The Role And Mechanism Of Canonical Transient Receptor Potential Channel In Nasal Polyps

BackgroundNasal polyps (NP) are a common disease of the upper airway; the incidence rate of NP is about1%-2%. The main pathological changes were:severe edema of loose connective tissue and chronic inflammation, however its pathogenesis has not been clarified.Recent studies have found that intracellular calcium plays a key role in immune cell activation, proliferation, secretion of cytokines and degranulation reaction function. However, its mechanism is not yet clear. TRPC channels are mammalian homologue proteins to transient receptor potential (TRP) of the Drosophila. TRPC can be activated by IP3and induce continuous calcium influx. Previous studies found TRPC channels were expressed in immune cells such as, neutrophils, eosinophils, and T lymphocytes, which mediated immune cell store-operated calcium influx. TRPC6deficiency inhibited specific allergic immune responses and eosinophil infiltration. However, the role of TRPC channels in nasal polyps development and mechanism are not in-depth study. So we put forward the following hypothesis:the expression of TRPC5channel in nasal polyps are increased, TRPC5channel might play an important role in the development of NP through the activation of NF-KB signal transduction pathway, increase of eosinophil infiltration and inflammatory cytokines.Objectives1. To assess the expression of classical transient receptor potential channel family in patients with nasal polyps; 2. To study the relationships between TRPC channel expression and eosinophil infiltration and inflammatory reaction in nasal polyps;3. To study the relationship between TRPC channel expression and nuclear transcription factor NF-κB in nasal polyp.Methods1. The inclusion of patients with nasal polyps and specimen collection：From2011May to2011December in Department of Otolaryngology, Qilu Hospital of Shandong University, we randomly selected58patients with nasal polyps to undergo polypectomy and35patients who received correction of the deviation of nasal septum. Inferior turbinate mucosa specimens were considered as normal control.2. Real-time RT-PCR was used to detect mRNA expression of TRPC channels in patients with nasal polyps.3. Histopathology and immunohistochemistry techniques were used to detect distribution of TRPC channel, IL-6, eosinophil number and nuclear transcriptionfactor NF-κB in nasal polyp tissues.4. Western blot was used to detect protein expression of TRPC channel, inflammatory factor IL-6and nuclear transcriptionfactor NF-κB in nasal polyp tissues. Analyze the relationship between them.Results1. Analysis of clinical data Nasal polyps patients and control groups did not differ in age and gender.2. mRNA and protein expressions of TRPC channel in nasal polypsTRPC5channel mRNA expression level was significantly higher than that of control group (P<0.01). Other members of TRPC were not obviously changed (P>0.05). Compared with normal mucusa, TRPC5channel protein expression was significantly increased in NP (P<0.01).3. Esinophils infiltration analysisEosinophils showed obviously eosinophilic cytoplasm and a strong reflection of bright red granules in HE staining. Compared with normal mucusa, eosinophil number was significantly raised in NP tissues (P<0.01).4. Protein expression of IL-6Compared with normal mucusa, IL-6protein expression was significantly increased in nasal polyps (P<0.01). Immunohistochemistry showed that IL-6positive products were mainly located in the cytoplasm and intercellular substance, with buffy granules stain.5. NF-κB expressionWerstern blot results also confirmed the expression of protein p65tion and normal control group no difference (P>0.05), but the phosphorylation level of p65expression was significantly higher than that in normal mucusa (P<0.01).6. Correlation analysis between expressions of TRPC channel with characteristics in nasal polypsTRPC5channel expression was positively correlated with eosinophil infiltration, inflammatory factor and the phosphorylation level of p65(P<0.01).Conclusions1. TRPC5channel is upregulated in nasal polyps;2. TRPC5channel plays an important role in the development of nasal polyps through the activation of NF-κB signal transduction pathway, increase of eosinophil infiltration and inflammatory cytokines. BackgroundMany factors participated in the pathogenesis of nasal polyps(NP). The patho-physiological basis is allergic reaction and chronic inflammation of nasal