dawley rats were sensitized and challenged with ovalbumin to make AR models. During the challenge period, the rats were treated intranasally with NK-1R-specific small interfering RNA (siRNA) for NKR group, negative siRNA for NCS group, rats in NSAR group and NS group were given saline. The amount of nasal secretion and the numbers of nose rubs and sneezes were measured in each rat. The levels of NK-1R and RANTES in the nasal mucosal tissues were determined through real-time fluorescence quantitative RT-PCR and immunohistochemical staining. The numbers of eosinophils in the collected nasal lavage fluid (NLF) were counted, and the concentration of RANTES in NLF was determined by enzyme-linked immunosorbent assay. Compared with that in the NS group, the expression of NK-1R and RANTES was significantly higher in the nasal mucosa of NSAR and NCS group rats. The sneezing and nose rubbing counts and the amount of nasal secretions were increased significantly in the NSAR and NCS groups. Rats in the NKR group experienced greater relief from AR symptoms than rats in the NSAR and NCS groups. Furthermore, knockdown of NK-1R expression also significantly eliminated RANTES expression and eosinophil infiltration in the nasal mucosa of NKR group rats. For the first time, we show that intranasal treatment with NK-1R-specific siRNA can significantly decrease RANTES expression, AR-related symptoms, and eosinophil inflammation, suggesting that the regulating effect of NK-1R in the development of AR occurs via alteration of RANTES expression.

Mucoadhesive pickering nanoemulsions via dynamic covalent chemistry

Hypothesis. Submicron oil droplets stabilized using aldehyde-functionalized nanoparticles should adhere to the primary amine groups present at the surface of sheep nasal mucosal tissue via Schiff base chemistry. Experiments. Well-defined sterically-stabilized diblock copolymer nanoparticles of 20 nm diameter were prepared in the form of concentrated aqueous dispersions via reversible addition-fragmentation chain transfer (RAFT) aqueous emulsion polymerization of 2,2,2-trifluoroethyl methacrylate (TFEMA) using a water-soluble methacrylic precursor bearing cis-diol groups. Some of these hydroxyl-functional nanoparticles were then selectively oxidized using an aqueous solution of sodium periodate to form a second batch of nanoparticles bearing pendent aldehyde groups within the steric stabilizer chains. Subjecting either hydroxyl- or aldehyde-functional nanoparticles to high-shear homogenization with a model oil (squalane) produced oil-in-water Pickering macroemulsions of 20–30 µm diameter. High-pressure microfluidization of such macroemulsions led to formation of the corresponding Pickering nanoemulsions with a mean droplet diameter of around 200 nm. Quartz crystal microbalance (QCM) experiments were used to examine adsorption of both nanoparticles and oil droplets onto a model planar substrate bearing primary amine groups, while a fluorescence microscopy-based mucoadhesion assay was developed to assess adsorption of the oil droplets onto sheep nasal mucosal tissue. Findings. Squalane droplets coated with aldehyde-functional nanoparticles adhered significantly more strongly to sheep nasal mucosal tissue than those coated with the corresponding hydroxyl-functional nanoparticles. This difference was attributed to the formation of surface imine bonds via Schiff base chemistry and was also observed for the two types of nanoparticles alone in QCM studies. Preliminary biocompatibility studies using planaria indicated only mild toxicity for these new mucoadhesive Pickering nanoemulsions, suggesting potential applications for the localized delivery of hydrophobic drugs.

Cineole inhibits the biosynthesis of leukotrienes and prostaglandins to alleviate allergic rhinitis: Insights from metabolomics

