| PART Ⅰ: TRPM8 promotes epithelial-mesenchymal transition of normal human nasal epithelial cells Objective: As a subtype of TRP,the main function of TRPM8 is the temperature sensing channel of cold stimulation.It’s found that TRPM8 expressed in both bronchial epithelial cells and nasal epithelial cells.The overexpression of TRPM8 in tumor cells of breast cancer can induce the epithelial-mesenchymal transition(EMT)process and promote the occurrence and development of the disease.Whether TRPM8 can induce EMT in airway epithelial cells when the expression of TRPM8 is up-regulated has not been confirmed.This study aims to investigate whether epithelial-mesenchymal transformation will occur when TRPM8 is overexpressed in normal nasal epithelial cells by specific stimulation.Methods: Therefore,the TRPM8-specific agonist WS-12 was used to stimulate the upper airway epithelial cell line RPMI 2650.After setting different concentrations and different time gradients to determine the experimental conditions,the expression of TRPM8 and EMT-related markers were detected by real-time quantitative PCR and western blot and immunofluorescence.The normal primary epithelial cells of human nasal mucosa cultured in vitro were stimulated by WS-12 under the same conditions,and the morphological changes of the cells were observed by microscope,and the expression of TRPM8 and EMT-related markers by western blot.The expression levels of TRPM8 and EMT-related markers and Smad3 and P-Smad3 were detected by western blot after ws-12,Smad3 inhibitor SIS3 and TGF-β1 stimulation,respectively.The expression levels of TGF-β1 in epithelial cell culture medium supernatant after WS-12 stimulation were detected by ELISA.Results: After WS-12 stimulation,the expression of TRPM8 was increased in both RPMI 2650 cell lines and human nasal normal primary epithelial cells.Meanwhile,the expression of E-cad which was epithelial markers associated with EMT was decreased,while the expression of α-SMA and vimentin which were interstitial markers was increased,suggesting that EMT process was promoted.The results of immunofluorescence also further confirmed that when the expression of TRPM8 in RPMI 2650 was increased,the expression of EMT marker E-cad was down-regulated.The primary nasal epithelial cells would change from oval shape to long spindle shape after WS-12 stimulation,further confirming the EMT process was promoted.After the expression of TRPM8 was upregulated,the EMT process was not promoted after the addition of SIS3,suggesting that Smad3 pathway may be the downstream of TRPM8 regulating EMT.However,the expression of TGF-β1 was not affected after the expression of TRPM8 was up-regulated,suggesting that TRPM8 may regulate EMT by affecting Smad3 phosphorylation through other signal pathways.Conclusion: The stimulation of TRPM8 specific agonist can induce EMT in normal nasal epithelial cells in vitro experiments.Meanwhile,TRPM8 may regulate EMT in nasal epithelial cells through the Smad3 pathway.The characteristic may be related to the pathogenesis of nasal diseases closely related to EMT.PART Ⅱ: Research on regulation mechanism of TRPM8 on human nasal epithelial cells of chronic rhinosinusitis with nasal polypsObjective: The nasal epithelial cells(NEp Cs)are a powerful barrier against external irritation of the upper respiratory airway.When pathogens or harmful substances invade the nasal mucosal epithelium and damage its integrity,it will cause inflammatory changes of nasal cavity and sinus mucosa.In recent years,melastatin-related transient receptor potential 8(TRPM8)has been found to be the protein of cationic channels located on cell membranes and it is widely distributed.TRPM8 protein participates in the abnormal changes of cell molecular biology by mediating calcium ion influx.We found that TRPM8 can enhances the EMT of nasal epithelial cells in previous study.Chronic rhinosinusitis with nasal polyps(CRSw NP)is one of the most common diseases in otolaryngology.Studies have suggested that nasal epithelial cells of nasal polyps in CRSw NP also undergo epithelial-mesenchymal transition,but the mechanism of that has not been clarified.This study aims to investigate the localization and expression of TRPM8 in CRSw NP and verify whether it is related to EMT in nasal epithelial cells of CRSw NP.The effect of TRPM8 on the formation of nasal polyps was further verified by animal model.Methods: Cluster analysis was performed on nasal polyp-related chips screened from GEO database by R language to observe