A Study Of BDNF Effection In The Basal Cells Of Nasal Polyp Epithelium Through TRKB/PI3K/AKT Signaling Pathway And The Pathogenesis Of Nasal Inverted Papilloma

1.BackgroundNasal polyps（NP）is the most common benign tumor in nasal cavity.The tumor presents exogenous growth into the nasal cavity or sinuses,causing the blockage of nasal cavity or mouth of the sinuses.As a result,patients with nasal polyps are often accompanied by chronic rhinosinusitis（CRS）.The clinical manifestations are presented as nasal congestion,runny nose,headache,hyposmia,and some other symptoms.The histopathological characteristics of nasal polyps showed a large amount of inflammatory cell infiltration,high edema of the stroma of nasal polyps,and hyperplasia of polyps’ epithelium,often accompanied by squamous metaplasia and goblet metaplasia.The pathogenesis of nasal polyps is still not fully understood.This high recurrence rate of benign nasal tumors caused by multiple factors has a great impact on the life quality of patients,and also caused a high burden on the clinical medical management.Chronic rhinosinusitis with nasal polyps（NP）often accompanied by hyperplasia of the epithelium,the epithelial proliferation of NP is mainly concentrated in the basal cells of epithelium,but the cause of the excessive proliferation of epithelial basal cells is not clear.The brain-derived neurotrophic factor（BDNF）is upregulated in the nasal lavage fluid of patients with allergic rhinitis,and its expression in inflammatory diseases of the central nervous system is upregulated compared with normal tissues.Moreover,in lung tumors,the expression of BDNF can be detected significantly higher than that of normal tissues.BDNF has a variety of physiological functions,which can promote cell proliferation and inhibit cell apoptosis through growth factor receptors and PI3K/AKT signaling pathway.In this study,we explore the expression site of BDNF in the epithelial cells of nasal polyps and its abnormality to epithelial cells,also the effect of BDNF in the proliferation of epithelial cells in nasal polyps.2.Methods24 healthy control and 24 nasal polyps were collected from Shandong Provincial ENT Hospital.All samples were identified by HE staining,which conformed to the histopathological characteristics of nasal polyps and were diagnosed as nasal polyps by pathologists.We divided the collected samples into three equal parts,one of which was soaked in 4%PFA for tissue sections.Total ribonucleic acid（RNA）and tissue protein were extracted from the remaining two samples respectively.2.1 The differentiation model of human nasal epithelial stem cells in vitro was established in the laboratory.The epithelial cells were obtained by dissociating the mucosal epithelium of the healthy control inferior turbinate with disperse enzyme and trypsin.The epithelial cells were cultured in airway epithelial proliferation medium to obtain nasal epithelial stem cells.When the epithelial stem cells proliferated to a sufficient amount,the air-liquid interface cell differentiation model was established by using Transwell chamber.The differentiated nasal epithelial cells were obtained after being cultured in airway epithelial differentiation medium for 4 weeks.The cells were collected for immunofluorescence detection.2.2.Human nasal epithelial cells transfactionThe nasal epithelial cell line was transfected with BDNF overexpression plasmid by transfection and drug stimulation,and high concentration of LPS was added to stimulate the cells to simulate the injury effect of inflammation on the cells.The cells were divided into four groups:normal control group,BDNF Vector group,BDNF Vector + LPS group and LPS group.After 48 hours of stimulation,the cells were collected for immunofluorescence,qPCR and WB detection.2.3.Immunohistochemical（IHC）staining and immunofluorescence（IF）stainingIHC and IF were performed on paraffin sections of healthy controls and nasal polyps by immunohistochemical（Immunohistochemical,IHC）staining and immunofluorescence（Immunofluorescences,IF）staining to determine the expression of BDNF in NP and healthy controls.The optical density of BDNF in healthy controls and nasal polyps was analyzed by software to calculate its expression in healthy controls and nasal polyps.Through immunofluorescence staining,BDNF was co-stained with the specific markers of basal cells,ciliated cells and goblet cells of polyp epithelium to determine the expression pattern of BDNF in nasal polyp epithelium.Phosphorylated AKT was co-stained with basal cell markers to analyze the expression of phosphorylated AKT in nasal polyp epithelial basal cells.The IF staining of nasal epithelial cells in vitro was used to determine the expression of BDNF in different cells.Immunofluorescence staining of HNECs was used to analyze the expression of BDNF under different experimental conditions.2.4 qPCRdetected the expression of TRKB/PI3K/AKT signaling pathway related molecules TRKB,PI3K and AKT by qPCR,and the expression of proliferation-related cytokines KI67,CDK2,3CyclinE1 and CyclinA2 mRNA in healthy control and NP tissues downstream of AKT by extracting tissue total RNA,and detected the mRNA expression of apoptosis-related cytokines such as Caspase-9,Caspase-3 and BAX.The expression of antiapoptotic BCL2 mRNA in healthy controls and NP was detected.The total RNA of nasal epithelial cell line（HNECs）was extracted and detected by qPCR.The proliferation activity of HNECs was analyzed under different stimulation conditions.The mRNA expression of proliferation-related cytokines KI67,CDK2,CyclinE1 and CyclinA2 was detected,and the mRNA expression of apoptosis-related cytokines such as Caspase-9,Caspase-3 and BAX was detected.2.5 Western BlottingWB was used to detect the expression of KI67,CDK2,CyclinE1 and CyclinA2 related to cell proliferation in healthy control and NP tissues,and to detect the expression of apoptosis-related factors.The expression of the above cytokine proteins related to cell proliferation and apoptosis was detected.Results3.1BDNF