IL-13 Regulates The Proliferation Of Nasal Epithelial Through MTOR/p70S6K1 Pathway And Predictive Value Of Adenoid-nasopharyngeal Ratio In The Diagnosis Of Otitis Media With Effusion

Part Ⅰ IL-13 regulates the proliferation of nasal epithelial through mTOR/p70S6K1 pathwayBackground and Objectives:Chronic rhinosinusitis(CRS)is typically divided into two types,chronic rhinosinusitis with nasal polyps(CRSwNP)and chronic rhinosinusitis without nasal polyps(CRSsNP).According to the endotype of CRS,it is divided into type 1,type 2 and type 3 inflammation.Compared with non-type 2 inflammation,type 2 inflammation has more serious symptoms and higher recurrence rate.The pathogenesis of CRSwNP was mainly focused on type 2 inflammation.The development of nasal polyps(NP)is associated with abnormal tissue remodeling of the nasal epithelium.One of the histopathological characteristics of NP remodeling is the excessive proliferation of basal cells.The overexpression of basal cell marker P63 is associated with epithelial hyperplasia in patients with NP.The expression of the proliferation marker Ki67 in NP is up-regulated,and IL-13,as the main factor of type 2 inflammation,can promote the proliferation of various cells.However,the function of IL-13 in promoting the proliferation of nasal epithelium has yet to be clearly identified.PI3K/Akt/mTOR pathway plays an essential biological function in the physiological processes of cell growth,proliferation,differentiation,and apoptosis.70 kDa ribosomal protein S6 kinase 1(p70S6K1),one of the direct downstream substrates of mTOR,plays an important role in cell proliferation and cell cycle progression and may perform its physiological function by promoting G1/S phase transition.This study aimed to explore the expression level and functions of mTOR and p70S6K1 in CRSwNP,and verify that IL-13 promote the overexpression of mTOR/p70S6K1 pathway,increasing the degree of proliferation of NP and ultimately leading to the occurrence of CRSwNP.Methods:Tissue studies:1)67 cases of CRSwNP and 29 cases of healthy control patients were included in this study.NP samples and turbinate mucosa samples were obtained through nasal endoscopic surgery.2)Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to detect the mRNA expressions of IL-13,mTOR,p70S6K1,P63,Ki67,cyclin E1 and CDK2 between healthy control and NP.3)Western blotting and immunohistochemical(IHC)staining were performed to detect the protein expressions of mTOR,p-mTOR,p70S6K1,p-p70S6K1,P63,Ki67,cyclin E1 and CDK2 in the healthy control and NP.Cell studies:1)HNEpCs cell lines were divided into four groups:a,control group;b,IL-13 stimulation group;c,IL-13+mTOR inhibitor treatment group;d,IL-13+p70S6K1 inhibitor treatment group.Cells were collected for western blotting to detect the protein expressions of mTOR,p-mTOR,p70S6K1,p-p70S6K1,P63,Ki67,cyclin E1 and CDK2.EdU proliferation assay were performed to detect the proliferation ability after 48h cultured.2)Human nasal epithelial stem/progenitor cells(HNESPCs)were isolated from the turbinate mucosa of the healthy controls and transplanted into transwell membranes for culture.The cells were divided into 4 groups in expansion phase:a,control group;b,IL-13 stimulation group;c,IL-13+mTOR inhibitor treatment group;d,IL13+p70S6K1 inhibitor treatment group.Cells were collected 5 days later,western blotting to detect the protein expressions of mTOR,p-mTOR,p70S6K1,p-p70S6K1,P63,Ki67,cyclin E1 and CDK2,immunofluorescence(IF)co-staining with P63 and Ki67 after 5 days cultured.3)The HNESPCs were transplanted into transwell membranes to construct an air-liquid interface(ALI)system and stimulated with IL-13 in the phase of expansion and expansion+differentiation phase.The cells were collected for western blotting to detect the protein expressions of mTOR,p-mTOR,p70S6K1,p-p70S6K1,P63,Ki67,cyclin E1 and CDK2.,and IF co-staining with P63 and Ki67 until the cells were fully differentiated into human nasal epithelial cells.Results:1.The expression of mTOR/p70S6K1 signaling pathway-related molecules such as p-mTOR,p-p70S6K1,and P63 in NP tissues were significantly up-regulated compared with the control group.2.The expressions of Ki67,cyclin E1 and CDK2 in NP were up-regulated compared with the control group.3.After IL-13 stimulation,the expressions of p-mTOR,p-p70S6K1,P63,Ki67,cyclin E1 and CDK2 in HNEpCs cell lines and HNESPCs were significantly up-regulated compared with the control group.In the IL-13+mTOR inhibitor treatment group and IL13+p70S6K1 inhibitor treatment group,the expressions of P63,Ki67,cyclin E1,and CDK2 decreased significantly compared with the IL-13 stimulation group.3.After adding IL-3 stimulation into the ALI,the expressions of p-mTOR,p-p70S6K1,P63,Ki67,cyclin E1,and CDK2 were significantly up-regulated compared with the untreated group.However,there was no statistical significance between the proliferative stage stimulation and the whole stage with IL-13 stimulation.Conclusions:IL-13 could promote the proliferation of nasal epithelial cells through the mTOR/p70S6K1 signaling pathway,which may affect the development of CRSwNP.mTOR/p70S6K1 signaling pathway can be used as a clinical target for treating CRSwNP.Part Ⅱ Predictive value of adenoid-nasopharyngeal ratio in the diagnosis of otitis media with effusionObjectives:Adenoid hypertrophy is one of the most common diseases in children.It can compress the eustachian tube,affect the fluid-air exchange of the middle ear,and lead to negative pressure and outflow of the tympanum.Otitis media with effusion(OME)may occur secondarily.OME is the most common childhood