Allergic rhinitis (AR) is a common allergic disease characterized by nasal congestion, rhinorrhoea, and sneezing. Cineole, a monoterpenoid compound widely present in various volatile oils, has a wide range of pharmacological activities and is of interest in allergic airway diseases for its anti-inflammatory and anti-mucus production abilities. However, the protective effects of cineole in mice with allergic rhinitis and its mechanisms have not been well investigated. In this study, the protective effect of cineole against ovalbumin-induced (OVA-induced) allergic rhinitis and its molecular mechanism is investigated by metabolomic analysis based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). OVA combined with aluminum hydroxide adjuvant is used to sensitize and establish the allergic rhinitis (AR) mouse model. The mice are randomly divided into groups of control, AR, cineole (30 mg/kg), and budesonide (38.83 μg/kg). The pharmacodynamic results show that cineole significantly reduces the levels of Th2-type cytokines and OVA-specific IgE (OVA-sIgE) in AR mice, improves nasal mucosal tissue damage and alleviates nasal symptoms compared to the untreated AR group. Metabolomic results show that arachidonic acid (AA) metabolism and tryptophan (Trp) metabolism are reprogrammed on the basis of 27 significantly altered metabolites. Further studies show that cineole inhibits the biosynthesis of pro-inflammatory lipid mediators leukotrienes (LTs) and prostaglandins (PGs) in mice by inhibiting the activity of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) in the arachidonic acid metabolic (AA metabolic) pathway. It also inhibits the production of Th2 cytokines and inflammatory cell infiltration, thereby alleviating symptoms such as nasal congestion and nasal leakage. These results reveal the action and molecular mechanism of cineole in alleviating AR and provide a theoretical basis for the clinical application of cineole in treating AR.

Integration of network pharmacology and proteomics to elucidate the mechanism and targets of traditional Chinese medicine Biyuan Tongqiao granule against allergic rhinitis in an ovalbumin-induced mice model

Ethnopharmacological relevanceBiyuan Tongqiao granule (BYTQ) is a traditional Chinese medicine that has been used in China to clinically treat patients with allergic rhinitis (AR), yet its underlying mechanism and targets remains unclear. Aim of the studyThe study aimed to investigate the potential mechanism of BYTQ against AR using the ovalbumin (OVA) -induced AR mice model. Integrating network pharmacology and proteomics to investigate possible targets of BYTQ for AR. Materials and methodsThe compounds in BYTQ were analyzed using UHPLC-ESI-QE-Orbitrap-MS. The OVA/Al(OH)3 were used to induce the AR mice model. The nasal symptoms, histopathology, immune subsets, inflammatory factors, and differentially expressed proteins were examined. Proteomics analysis elucidated the potential mechanisms of BYTQ to improve AR, which was further validated by Western blot (WB) assay. The compounds and potential targets of BYTQ were systematically elucidated by integrating network pharmacology and proteomics analysis to explore the mechanism. The binding affinity between key potential targets and corresponding compounds was then validated using molecular docking. Molecular docking results were verified by a western blotting and cellular thermal shift assay (CETSA). ResultsA total of 58 compounds were identified from BYTQ. BYTQ significantly suppressed AR symptoms by inhibiting the release of OVA-specific immunoglobulin E (IgE) and histamine, improving the pathological injury of nasal mucosal tissue, and regulating the proportions of lymphocytes to maintain immune balance. Proteomics analysis showed that the cell adhesion factors and focal adhesion pathway might be potential mechanism of BYTQ against AR. The levels of E-selectin, vascular endothelial cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) proteins in the nasal mucosal tissue were significantly downregulated in the BYTQ-H group compared to the AR group. Integrating network pharmacology and proteomics analysis identified that SRC, PIK3R1, HSP90AA1, GRB2, AKT1, MAPK3, MAPK1, TP53, PIK3CA, and STAT3 may be potential protein targets for BYTQ to treat AR. Molecular docking analysis indicated that the active compounds of BYTQ could bind tightly to these key targets. In addition, BYTQ could inhibit OVA-induced phosphorylation levels of PI3K, AKT1, STAT3 and ERK1/2. The CETSA data suggested that BYTQ could improve the heat stability of PI3K, AKT1, STAT3 and ERK1/2. ConclusionsBYTQ suppresses E-selectin and VCAM-1 and ICAM1 expression by regulating PI3K/AKT and STAT3/MAPK signaling pathways, thus alleviating inflammation in AR mice. BYTQ is the aggressive treatment for AR.

CircARF3 Mitigates Allergic Rhinitis through Targeting microRNA-205-5p/Sirtuin 5 Axis

