Objective:The mucosa of Chronic rhinosinusitis with nasal polyps（CRSwNP） is accompanied by tissue remodeling. Epithelial-mesenchymal transition（EMT） plays an important role in tissue remodeling, but the mechanism of EMT is not yet clear. The purpose of this study is to further clarify the pathogenesis of CRSwNP and provide another idea and theoretical basis for the treatment of CRSwNP. Methods:①The expression of NLRP3 and EMT-related protein（E-cadherin, Vimentin） in the nasal mucosa of the CRSwNP group and the normal control group were detected by immunohistochemistry（IHC）. ②Primary human nasal epithelial cells（HNECs） were cultured in vitro, and HNE-intervened cells with different concentrations（0, 10, 25, 50, 100 ng/mL） were used. After stimulation for 24 h, mRNA and protein expressions of E-cadherin, Vimentin, NLRP3 were detected by qRT-PCR and western blotting. ③Cells were collected at 0, 24, 36, 48 and 72 hours later after incubation with HNE with the optimal concentration, and the mRNA and protein expressions of E-cadherin, Vimentin and NLRP3 were detected by qRT-PCR and western blotting. ④Primary human nasal epithelial cells were pretreated with NLRP3 inhibitor MCC950, then stimulated with HNE, and EMT-related proteins（E-cadherin, Vimentin） and NLRP3 expression were detected by qRT-PCR and western blotting. Results:①The expression levels of NLRP3 and Vimentin in nasal polyps of CRSwNP patients were higher than those of control group, and the expression of E-cadherin was lower（P<0.05）. The mRNA and protein expression levels of NLRP3 and Vimentin increased when HNE stimulated primary human nasal epithelial cells, while the expression of E-cadherin decreased. ②The effect was most significant when the HNE stimulated nasal mucosal epithelial cells were exposed to 50 ng/mL（P<0.05）. The primary human nasal epithelial cells were stimulated with 50 ng/ml HNE, and the effect was most significant when the duration of HNE exposure was 36 h（P<0.05）. ③Primary human nasal epithelial cells were pretreated with MCC950 and then stimulated with HNE. The mRNA and protein expression levels of E-cadherin in the NLRP3 inhibitor pretreated group were increased, while the mRNA and protein expression levels of Vimentin and NLRP3 were decreased（P<0.05）. Conclusion:ln CRSwNP, HNE promotes EMT in human nasal mucosal epithelial cells by activating NLRP3.
Humans ; Nasal Polyps/metabolism* ; Epithelial-Mesenchymal Transition ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Sinusitis/metabolism* ; Cadherins/metabolism* ; Vimentin/metabolism* ; Chronic Disease ; Nasal Mucosa/cytology* ; Rhinitis/metabolism* ; Epithelial Cells/metabolism* ; Cells, Cultured ; Rhinosinusitis

OBJECTIVETo investigate the regulation of adipose-derived mesenchymal stem cells (ADSC) on helper T cells and regulatory T cells in allergic rhinitis(AR) mouse model and the underlying mechanisms.

METHODSUsing random number table, 60 Balb/c mice were divided into 6 groups (represented by: sensitized/challenged/treated ), they were the experimental group 1(OVA/OVA/high dose ADSC), the experimental group 2(OVA/OVA/low dose ADSC), the experimental group 3(OVA/OVA/PBS), the experimental group 4(OVA/OVA/0), the control group 1(PBS/PBS/0) and the control group 2(0/0/0). The mouse ADSC were isolated and cultured through conventional method, and AR mouse model was built with OVA and aluminum. The mice were injected with high (3×10(6)), low (1×10(6)) ADSC respectively labeled by CM-Dil for 3 consecutive days via tail-vein injection and sacrificed 48 hours later. Finally, levels of IL-4, IL-6, IL-10 and IFN -γ in serum were examined by ELISA; expressions of the four cytokines in spleen were examined by q RT-PCR; migration of ADSC to mouse model nasal mucosa were observed through fluorescence microscope; eosinophil infiltration were observed by the nasal HE staining.

RESULTSMouse ADSC was isolated, cultured and identified successfully. There was significant difference in symptom scores of AR models (compared with 0/0/0 group, P<0.01). The IL-4 and IL-6 levels of OVA/OVA/high ADSC group were significantly lower than OVA/OVA/0 group (group 1: (17.95±7.78), (27.51±5.93) pg/ml; group 4: (56.82±9.12), (70.03±7.22) pg/ml), the IFN-γ and IL-10 levels increased significantly (group 1: (367.74±13.79), (417.10±72.40) pg/ml; group 4: (199.46±11.25), (122.50±15.57) pg/ml) in serum. These differences were statistically significant(P<0.01). Compared with OVA/OVA/low ADSC group, the IL-4 and IL-6 levels decreased significantly (group 1: (17.95±7.78), (27.51±5.93) pg/ml; group 2: (41.57±12.27), (56.21±9.23)pg/ml) of OVA / OVA / high ADSC group, and the IFN-γ and IL-10 increased significantly (group 1: (367.74±13.79), (417.10±72.40)pg/ml; group 2: (281.77±30.41), (203.45±87.10) pg/ml). These differences were statistically significant(P<0.01). At the same time, the corresponding changes observed at the levels of the cytokines' mRNA. ADSC labeled by CM-Dil could migrate to the mouse nasal mucosa. OVA/OVA/high ADSC group showed the more red fluorescence than the OVA/OVA/low ADSC group. The eosinophils in nasal mucosa of the two groups reduced compared with the normal control.

