Mechanisms Of The Regulation Of Epithelial Barrier Function By ZEB1 In Antrochoanal Polyp

Objective:Antrochoanal polyp(ACP)is a kind of benign hyperplasia of the nasal and paranasal sinuses with unknown etiology.Recently,a study confirmed that ACP was dominated by T1/3-type inflammation,leading to tissue remodelling processes.Tight junctions(TJs)and adherent junctions(AJs)are the main components of the protective epithelial barrier and form the key structure that shields the body from harmful external substances.Damaging this structure accelerates tissue remodelling in disease.ZEB1 is an important regulatory factor in epithelial remodeling.Whether ACP is involved in the mechanism of epithelial barrier damage is not clear.We aimed to observe the abnormal expression of epithelial barrier-associated proteins(ZO-1,Claudin-3,Occludin and Ecadherin)in ACP,and the regulatory effect of ZEB1 on IL-17A-mediated tight junctions was investigated.Methods:The samples were collected from 20 controls,37 patients with ACP and 45 patients with nasal polyposis(NP).Quantitative real-time PCR(qRT-PCR)and immunefluorescence staining(IF)were performed to analyze the mRNA expressions of cell junction markers(ZO-1,Claudin-3,Occludin and E-cadherin)and ZEB1 in the nasal tissues.In addition,the level of IFN-γ and IL-17A expression in control and ACP groups was examined using qRT-PCR.In human nasal epithelial cell line(hNEpCs),ZEB1 small interference RNA(siZEB1)and ZEB1 over-expression plasmid(OE-ZEB1)were used to stimulate,respectively,to detection expression levels of ZO-1、Claudin-3、Occludin and Ecadherin by qRT-PCR and Western blotting.Primary human nasal epithelial cells(hNECs)were cultured for 2-4 generations and then stimulated with si-ZEB 1 and rhIL-17A at 8h intervals to assess the expression levels of TJs and AJs-related markers,respectively.The expression levels of ZO-1,Claudin-3,Occludin and E-cadherin were detected.Results:1.qRT-PCR showed that mRNA expression levels of ZO-1,Occludin and Ecadherin in ACP group were significantly down-regulated compared with control and NP group;The expression level of Claudin-3 mRNA in ACP group was significantly lower than that in control group.IF staining displayed that the staining intensity of TJsrelated markers and AJs-related markers in the ACP and NP groups was significantly decreased,and no difference was observed between the NP and ACP groups.2.In terms of age at disease onset and BMI,ACP group were generally lower than healthy control group and NP group.Spearman analysis showed that mRNA levels of ZO-1,Claudin-3 and Occludin were negatively correlated with Lund-Mackay score,respectively.3.ZEB1 was significantly elevated in the ACP group compared to the control group based on qRT-PCR and IF staining.4.In hNEpCs,OE-ZEB1 group were down-regulated mRNA and protein expression levels of ZO-1,Claudin-3,Occludin and E-cadherin compare with NC group,while si-ZEB1 group were up-regulated celll junction markers mRNA and protein expression.5.Significantly higher levels of IL-17A and IFN-γ expression in patients with ACP were detected by qRT-PCR.6.In hNECs,cells were stimulated by IL-17A resulted in decreasing expression of cell junction related markers(ZO-1,Claudin-3,Occludin and E-cadherin),and the damage degree of cell junction markers were partly recovered after stimulation with IL-17 and si-ZEB1 group compared with IL-17A group alone.Conclusion:We preliminary detected that the epithelial barrier was severely damaged in ACP,and the damage degree related to the severity of the disease.But no significant difference was observed between ACP and NP groups.Higher expression of ZEB1 in ACP.Furthermore,ZEB1 upregulation was found to disrupt cell tight junctions in hNEpCs.And downregulation of ZEB1 alleviated IL-17A-mediated disruption of epithelial tight junctions in hNECs.In conclusion,ZEB1 regulates IL-17A mediated tight junctions and is involved in the pathogenesis of ACP.