Introduction: Circular RNAs (circRNAs) are essential in the progression of allergic rhinitis (AR). The purpose of this research was to examine the role of circRNA ADP-ribosylation factor 3 (circARF3) in the pathogenesis of AR. Methods: To generate an animal model of AR, mice were treated with house dust mite (HDM), and mice nasal epithelial cells (NEpCs) were treated with IL-4/IL-13 to imitate the inflammatory damage of AR in vitro. Sanger sequencing, qRT-PCR, and RNAse R digestion assays all validated the circularization structure of circARF3. The levels of circARF3, miR-205-5p, and sirtuin 5 (SIRT5) were determined by qRT-PCR or Western blotting. Luciferase reporter, RNA immunoprecipitation, and pull-down experiments were used to investigate the regulatory network. Flow cytometry was used to investigate the rate of cell apoptosis, and Western blotting was used to determine the levels of apoptotic-related proteins (cleaved caspase 3, cleaved polyadenosine-diphosphate-ribose polymerase) and HMGB1, TLR4, and MyD88. Enzyme-linked immunosorbent assay was used to assess the inflammatory response. Hematoxylin-eosin staining and TUNEL were used to detect the histology of injury and apoptosis of nasal mucosa tissues. Results: CircARF3 and SIRT5 levels were reduced in HDM-treated animals and IL-4/IL-13-treated NEpCs, while miR-205-5p expression was increased. CircARF3 was generated by back-splicing exons 3–5 with a stable circular shape. CircARF3 overexpression mitigated IL-4/IL-13-induced apoptosis in NEpCs by inhibiting miR-205-5p. SIRT5 upregulation attenuated IL-4/IL-13-induced inflammatory injury in NEpCs, and SIRT5 knockdown induced opposite effects. miR-205-5p silencing reversed the effects of SIRT5 knockdown on IL-4/IL-13-induced inflammatory injury. Furthermore, circARF3 overexpression alleviated histological abnormalities, apoptosis, inflammatory response, and HMGB1/TLR4 signaling activation in HDM-treated animals. Conclusion: CircARF3 inhibited cell apoptosis and inflammation via the miR-205-5p/SIRT5 axis in IL-4/IL-13-treated NEpCs and HDM-treated mice.

Therapeutic effects of Pulsatilla koreana Nakai extract on ovalbumin-induced allergic rhinitis by inhibition of Th2 cell activation and differentiation via the IL-4/STAT6/GATA3 pathway

Allergic rhinitis (AR), caused by immunoglobulin E (IgE)-mediated inflammation, generally occurs in the upper respiratory tract. T helper type 2 (Th2) cell-mediated cytokines, including interleukin (IL)-4, IL-5, and IL-13, are important factors in AR pathogenesis. Despite various treatment options, the difficulty in alleviating AR and pharmacological side effects necessitate development of new therapies. The root of Pulsatilla koreana Nakai (P. koreana), a pasque flower, has been used as a herbal medicine. However, its effects on AR remain unclear; therefore, we aimed to explore this subject in the current study. The therapeutic effects of P. koreana water extract (PKN) on the pathophysiological functions of the nasal mucosa was examined in ovalbumin (OVA)-induced AR mice. The effect of PKN on Th2 activation and differentiation was evaluated using concanavalin A-induced splenocytes and differentiated Th2 cells from naïve CD4+ T cells. We also investigated the effect of changes in JAK/STAT6/GATA3 signaling on IL-4-induced Th2 cells. In OVA-induced AR mice, PKN administration alleviated allergic nasal symptoms and decreased the total number of immune cells, lymphocytes, neutrophils, and eosinophils in nasal lavage fluid; serum levels of OVA-specific IgE, histamine, and IL-13 were also significantly reduced. PKN also ameliorated OVA-induced nasal mucosal tissue thickening by inhibiting inflammation and goblet cell hyperplasia. PKN treatment significantly inhibited Th2 activity and differentiation through the IL-4/STAT-6/GATA3 pathway in Th2 cells. PKN is an effective AR treatment with the potential to improve patients' daily lives by regulating the allergic inflammatory response induced by Th2 cells.

Experimental observation of the effect of immunotherapy on CD4+ T cells and Th1/Th2 cytokines in mice with allergic rhinitis