CONCLUSIONADSC injected via tail-vein can migrate to nasal mucosa and play non-specific immune effects, that may to effect the releases of some cytokines then to regulate the Th1/Th2 imbalance and the function of Treg cell, finally that be dose-related in a certain extent.

Adipose Tissue ; cytology ; Animals ; Cytokines ; blood ; Disease Models, Animal ; Eosinophils ; immunology ; Inflammation ; Mesenchymal Stem Cell Transplantation ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Rhinitis, Allergic ; immunology ; therapy ; T-Lymphocytes, Helper-Inducer ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVE: To study the role of P-JNK and P-c-Jun of JNK (c-Jun N-terminal kinase) on nasal mucosa remodeling in allergic rhinitis rats. METHOD: Sixty male Wistar rats (weighing about 200-250 g) were randomly divided into AR group (A group) and B group(control group). The rats in A group were sensitized for inducing AR by intraperitoneal injection of ovalbumin and Al(OH)₃. Rats in group A were randomized into A4, A8 and A12 group (each had 10 rats). Ovalbumin was dropped in each nasal cavity of every rat for 4,8,12 weeks, respectively. Rats in group B were sensitized by saline instead of OVA, and were also divided into B4, B8 and B12 group. Each group had 10 rats. Pathological changes of nasal mucosa in each period were observed by hematoxylin and eosin stain dyeing. The phosphorylation of JNK and c-Jun were tested by immunohistochemistry. RESULT: In A8 group, mucosal congestion and edema thickening with inflammatory cells infiltration of eosinophils were observed in the eighth week, and the inflammatory changes were significantly increased as time went on. The mean absorbance values of P-JNK and P-c-Jun in A group were significantly higher than those in the corresponding B group (all P < 0.01). Moreover, the mean absorbance values of A12 group were significantly higher than A4 group and A8 group (all P < 0.01 ). CONCLUSION The expression of P-JNK and P-c-Jun in the process of nasal mucosa remodeling in allergic rhinitis rats were increased, which suggested that P-JNK and P-c-Jun played important roles in nasal mucosa remodeling of the allergic rhinitis rats.
Airway Remodeling ; Animals ; Disease Models, Animal ; Eosinophils ; cytology ; Injections, Intraperitoneal ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Nasal Mucosa ; metabolism ; Ovalbumin ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Rhinitis, Allergic ; metabolism ; Signal Transduction

OBJECTIVE: To discuss the effect of different doses intranasal corticosteroids on remodeling of allergic rhinitis (AR) mice nasal mucosa and expression level of matrix metalloproteinase-9 (MMP-9). METHOD: Thirty BALB/c female mice were divided into five groups randomly and received OVA or normal saline (NS) with intraperitoneal injection or nasal challenge, respectively. The treatment groups received additional different doses of budesonide (0.6 μg/20 g, 3.0 μg/20 g and 15.0 μg/20 g) daily for 16 weeks. We assessed the nasal symptoms at 4 and 16 weeks. Collected the mice nasal tissue, and then stained with hematoxylin-eosin, Masson's Trichrome, and periodic acid-schiff respectively to evaluate airway remodeling at 16 weeks. MMP-9 was measured with enzyme-linked immunosorbent assay (ELISA). Result: Times of rubbing, sneezes and infiltrate of eosinophil increased more in B group than in A group, and subepithelial fibrosis, collagen deposition, goblet cell hyperplasia, and submucosal gland hypertrophy were only observed in B group at 16 weeks. The nasal symptoms and eosinophil infiltration were inhibited by treatment with budesonide from a dose of 0.6 μg onwards, while the prevention of structure changes was only observed with 3.0 μg onwards. In addition, intranasal budesonide reduced MMP-9 in the nasal of AR mice. CONCLUSION The study suggests that higher dose intranasal corticosteroids might inhibit the airway remodeling of nasal mucosa by reducing MMP-9.
Airway Remodeling ; Animals ; Budesonide ; pharmacology ; Disease Models, Animal ; Eosinophils ; cytology ; Female ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; drug effects ; Rhinitis, Allergic ; drug therapy ; metabolism

