The Effect Of NF-κB Pathway On The Expression Of AQP5 On The Nasal Epithelial Cell Of Rats With Allergic Rhinitis And The Study On The Hyper-secretion Of Allergic Rhinitis

Objective1.The purification,culture and identification of rat primary nasal epithelial cells.2.To detect the mechanism by which Cyclic adenosine monophosphate-dependent protein kinase-A (cAMP-PKA) pathway regulates aquaporin 5(AQP5) on rat nasal epithelial cells.3.The establishment and evaluation of the rat model with allergic rhinitis (AR).4.To detect the effect of NF-κB pathway on the expression of AQP5 in the nasal mucosa of AR rats.5.To detect the possible mechanism on the hyper-secretion of AR.6. To detect the cross-talk between the NF-κB and cAMP-PKA pathways in AR.Methods1.The primary nasal epithelial cells were purified and cultivated by differential adhesion and defined medium,and were identificated by immunocytochemical staining,and estimated their purity2.The nasal epithelial cells were treated with PKA inhibitor H89 (3μM) or the cAMP activator forskolin (1μM) for 12h and 24h. CCK-8 was applied to detect the growth of the cultured nasal epithelial cells.AQP5 and p-CREB(ser133) were detected by immunocytochemical staining, western-blotting or Real-time PCR.3.The animal model of rats with allergic rhinitis was established by the general and local sensitization with ovalbumin (OVA).Eosinophils in the nasal secretions and the nasal mucosa of model group were detected by Wright's staining.The level of IgE in peripheral blood was detected by enzyme-linked immunosorbent assay. Theanimal model with AR was evaluated by behavioral scores.4.AR rats were treated with NF-κB inhibitors pyrrolidinedithiocarbamate ammonium (PDTC 100mg/kg/d and 50mg/kg/d). the NF-κBp65,p-CREB(ser133),AQP5, IL-1β, TNF-αand IL-6 in the nasal mucosa were detected by immunohistochemistry, Real-time PCR or western-blotting.5.AR rats were treated with H89(5mg/kg/d) or forskolin(5mg/kg/d). p-CREB(ser133), NF-κBp65,AQP5,IL-1β,TNF-αand IL-6 in the nasal mucosa were detected by immunohistochemistry, Real-timePCR or western-blotting.6.The protein expression of MUC5AC和MUC5B of each group were detected by western-blotting.Results1.The rat nasal epithelial cells were purified and cultured by differential adherence and defined culture medium,which were identificated by immunocytochemical staining.The purity of the cells were more than 95%.2.H89 or forskolin made no difference on the growth of the rat nasal epithelial cells, After the treatment with 3μM H89,compared with group C,the positive cells of p-CREB(Ser133) and AQP5 decreased, the expression of p-CREB(Ser133) and AQP5 protein was decreased,and the mRNA level of AQP5 decreased;After the treatment with 1μM forskolin,compared with group C, the positive cells of p-CREB(Ser133) and AQP5 increased,and the expression of p-CREB(Ser133) and AQP5 protein was decreased,and the mRNA level of AQP5 increased.3.The model of SD rat with AR was established by by the general and local sensitization with ovalbumin (OVA).Compared with group C,the cilias on nasal mucosa tissue was off or flattened,the number of vessels,glands and eosinophile granulocytes increased,the level of IgE in peripheral blood was elevated,and the behavioral scores increased.4.Compared with group C,the mean optical density(MOD) and protein expression of NF-κBp65 of group M increased significantly,the mRNA level of IL-1β,TNF-αand IL-6 of group M increased,the MOD and protein expression of p-CREB of group M decreased,and the MOD, protein expression and mRNA level of AQP5 of group M decreased significantly.After the treatment with PDTC(100mg/kg/d and 50mg/kg/d) for ld,3d or 5d,compared with group M,the MOD and protein expression of NF-KBp65 decreased significantly,the mRNA level of IL-1β,TNF-αand IL-6 decreased,the MOD and protein expression of p-CREB(ser133) increased,and the MOD,protein expression and the mRNA level of AQP5 increased significantly.5.After the treatment with H89(5mg/kg/d) for 1d,3d or 5d,compared with group M,the MOD and protein expression of NF-κBp65 increased significantly,the mRNA level of IL-1β,TNF-αand IL-6 were up-regulated,the MOD and protein expression of p-CREB(Ser133) were reduced, the MOD,and protein expression and mRNA level of AQP5 decreased.After treatment with forskolin (5mg/kg/d) for for 1d,3d or 5d,compared with group M,the MOD and protein expression of NF-κBp65 decreased significantly,the mRNA level of IL-1β,TNF-αand IL-6 were down-regulated,the MOD and protein expression of p-CREB were elevated,the MOD,protein expression and mRNA level of AQP5 increased.6.The protein expression of MUC5AC or MUC5B in group M increased significantly compared with group C,but decreased after the treatment with PDTC(100mg/kg/d and 50mg/kg/d) for 1d,3d or 5d.After the treatment with H89 (5mg/kg/d)for 1d,3d or 5d, The protein expression of MUC5AC and MUC5B protein expression increased significantly,while they decreased in a time-dependent manner after the treatment with forskolin(5mg/kg/d) for ld,3d or 5d.Conclusions1.Rat nasal epithelial cells with high purity of more 95% were cultured by differential adherence and defined culture medium.2.The cAMP-PKA pathway is involved in the regulation of AQP5 in rat nasal epithelial cells by phosphorylating CREB at serine 133.3.The model with allergic rhinitis can be successfully established by general and local sensitization with OVA.4.The NF-κB pathway can down-regulate the expression of AQP5 by inhibiting CREB phosphorylation at serine 133 or by competitive binding to CBP with p-CREB(Serl33).5.The NF-κB pathway can up-regulate the protein expression of the MUC5AC and MUC5B,which maybe be involved in the hyper-secretion of AR, and the decrease of AQP5 results in the clearance dysfunction of the secretions,which promotes the hyper-secretion of AR 6.The cAMP-PKA pathway can inhibit the NF-κB pathway.