the expression of TRP family proteins in which TRPM8 was located.The expression and localization of TRPM8 in nasal polyps tissues were detected by IHC.The control group,chronic rhinosinusitis without nasal polyps(CRSs NP)group and the CRSw NP group were collected and grinded into tissue homogenized.The expression of TRPM8 protein was detected by western blot after 24 h stimulation after homogenate was added into the medium of RPMI 2650.The human nasal primary epithelial cells of CRSw NP patients were cultured in vitro and verified by immunofluorescence.After knocking down the expression level of TRPM8 in cultured polyp epithelial cells,the expression levels of p-Smad3 were detected by western blot and the EMT-related markers were detected by quantitative real-time PCR and western blot.C57/BL6 J mice aged 6-8 weeks were randomly divided into 5 groups with 5-7 mice in each group.One group(group A)set as the control group and did not receive drug stimulation.The other four groups were intraperitoneally injected with a PBS suspension of ovalbumin(OVA)+ aluminum hydroxide(ALUM)for 5 days,followed by nasal drip treatment using OVA once a day for a week and followed by 3 times a week for 4 weeks.One group(group B)was then given PBS by nasal drip treatment 3 times a week for 8 weeks;one group(group C)received OVA+ Staphylococcus aureus bacteriotoxin(SEB)by nasal drip treatment 3 times a week for 8 weeks;one group(group D)received intraperitoneal injection of OVA+SEB by nasal drip treatment + TRPM8-specific inhibitor AMG-333 by injecting 3 times a week for 8 weeks;one group(group E)received intraperitoneal injection of OVA+SEB by nasal drip treatment + dexamethasone by injecting 3 times a week for 8 weeks.After modeling,mucosal tissues of mice were taken out completely,and histological changes between each group were observed by hematoxylin-eosin staining.In addition,the number of polypoid changes between each group was counted.The serum OVAspecific immunoglobulin E expression level was detected by ELISA.Results: Microarray analysis showed that the expression of TRPM8 in polyps of CRSw NP was significantly higher than in uncinate tissues of CRSw NP(P<0.01).Compared with the control,the expression of TRPM8 was significantly up-regulated in nasal polyps(P<0.05).TRPM8 was mainly expressed in epithelial cells.The expression of protein of TRPM8 in the epithelial cells of CRSs NP had no difference with the control group(P>0.05).By adding the homogenization of control and CRSs NP and CRSw NP tissues,the expression of TRPM8 in RPMI 2650 cells was changed.Compared with adding the homogenization of the group of control and CRSs NP,the expression of TRPM8 in RPMI 2650 cells was significantly increased after adding the homogenization of the group of CRSw NP.It suggested that the inflammatory environment of CRSw NP promoted the expression of TRPM8 in epithelial cells.After down-regulation of TRPM8 expression in human primary nasal epithelial cells derived from CRSw NP,it was found that the expressions of E-cad was increased,while the expressions of α-SMA and vimentin were decreased,suggesting that the epithelialmesenchymal transformation phenomenon occurred during the development of polyps was inhibited.Meanwhile,the expressions of p-Smad3 were decreased.In animal experiments,compared with the control mice,nasal mucosal tissues of nasal polyps model mice showed obvious polypoid changes,mucosal thickening and fold increasing,and epithelial ciliary arrangement disorder.Serum OVA-s Ig E levels in nasal polyps model mice were also elevated.Although inflammatory changes were also observed in the TRPM8 inhibitor treatment group and the dexamethasone treatment group,the number of polypoid lesions was less than that in the nasal polyp model group.The expression level of serum OVA-s Ig E in mice treated with TRPM8 inhibitor and dexamethasone was also lower than that in mice treated with polyps.Inhibition of TRPM8 expression in epithelial cells can decrease the formation of nasal polyps.Conclusion: The expression of TRPM8 was up-regulated in nasal primary epithelial cells with chronic rhinosinusitis with nasal polyps,and down-regulation of TRPM8 may inhibit the EMT process in nasal polyps epithelial cells.Animal experiments verified that inhibition of TRPM8 expression did have a certain inhibitory effect on the development of polyps.TRPM8 may provide a new direction for the treatment strategy of chronic sinusitis with nasal polyps. |
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