OBJECTIVE: To investigate method established and system evaluated in the model of SD rat with AR. METHOD: To establish AR model of SD rats by ovalbumin (OVA), 20 cases of SD rats were randomly divided into two groups, namely control group (10 cases) and AR group (10 cases). AR models were sensitized and challenged by OVA. Control group were used with normal saline instead of OVA. The score of pathology and praxiology were observed when the SD rats in AR group appeared typical symptom of allergic rhinitis, and levels of IL-4, IFN-γ, IgE in the serum were examined by ELISA. According to the behavioral score, nasal histology and content of IL-4, IFN-γ, IgE of serum, Rat allergic rhinitis model were judged successfully established or not. RESULT: Behavioral scores were significantly increased in OVA-challenged rats compared with the control group, P<0.05. Nasal epithelial goblet cells, eosinophils and lymphocytes in nasal mucosa in the AR rats exhibited obvious increase relative to the control group. IL-4, IgE levels in the AR rat exhibited obvious increase relative to control group while INF-γ levels exhibited obvious reduction (P<0.05). CONCLUSION The allergic rhinitis models in SD rat by OVA were successfully established. The levels of IgE, INF-γ and IL-4 in Serum can be used as objective evaluation of animal models of allergic rhinitis established successfully or not.
Animals ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; immunology ; Goblet Cells ; immunology ; Immunoglobulin E ; blood ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Nasal Mucosa ; cytology ; pathology ; Ovalbumin ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic ; physiopathology

BACKGROUNDAberrant epithelial repair has been observed in chronic rhinosinusitis (CRS) patients; however, the mechanism of epithelial cell repair regulation is unclear. Epidermal growth factor (EGF) plays an important role in regulating epithelial cell repair in lower airway and may be a critical factor in the remodeling processes of CRS. The objective of our research is to evaluate the differences between CRS and normal subjects and between chronic rhinosinusitis without nasal polys (CRSsNP) and chronic rhinosinusitis with nasal polys (CRSwNP) in the regulation of EGF pathways and the regulating proliferative position of classic Ras/Raf/MEK/ERK pathways.

METHODSWe evaluated the proliferation rates of ethmoidal mucosal cells before and after stimulation with EGF, epidermal growth factor receptor (EGFR) kinase inhibitor AG1478, and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 using MTT assays. We also analyzed the sinonasal epithelial cells collected from control subjects and patients with CRS subtypes CRSsNP and CRSwNP for the expression of ERK1/2, phosphorylated ERK1/2, P21, P15, and P27 using western blotting analyses.

RESULTSThe proliferation rates of sinonasal epithelial cells before and after EGF stimulation were lower in CRS patients than in the controls. AG1478 or PD98059 inhibitor treatment of control epithelial cells did not result in a significant difference in proliferation. Although, AG1478 and PD98059 inhibited the proliferation of CRS cells, the degree of proliferation inhibition was markedly different in CRSsNP. AG1478 suppressed the proliferation of CRSwNP epithelial cells, whereas PD98059 had no effect. The ratio of ERK1/2 phosphorylation in CRS cells was lower than that of the control cells. Cyclin-dependent kinase inhibitors were highly expressed in CRS cells compared with that of control cells. ERK1/2 and P27 showed differential expression in CRSsNP and CRSwNP.

CONCLUSIONSDifferences existed in EGF pathways in CRS patients and normal subjects as well as in CRSsNP and CRSwNP. Classical Ras/Raf/MEK/ERK pathway may assume absolute superiority in control cells. Ras/Raf/MEK/ERK classical pathway and other pathways might be active at the same time to stimulate epithelial cell proliferation in CRSsNP. The function of Ras/Raf/MEK/ERK classical pathway was weaker in CRSwNP than in CRSsNP and when the classical pathway was blocked in CRSwNP, some other pathway could have completely compensated the proliferation induced by the Ras/Raf/MEK/ERK pathway.

Adult ; Aged ; Cell Proliferation ; drug effects ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; Female ; Humans ; Male ; Middle Aged ; Nasal Mucosa ; cytology ; Sinusitis ; metabolism

OBJECTIVE: To investigate the distribution of mast cells in nasal polyps. METHOD: Biopsy specimens from patients with nasal polyps (n = 20) and control patients (n = 8) were obtained and included in this study. The distribution of mast cells in nasal polyps and the expression of chemokines (CCL5, CCL11, CX3CL1, IL-8, IL-6) in the epithelial cells of normal nasal mucosa and nasal polyps was determined by immunohistochemistry. RESULT: Mast cells migrate to intraepithelial in nasal polyps and the expression of chemokines (CCL5, CCL11, CX3CL1, IL-8) was up regulated in the epithelial cells of nasal polyps compare to normal nasal mucosa. CONCLUSION Our findings showed that mast cells migrate to intraepithelial in nasal polyps and the over expression of chemotaxins (CCL5, CCL11, CX3CL1, IL-8) may be response for mast cells' migration in nasal polyps. Mast cells might be associated with the development of nasal polyps.
Chemokine CCL11 ; metabolism ; Chemokine CCL5 ; metabolism ; Chemokine CX3CL1 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Immunohistochemistry ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Mast Cells ; metabolism ; pathology ; Nasal Mucosa ; cytology ; metabolism ; Nasal Polyps ; metabolism ; pathology ; Up-Regulation