The present study aims to investigate the effect of immunotherapy in a mouse model of allergic rhinitis (AR) and to explore the possible molecular mechanisms of action. An animal model of AR was established by sensitization and challenge of BALB/c mice with house dust mite (HDM) extract. The mice were injected subcutaneously with HDM for immunotherapy. AR nasal symptoms were evaluated according to the frequencies of nose rubbing and sneezing and the degree of rhinorrhea. The nasal mucosa and lung tissue architecture and inflammatory status by histological analysis; the infiltration of eosinophils in nasal lavage fluid (NALF) of mice was observed by Diff-Quik stain; ELISA-based quantification of serum HDM-specific IgE and TH1/TH2 cytokine concentration; and flow cytometry detected the number of serum CD4+/CD8+ cells to evaluate the mechanism of immunotherapy. It was found that after immunotherapy, the AR symptom score was reduced, the number of eosinophils in NALF was reduced, and the infiltration of inflammatory cells and tissue damage in the nasal mucosa and lung tissue were alleviated. Immunotherapy can increase the number of CD4+ T cells in the peripheral blood, increase the ratio of CD4+/CD8+ cells, increase the expression of Th1 cytokines such as IL-2 and IFN-γ, reduce the expression of Th2 cytokines such as IL-4 and IL-5. The results showed that repeated intraperitoneal injection of crude extract of HDM for sensitization, followed by nasal drops can effectively construct a mouse model of AR, and subcutaneous injection of immunotherapy in mice can reduce allergic inflammation in model mice and improve the inflammatory infiltration of the nasal cavity in allergic rhinitis. Immunotherapy can reduce the expression of inflammatory factors in AR, improve Th1/Th2 balance, and may play a role in the treatment of AR by improving the function of immune cells.

Involvements of Nrf2 and oxidative stress in the ozone-elicited exacerbation in an allergic rhinitis model

Objective and designAn experimental rat allergic rhinitis(AR) model was made to explore the effect of different concentrations of ozone exposure and evaluate the roles of nuclear factor erythroid 2-related factor 2(Nrf2) and oxidative stress in ozone exposure. MethodSprague-Dawley rats were sensitized with ovalbumin (OVA). Three groups of AR rats were exposed respectively to different concentrations of ozone for 2 h on 6 weeks. Nasal symptoms and OVA- specific Ig E in the serum were evaluated. The pathological changes in the nasal mucosa were examined. Malondialdehyde (MDA) level and activity of superoxide dismutase(SOD) and glutathione peroxidase (GSH-Px,GPX) in the nasal mucosa tissue were measured through a spectrophotometry-based method. Nrf2、Kelch-1ike ECH- associated protein-l (Keap1) proteins was measured by western blotting. GPX1、GPX2 mRNA were detected by quantitative real time-PCR(qRT-PCR). ResultsOur results showed that ozone exposure induced a significant increase of the number of sneezes, nasal rubs, amount of nasal secretion and OVA-sIgE in the serum of AR model. Ozone effected oxidative stress in different concentration. The content of MDA in AREH group was significantly higher than AR groups. The activities of SOD and GSH-Px in nasal mucosa showed different trends in different concentration groups. The activities of SOD and GSH-Px in AREL and AREM groups were higher than AR group, but decreased at AREH group. The nucleoprotein level of Nrf2 in AREL and AREM groups was higher than AR groups. However, in AREH group, it was significantly decreased, compared with AREL and AREM groups. GPX1 and GPX2 mRNA levels in nasal mucosa showed the same trend in different exposure groups. ConclusionsDifferent concentrations of ozone inhalation causes changes of the expression of Nrf2 nuclear protein and its target genes in nasal mucosa of AR. High concentration ozone breaks the redox balance and aggravates oxidative damage in AR. This study suggests that inhibiting oxidative stress might be a solution for ozone-elicited detrimental effects on AR.

Effects of Pendrin Protein in Nasal Epithelial Cells on Mucin Production in the Context of Type 2 Inflammation.

Chronic rhinosinusitis (CRS) is a heterogeneous disease. The pathogenesis of chronic sinusitis is still unclear; however, the nasal cavity and paranasal sinuses are commonly affected by type 2 inflammation, which is caused by Th2 cytokines such as interleukin (IL)-5, IL-4, and IL-13. Previous studies have shown that pendrin promotes local infiltration of neutrophils through the production of human neutrophil elastase (HNE), which is essential for the secretion of mucin 5AC (MUC5AC) in chronic inflammatory diseases of the lower respiratory tract. This study investigated pendrin expression and its relationship to mucin in type 2 inflammation. A total of 40 patients (10 CRS patients with nasal polyps,10 CRS patients without nasal polyps, and 20 nasal septum deviation patients) were included in this study and were divided into the CRS group and the NC group. A normal nasal mucosa tissue culture model was established in vitro. IL-13 was used to stimulate primary cultures of human nasal epithelial cells (HNECs). Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of pendrin, MUC5AC, and MUC5B. After transfecting HNECs with siRNA pendrin or negative control (NC), EGF receptor (EGFR), HNE, MUC5AC, and MUC5B expression were analyzed using qRT-PCR, WB, or ELISA in terms of their relationships with pendrin. Pendrin expression in the tissue was also analyzed. After IL-13 stimulation, pendrin, MUC5AC, and MUC5B expression levels were upregulated; the optimal concentration of IL-13 was 50 ng/mL. The expression levels of HNE, EGFR, MUC5AC, and MUC5B were downregulated after transfection with siRNA pendrin-1650. Pendrin expression in the NC group was lower than in the CRS group. IL-13 is implicated in the inflammation of nasal mucosa, and pendrin is closely related to the excessive secretion of mucin. The expression of mucin is downregulated after transfection with siRNA pendrin. There is a positive relationship between pendrin and EFGR/HNE. Moreover, pendrin plays an important role in type 2 inflammation.