ear disease and one of the leading causes of hearing loss in children.Long-term failure can lead to speech retarding and decreased academic performance in children.And the disease can lead to adhesive otitis media,cholesterol granuloma and the formation of inverted tympanic pockets,unopen tympanic membranes,and then the construction of cholesteatoma.Adenoid hypertrophy is often examined by nasal endoscopy and lateral nasopharyngeal X-ray or CT.Adenoid-nasopharyngeal(A/N)ratio means the adenoid size takes the proportion of the nasopharyngeal and is an objective index to evaluate adenoid hypertrophy.The size of the adenoid and the proportion of the adenoids in the nasopharyngeal airway can be well reflected.Tympanometry is commonly used to detect middle ear pressure in clinical practice.It can be used to evaluate the abnormal function of the eustachian tube and is also of high value for the diagnosis of OME.Curve B is typical of OME,with a positive predictive value of 49%to 99%.Most children with OME cannot express properly or are not detected by their parents.Portion of them can heal themselves,but the rest of children with a long history may lead to further serious consequences.Therefore,detecting the abnormality of middle ear function in children and avoiding OME is of great clinical significance.However,to what extent adenoid volume growth will lead to abnormal middle ear function in children has yet to be proposed.This study analyzed and compared the risk factors between patients with abnormal middle ear function and those without abnormal middle ear function to determine whether the A/N ratio can be used as a clinical indicator for predicting abnormal middle ear function in children with adenoid hypertrophy.The cut-off value of the A/N ratio was calculated to predict whether patients with adenoid hypertrophy were complicated with middle ear dysfunction to improve the hearing attention of doctors and parents of patients with adenoid hypertrophy.Methods:Clinical data of 316 children with adenoid hypertrophy who underwent adenoidectomy in the nasal department of Shandong Province ENT Hospital from 2019 to 2022 were collected.Primary data(age and sex)were collected,the Lund-Mackay score was performed using Cone Beam Computerized Tomography(CBCT),and results of tympanometry,behavior audiogram,nasal endoscopy,allergen detection,and blood analysis were collected.Patients were divided into two groups according to Liden/Jerger classification.Type B and type C were the groups with the tympanogram abnormal group(TAG),and the rest were those with the tympanogram normal group(TNG).SPSS was used for statistical analysis,and the Mann-Whitney U test was used to compare whether there were differences in the A/N ratio between the two groups.The correlation between middle ear dysfunction and the A/N ratio and other factors,such as gender,age,blood eosinophils,and Lund-Mackay scores,were verified by binary logistics regression analysis.The diagnostic value and cut-off value of the A/N ratio and other possible factors related to middle ear dysfunction were determined by the Receiver Operating Characteristic curve(ROC)curve.Results:1.The A/N ratio in the TAG was significantly higher than that in the TNG,and the A/N ratio in both the unilateral and bilateral tympanogram abnormal groups were significantly higher than that in the TNG.Still,the difference between the unilateral and bilateral tympanogram abnormal groups was not statistically significant.There was a statistical difference between the A/N ratio and the Visual obstruction ratio in the same patient,but these two ratios do not have too much disparity,and there was a positive correlation between the A/N ratio and Visual obstruction ratio.2.The age of children in the TAG was significantly younger than that in the TNG group,and the duration of adenoid hypertrophy was significantly shorter than that in the TNG.Age and disease duration were negatively correlated with the A/N ratio,respectively.3.The results of CBCT and Lund-Mackay scores showed that the Lund-Mackay scores in the TAG were significantly higher than that in the TNG.There was a negative correlation between the Lund-Mackay scores and the negative middle ear pressure of patients,indicating that patients with abnormal middle ear function were more likely to be complicated with chronic rhinosinusitis(CRS).4.Allergen detection results showed no significantly difference in the two group patients.Blood analysis showed that the eosinophil count and ratio in the TAG were significantly lower than those in the TNG.5.Multivariate logistics regression analysis showed that only the A/N ratio was significantly correlated with abnormal middle ear function.ROC curve was used to analyze the A/N ratio,and the cut-off value was 0.815,indicating that when the A/N ratio reached 0.815,children with adenoid hypertrophy may have abnormal middle ear function.The area under the curve(AUC)was 0.747,which had a medium diagnostic value—sensitivity:75.3%,specificity:64.3%.Conclusions:Patients with adenoid hypertrophy combined with middle ear dysfunction were more likely to have increased A/N ratio,get higher Lund-Mackay scores.Patients with chronic rhinosinusitis were more likely to get middle ear dysfunction.According to the multivariate logistics regression analysis results,the A/N ratio has the highest predictive value for adenoid hypertrophy combined with abnormal middle ear function among the above factors.When the A/N ratio is bigger than 0.815,it indicates that the patients with adenoid hypertrophy may be complicated by abnormal middle ear function.