mucosa. There are a lot of inflammatory cytokines, chemical medium and chemokines in NP, including IgE, which plays a key role in the eosinophil activation and degranulation. We found that TRPC5channel was upregulated in NP and that TRPC5channel could affect eosinophil infiltration, inflammatory cytokines and activation of NF-κB signal transduction pathway. However, the mechanism of canonical transient receptor potential channel5in NP is unclear.IgE levels are increased in peripheral blood and tissues in NP. IgE can stimulate the nasal polyps to produce more histamine, leukotrienes, PGD2, resulting in nasal mucosa edema and inflammatory reaction. Other studies have found that IgE can regulate the intracellular calcium concentration and ion channels. Recently, TRPC5channels can be stably expressed in HEK293cells, which provides a simple and feasible method to study the function and regulatory mechanism of TRPC5channel. So we hypothesized that expression of IgE was increased in nasal polyps, and that IgE induced intracellular calcium increasing and store operated calcium influx, which was medieated by TRPC5channel.Objectives1. To assess IgE expression in nasal polyps; 2. To study the function of TRPC5channel expressed in vitro;3. To explore whether calcium regulation in cells induced by IgE is mediated by TRPC5channels.Methods1. Detect the expression of IgE in nasal polyp tissue specimens by immunohistochemical technique.2. TRPC5stably expressed HEK293cells (HEK-TRPC5):Cells was incubulated in DMED-F12supplemented with10%fetal bovine serum,100μg/ml streptomycin,100U/ml penicillin and1.5%sodium bicarbonate at37℃,5%CO2and95%air. TRPC5expression was induced by1μg/ml tetracycline (Te), for HEK-TRPC5cells carried a tetracycline transcriptional repressor.3. Calcium imaging:Cells was incubated with Ca2+fluorescent marker Fura-2AM (2μM) at37℃for1h. The fixed emission wavelength was510nm, time series of fluoresce were recorded by a dual wavelength scanning in340nm and380nm with TILL Vision image acquisition software. Fluorescent signal ratio between340nm and380nm reflects the concentration of intracellular calcium. To study the effect of different concentrations of IgE on intracellular calcium concentration mediated by TRPC5.3The patch clamp electrophysiological recording:Cells were cultured on glass coverslips and placed in the perfusion chamber at room temperature. The recordings were made by the application of EPC-10patch clamp amplifier and Pulse software. TRPC5channel currents were recorded by whole-cell mode of patch clamp recording. To study the effect of10μg/ml IgE on TRPC5channel currents.Results1. Expression of IgE in NP:Compared with normal mucusa, IgE protein expression was significantly increased in nasal polyps (P<0.01).2. Intracellular calcium increase mediated by TRPC5channel:in Te induced cells (Te+), calcium imaging showed that TRPC5channel activator Gd3+(100μM) increased intracellular calcium (P<0.05), which could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μg/ml).3. Store-operated calcium influx mediated by TRPC5channel:In Te induced cells (Te+), thapsigargin (Tg) was used to deplete intracellular calcium stores in calcium-free solutions, and then the bath solution was changed to standard bath solution with2mM Ca2+, resulting in significantly intracellular calcium increasing which phenomenon was called store-operated calcium influx. In Te-cells, there was only a slighter store-operated calcium influx4. Effect of IgE on intracellular calcium increase mediated by TRPC5channel:In the Te induced cells (Te+), calcium imaging showed that different concentrations of IgE (0.1μg/ml,1μg/ml,10μg/ml) increased intracellular calcium (P<0.05), which could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μ g/ml).5. Effect of IgE on store-operated calcium influx mediated by TRPC5channel:In Te induced cells (Te+), there was store-operated calcium influx which could be strentherned by pre-stimulation with10μg/ml IgE for30min (P<0.05).6. Effect of IgE on TRPC5channel currents:In the Te induced cells (Te+), patch clamp recordings showed that TRPC5channel activator Gd3+(100μM) induced TRPC5channel currents (P<0.05) and that10μg/ml IgE also induced channel currents (P<0.05), which both could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μg/ml).Conclusions1. IgE expression was increased in nasal polyps.2. Increased IgE could activate TRPC5channels resulting intracellular calcium increase and store-operated calcium influx enhancement.