Significance of leukocyte-specific transcript 1 levels in nasal mucosal tissue to predict recurrence of nasal polyps.

Chronic Rhinosinusitis with Polyps (CRSwNP) is characterized by high heterogeneity and postoperative recurrence rate. This study aims to explore the clinical significance of tissue Leukocyte-Specific Transcript 1 (LST1) in predicting CRSwNP recurrence. We enrolled 62 CRSwNP patients including 30 primary CRSwNP and 32 recurrent CRSwNP patients, and 40 Healthy Controls (HC). Tissue samples were collected. Tissue LST1 expression was assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western Blotting (WB) and Immunofluorescence (IF) staining. The predictive values of LST1 expression for CRSwNP postoperative recurrence were assessed through the Receiver Operating Characteristic (ROC) curves. The tissue levels of LST1 were significantly increased in the CRSwNP group than the HC group, especially in the recurrent group, and the elevated LST1 mRNA levels were positively correlated with the peripheral eosinophil percentages, tissue eosinophil counts and percentages. IF staining results showed that the LST1 protein levels were higher in CRSwNP patients, especially in the recurrent patients than in the HC group. ROC curves highlighted that tissue LST1 levels were associated with recurrent CRSwNP and exhibited a higher predictive ability for postoperative CRSwNP recurrence. This was the first report suggesting that LST1 expression was upregulated and associated with mucosal eosinophil infiltration and CRSwNP recurrence. Tissue LST1 could be a promising biomarker for predicting postoperative recurrence in CRwNP patients. Level 5.

Mechanism of Kruppel-Like Factor 4 in Pyroptosis of Nasal Mucosal Epithelial Cells in Mice With Allergic Rhinitis.

Allergic rhinitis (AR) is a chronic nasal inflammation, characterized by nasal epithelial dysfunction. Gene therapy targeting transcription factors is a promising strategy for quenching allergic inflammation, including AR. This study sought to probe the mechanism of Kruppel-like factor 4 (KLF4) in pyroptosis of nasal mucosal epithelial cells (NEpCs) in AR mice and provide targets for AR treatment. AR mouse models were established using sensitization with ovalbumin, followed by injection with short hairpin RNA KLF4 (sh-KLF4). AR symptoms were assessed by the times of sneezing and nose rubbing, hematoxylin-eosin, and periodic acid-Schiff staining. Levels of KLF4, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), cleaved caspase-1, and N-terminal domain (GSDMD-N) in nasal mucosal tissues were determined by Western blot assay, and levels of interleukin (IL)-1β and IL-18 in nasal lavage fluid were determined by enzyme-linked immunosorbent assay. The binding of KLF4 to the NLRP3 promoter was verified using chromatin immunoprecipitation and dual-luciferase assays. The functional rescue experiment was performed with oe-NLRP3 and sh-KLF4 in AR mice. KLF4 was upregulated in nasal mucosal tissues of AR mice. KLF4 inhibition reduced the times of sneezing and nose rubbing, inflammatory cell infiltration, and goblet cell hyperplasia in nasal mucosal tissues, and levels of NLRP3, cleaved caspase-1, GSDMD-N, IL-1β, and IL-18. KLF4 was enriched on the NLRP3 promoter and improved NLRP3 expression. NLRP3 overexpression reversed the inhibition of sh-KLF4 on pyroptosis of NEpCs in AR mice. KLF4 bound to the NLRP3 promoter and promoted pyroptosis of NEpCs in AR mice via activating NLRP3.