OBJECTIVE: To observe 18β-glycyrrhetinic acid (GA) impact on ultrastructure of tight junctions (TJs) of nasal mucosa epithelial cells in rats models of allergic rhinitis (AR). METHOD: Ninety-six Wistar rats were randomly divided into control group, model group, loratadine group, and 18β-glycyrrhetinic acid group, and each group had 24 rats. Ovalbumin was used to establish a rat AR model. The behavioral changes and the tight junctions of nasal epithelial were observed and compared in different groups after 2,4,6 and 10 weeks intervention. RESULT: The length of TJs in allergic rhinitis model became shorter, electron-high-density plasma membrane became thicker, number of the integration loci reduced and gap of TJs widened or even ruptured. With the consistent effect of allergens,the changes of TJs in the model group aggravated gradually,and the changes of ultrastructure of TJs in 18β-glycyrrhetinic acid group was relieved apparently compared to model group and even were close to the control model with time. CONCLUSION 18β-glycyrrhetinic acid can recover the ultrastructure of the tight junctions of AR rat nasal epithelial cells.
Animals ; Cell Count ; Epithelial Cells ; ultrastructure ; Glycyrrhetinic Acid ; analogs & derivatives ; pharmacology ; Nasal Mucosa ; cytology ; Ovalbumin ; Rats ; Rats, Wistar ; Rhinitis, Allergic ; drug therapy ; Tight Junctions ; drug effects

OBJECTIVE: To establish an in vitro method to culture mesenchymal stem cells(MSCs) derived from human nasal mucosa, and explore their stemness and differentiation potential. METHOD: Based on the observation of distribution of MSCs in human nasal mucosa, we cultured and proliferated MSCs in vitro and identified the expression of stem cell markers on them including Nestin, CD133, Vimentin and Sa114 with immunofluorescence. The MSCs were induced to differentiate to osteoblasts with medium containing dexamethasone, ascorbic acid and beta sodium glycerophosphate, and to neurons with Neurobasal medium containing B27, ATRA and TSA. Histochemistry and immunofluorescence were applied to evaluate the differentiation. RESULT: The nestin and vimentin immunofluorescence-positive MSCs existed extensively in human nasal mucosa. While the MSCs were cultured in the osteogenic-inducing medium, activities of alkaline phosphatase were increased significantly, and bone nodules were found on the surface of the osteoblasts by alizarin red staining. After the induction by neural-inducing medium, the MSCs adopted neuron like appearance with many slim protrusions interconnected as a network. The induced cells expressed neural markers NF-200 and BM88 strongly. CONCLUSION The MSCs derived from human nasal mucosa are multipotent stem cells and can be utilized as seed cells to repair bone or neural injury.
Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; cytology ; metabolism ; Multipotent Stem Cells ; Nasal Mucosa ; cytology ; Neurons ; Osteoblasts ; cytology

OBJECTIVE: To explore the expression and distribution of vimentin in different types of chronic rhinosinusitis and its significance. METHOD: There were four groups including control (10 samples), Eos CRSwNP (10 samples), non-Eos CRSwNP (12 samples) and CRSsNP (10 samples). The expression of vimentin in chronic rhinosinusitis were detected by immunohistochemistry technique. The double-immunofluorescence was used to detect the positive staining of both vimentin and E-cadherin, both of which were the marker of epithelial cells. RESULT: The positive staining of vimentin were observed both in epithelium and lamina propria. The expression of vimentin were found in myofibroblast, endothelium and other mesenchymal cells. The vimentin positive cells in epithelium were epithelial cells but not mesenchymal cells, as they also expressed E-cadherin. CONCLUSION The vimentin positive staining cells distribute in lamina propria and epithelium of both normal nasal mucosa and chronic rhinosinusitis. The positive staining epithelial cells may generate from epithelial-mesenchymal transition. So the vimentin may play an important role in the development of chronic rhinosinusitis.
Adult ; Antigens, CD ; Cadherins ; metabolism ; Chronic Disease ; Epithelial Cells ; metabolism ; Female ; Humans ; Male ; Mesoderm ; cytology ; metabolism ; Middle Aged ; Nasal Mucosa ; metabolism ; Sinusitis ; metabolism ; pathology ; Vimentin ; metabolism ; Young Adult