can be expressed in the epithelium and subepithelium of healthy controls and NP tissues.And the expression of BDNF was upregulated in NP.The basal cells of NP epithelium account for the highest proportion of BDNF positive cells,accounting for 60%,ciliated cells account for about 30%,and goblet cells account for about 10%.3.2 In NP tissue,the expression of cytokines related to TRKB/PI3K/AKT signaling pathway such as TRKB,PI3K,pAKT was significantly up-regulated compared with healthy controls,the expression of CyclinA2,CyclinE1,CDK2,KI67 was up-regulated,and the expression of P21 was down-regulated.3.3We further analyzed the expression of apoptosis-related cytokines Caspase-9 and Caspase-3 directly affected by AKT,and found that the expressions of Caspase-9 and Caspase-3 in NP were down-regulated compared with healthy controls.And the expression of BAX,which is related to apoptosis,was down-regulated and the expression of BCL-2 was up-regulated in NP.3.4The correlation analysis between BDNF and KI67 showed that there was a significant positive correlation between them.After gene overexpression and apoptosis induced by high concentration of LPS in HNECs cell line.we found that BDNF could inhibit apoptosis induced by high concentration of LPS and stimulate the activation of TRKB/PI3K/AKT signal pathway.The secretion of proliferation-related cytokines such as KI67,CDK2 and CyclinE1 increased,while the expression of programmed cell death-related cytokines such as Caspase-3 and Caspase-9 decreased.BDNF can also inhibit the expression of P21 and promote cell cycle.4.ConclusionBDNF promotes the proliferation of epithelial basal cells in nasal polyps and inhibits their apoptosis,and plays an important role in the pathogenesis of nasal polyps.It can be used as a clinical target for the treatment of nasal polyps to inhibit the occurrence and development of polyp tissue.Background Nasal inverted papilloma（NIP）,as a common borderline tumor of nasal cavity next to nasal polyps,accounts for about 0.5% of nasal tumors,and it is easy to recur after surgical resection.About 10% of nasal inverted papilloma will turn into squamous carcinoma in the nasal cavity,seriously affecting the quality of life of patients.The pathogenesis of nasal inverted papilloma is still unclear,its pathological features are often described as: obvious proliferation of epithelial cells and fmger-like projections protruding into the sub-epithelium;a large number of inflammatory cell infiltration,the largest proportion of neutrophils;there is a complete basement membrane between the epithelium and the subepithelial matrix to separate the epithelium from the matrix.However,there are few studies on the pathological mechanism,occurrence and development of NIP.We systematically analyzed the pathological features of NIP and found that the basement membrane of its epithelium was destroyed and inflammatory cells infiltrated into the epithlium of nasal inverted papilloma,suggested that neutrophils may be an kay factor in the pathogenesis of NIP.The main components that can dissolve matrix and basement membrane are matrix metalloproteinases（MMP）,and the cytokines that can affect the expression of matrix metalloproteinases including hypoxia inducible factor（HIF-1 alpha）.Therefore,we want to further clarify the pathogenesis of nasal inverted papilloma through the study of MMP and HIF-1 alpha in nasal inverted papilloma.Methods We collected 37 samples of nasal inverted papilloma and 24 healthy control.The infiltration and proportion of various inflammatory cells such as neutrophils,eosinophils,macrophages and other inflammatory cells in nasal inverted papilloma were systematically analyzed by HE staining,immunohistocheraical staining,immunofluorescence staining,qPCR and Western blotting.At the same time,the expression levels of matrix metalloproteinases in neutrophils,eosinophils,macrophages,mast cells,CD4/CD8 positive T cells and FOXP3 positive cells were analyzed.The expression status and expression of hypoxia inducible factor were also detected.Reasonably speculate the pathological mechanism of nasal inverted papilloma.Results Through HE staining,we found that the epithelium of nasal inverted papilloma proliferated,bent,squeezed and deformed,and the epithelial hyperplasia was obvious.Compared with the normal control tissue,the subepithelial basement membrane became thinner in nasal inverted papilloma.The subepithelial matrix protrudes into the epithelium to form multiple "finger-like projections" structures.Finger-like projections contains fibroblasts,inflammatory cells（mainly neutrophils）,new capillaries and matrix collagen.And in or around the top of the finger-like projections,there are a large number of basement membrane destroyed,especially in the neutrophils gathering site,there are discontinuous basement membrane,infiltrated inflammatory cells in the basement membrane.The main expression sites of matrix metalloproteinase-1 and matrix metalloproteinase-7 were in the epithelial cells of nasal inverted papilloma,and matrix metalloproteinase-9 was mainly secreted by neutrophils,their distribution was highly consistent with that of the finger-like projections,and the increasing trend of matrix metalloproteinase-9 was the most obvious in nasal inverted papilloma.The expression of matrix metalloproteinases is up-regulated in nasal inverted papilloma,whereas their inhibitor,tissue inhibitor of metalloproteinases,is down-regulated in nasal inverted papilloma.Hypoxia induce factor alpha is co-located with matrix metal loproteinases-9? and its expression is significantly up-regulated in nasal inverted papilloma.Conclusion The excessive infiltration of neutrophils in nasal inverted papilloma secretes a large amount of matrix metalloproteinase-9 and hypoxia-inducible factor alpha,destroys the basic structure of the basement membrane,makes the subepithelial matrix protrude into the epithelium and causes epithelial folding and curl.The nasal inverted papilloma shows a very characteristic pathological form.