Dexamethasone Affects Tight Junction Function By Nitric Oxide And Related Mechanisms In Chronic Rhinosinusitis With Nasal Polyps

ObjectiveNOS expression level in nasal mucosa tissue;The role and related molecular mechanisms of dexamethasone（Dex）impacts the expression of tight junctions（TJs）through nitric oxide（NO）production;the diagnose value of nasal nitric oxide（nNO）in eosinophilic chronic rhinosinusitis with nasal polyps（Eos CRSwNP）.Methods1)The protein expression level and m RNA expression level of TJs and inducible nitric oxide synthase（iNOS）and endothelial nitric oxide synthase（eNOS）in nasal mucosa tissues were detected by immunohistochemistry,qRT-PCR and Western blot.2)Human nasal epithelial cells（HNECs）were isolated from the inferior turbinate of control subjects.Then,the HNECs were stimulated with different concentrations IL-13 at different times,suitable stimulated conditions were used in TJs damage cellular model.3)The TJs damage cellular model were added Dex in different concentrations.Then,the expression level of TJs,NO production and PI3K/Akt/eNOS were performed by qRT-PCR and Western blot.Immunofluorescence were using to visualize the structure of HNECs and evaluate the mean fluorescence intensity of Occludin.Apoptosis was determined by flow cytometry.4)eNOS inhibitor or Akt inhibitors was added at the same time as Dex in TJs damage cellular model.The expression of TJs and PI3K/Akt/eNOS pathway were performed by qRT-PCR and Western blot;NO production was detected by grisess assay.5)Medical records of 84 non-Eos CRSwNP patients,55 Eos CRSwNP and 37control subjects were retrospectively reviewed.Clinical relevance of nNO and other clinical indexes were assessed.In addition,the diagnosed value of nNO in Eos CRSwNP were evaluated.Results1)Tissue experiments:The expression of TJs were decreased in the nasal poly of Eos CRSwNP,the differences were statistically significant（p<0.05）;The expression of iNOS was increased in nasal polyps compared with control group（p<0.05）;The expression of iNOS in Eos CRSwNP was not significantly changed compared with non-Eos CRSwNP group;The expressions of p-eNOS were significant elevated in Eos CRSwNP compared to the non-Eos CRSwNP group（p<0.001）.2)TJs damage cellular model:After 36h stimulation of IL-13（10ng/ml）,the expression of TJs decreased most obviously,the in vitro model was successfully established.3)The influence of stimulation of Dex in TJs damage cellular model:Stimulation of Dex(10^-5mol/L)promote NO production and the expression of TJs recovery;Fluorescence intensity of Occludin was decreased and loose structure in cellular model,Dex significantly recovered this phenomenon（p<0.05）;Expressions of p-Akt and p-eNOS were significantly evaluated by Dex（p<0.001）;In addition,Dex was resistant to IL-13 induced apoptosis in HNECs（p<0.05）.4)The inner mechanism of influence of stimulation of Dex in TJs damage cellular model:Akt inhibitors（MK-2206）and eNOS（L-NAME）inhibitor can decreased the expression of TJs and the NO production（p<0.05）.The effect of Dex in increasing the expression of TJs and NO production was significantly inhabited by MK-2206 and L-NAME（p<0.05）.5)Clinical retrospective analysis:Compared with non-Eos CRSwNP group,the nNO level of Eos CRSwNP group was significantly increased（p<0.001）.In Eos CRSwNP,nNO level has a positive correlation with EOS percentage（r=0.307,p=0.023）;nNO level has a negative correlation with Lund-Mackay score（r=-0.507,p<0.001）.Morover,the nNO diagnosed model（nNO+Posterior ethmoid score+EOS percentage）has potential diagnostic implication in Eos CRSwNP（AUC=0.869）.Conclusion1)Tissue experiments:The expression of TJs were decreased in nasal polyps compared with control group;The expression of TJs were significantly decreased in Eos CRSwNP compared with non-Eos CRSwNP group;The expression of p-eNOS was increased in Eos CRSwNP group compared with non-Eos CRSwNP group.2)In vitro experiments:TJs damage cellular model was successful established by IL-13;Dex participate in the regulatory processes of NO production through PI3K/Akt/eNOS pathway in HNECs and affects the expression of TJs;Dex can inhibit IL-13-induced apoptosis in HNECs.3)Clinical retrospective analysis:nNO has positive correlation with eosinophilic infiltration and negative correlation with degree of inflammation in Eos CRSwNP;nNO has some diagnose value in Eos CRSwNP.