| Allergic rhinitis(AR)is a type I allergic disease mediated by immunoglobulin E.The exact pathogenesis of AR is not fully understood,with multiple inflammatory mediators,immune cells and neuropeptides all involved in this complex process.The conventional therapies currently available are unable to achieve the desired outcome.A variety of factors can trigger or exacerbate AR.In clinical work,it has been noted that some AR patients have symptoms such as runny nose,nasal congestion,burning sensation in the nose,etc.,which can be triggered or exacerbated by cold air or cold stimuli,and are more sensitive to cold air stimuli than the average AR patient.The transient receptor potential(TRP)ion channel family has been reported in the literature to be the main sensor of cold and warmth in the body,among which the transient receptor potential melastatin 8(TRPM8)ion channel is an important channel for cold stimulation.The TRPM8 ion channel is an important channel for the body to perceive cold stimuli.It has been found that TRP ion channels can act as temperature sensors to "sense" cold stimuli and trigger acute exacerbations of asthma and COPD in the lower airways,but the exact mechanism is not known.The upper and lower airways are thought to be "one airway,one disease" and TRPM8 has been identified in the nasal mucosa,but it is not clear whether and how it functions in AR to sense cold stimuli.PartⅠ: Expression level and correlation analysis of TRPM8 in cold stimulation sensitive AR patientsObjective: To investigate the clinical features and expression of TRPM8 and some epithelium-derived cytokines in nasal mucosal epithelial cells in patients with cold stimuli-sensitive AR.Methods: A total of 18 AR patients(8 patients sensitive to cold stimuli and 10 patients insensitive to cold stimuli)and 10 healthy controls at the same time were included in this study.Patients with AR were divided into cold stimulus sensitive and cold stimulus insensitive groups according to their performance after exposure to cold stimuli.The criteria for determining cold stimulus sensitivity were whether exposure to cold stimuli induced or aggravated nasal hyperresponsiveness such as runny nose,nasal congestion,burning sensation in the nose and subjective cold air hyperresponsiveness(SCH)A score of ≥2.The distribution characteristics and expression differences of TRPM8 protein in nasal mucosal tissue specimens from the AR and normal control groups were analysed by immunohistochemistry.Clinical demographic data,Ig E,Visual Analogue Scale(VAS)and SCH were also collected from the two groups.The three groups of human nasal mucosa primary epithelial cells were cultured in vitro and the m RNA of TRPM8 and epithelial-derived cytokines IL-33 and TSLP were measured by Quantitatie Reverse Transcription-Polymerase Chain Reaction(q RT-PCR).The m RNA expression of TRPM8 and the epithelial-derived cytokines IL-33 and TSLP in the three groups of nasal mucosal epithelial cells were measured separately by q RT-PCR.Results:TRPM8 protein was expressed in epithelial cells and submucosal glands in human nasal mucosal tissue,and the expression was significantly higher in the AR group than in the normal control group;there was no significant difference in demographic information and Ig E expression between the cold stimulation-sensitive AR group and the cold stimulation-insensitive AR group(p>0.05),and the Multi-VAS score and SCH score of the cold-sensitive AR patients were significantly higher than those of the cold stimulation-insensitive The relative m RNA expression of TRPM8,IL-33 and TSLP in primary nasal mucosal epithelial cells was significantly higher in the cold-stimulation-sensitive AR group than in the cold-stimulation-insensitive group(p<0.05),and the TRPM8 m RNA levels in both groups were positively correlated with VAS scores.Conclusion: TRPM8 is highly expressed in the epithelial cells of the nasal mucosa of AR patients.The induction of nasal symptoms or exacerbation of symptoms in cold-sensitive AR patients after cold stimulation may be related to the high expression of TRPM8 and epithelial-derived cytokines.Part II: Effect of transient receptor potential M8 ion channel on inflammatory response of nasal mucosal epithelial cellsObjective: Exploring the role played by TRPM8 in the cold stimulation/menthol-induced production of epithelium-derived factors by nasal mucosal epithelial cells.Methods: Human nasal mucosal epithelial cells(HNEp Cs cell line)were treated with cold air stimulation(18℃)and menthol,a TRPM8 channel agonist,respectively.The cells were divided into control group(cultured at 37℃),cold stimulation group(stimulated at 18℃),cold stimulation + BCTC group,menthol stimulation group and menthol + BCTC group as the intervention.The cell viability under different stimulation was measured by MTT;Ca2+ concentration in each group was measured by calcium imaging technique;m RNA transcript levels of TRPM8,IL-33 and TSLP in each group were measured by RT-PCR;the expression of interleukin IL-33 and TSLP secreted by each group was measured by ELISA.Results: The MTT results showed that the experimental menthol concentration did not inhibit the cellular activity of HNEp Cs;the relative intracellular Ca2+ concentration increased rapidly after cold stimulation and menthol intervention,and the relative Ca2+ concentration decreased in the cold stimulation + BCTC and menthol + BCTC groups compared with the cold stimulation and menthol stimulation groups;TRPM8,IL-33,TSLP m RNA and protein content increased significantly in the cold stimulation group compared with the control group(all P < 0.05),and decreased significantly in the cold stimulation + BCTC group compared with the cold stimulation group(all P < 0.05).In the cold stimulation group,TRPM8,IL-33,TSLP m RNA and protein levels were significantly higher than in the control group(all P < 0.05),and in the cold stimulation + BCTC group,they were significantly lower than in the cold stimulation group(all P < 0.05);in the menthol group,TRPM8,IL-33,TSLP m RNA and protein levels were significantly higher than in the control group(all P < 0.05),and in the menthol + BCTC group,they were significantly lower than in the menthol group(all P < 0.05).Conclusion: In vitro experiments have demonstrated that TRPM8 isoforms expressed in human nasal mucosal epithelial cells retain the characteristics of being activated by cold stimulation and menthol,and that both cold stimulation and menthol induce Ca2+ inward flow by activating TRPM8 ion channels in nasal mucosal epithelial cells,leading to increased expression and production of the epithelial-derived cytokines IL-33 and TSLP. |
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