Effect of TFF3 on tight junction protein in eosinophilic chronic sinusitis and its related mechanism

Abstract

Objective: To study the effect of trefoil factor family (TFF) 3 on the expression of tight junctions (TJs) in the nasal mucosa epithelium of eosinophilic chronic rhinosinusitis (eCRS) and its mechanism. Methods: From September to December 2020, eligible patients from the Department of Otorhinolaryngology of the First Affiliated Hospital of Nanchang University were recruited, including 11 control patients and 37 patients with chronic rhinosinusitis with nasal polyps (CRSwNP), from whom nasal mucosa and nasal polyp tissue samples were collected. Immunohistochemistry (IHC) was used to detect the localization and expression intensity of TFFs (TFF1, TFF2 and TFF3) and TJs (occudin, claudin-1 and ZO-1) in nasal mucosa. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the mRNA and protein expression. A cell model of tight junction injury in human nasal epithelial cells (HNECs) through stimulation with interleukin (IL)-13 was also established. The optimal modeling concentration and time for HNECs were determined, which were subsequently treated with TFF3 and/or a phosphoinositide 3-kinase (PI3K)-specific inhibitor (LY294002). Finally, RT-qPCR and WB were used to assess the effects of TFF3 on tight junctions and the PI3K/serine/threonine kinase (Akt) signaling pathway. Data were analyzed statistically using GraphPad Prism 7 software. Results: IHC results showed that the expression of TFF1 and TFF3 in nasal mucosa of eCRS group was significantly higher than that of control group (t=4.62, P=0.002; t=5.89, P<0.001), respectively, mainly expressed in goblet cell. The expression of occludin, claudin-1 and ZO-1 in the nasal mucosa of the eCRS group was lower than that of the control group (occludin t=3.98, P=0.019; claudin-1 t=5.15, P=0.002; ZO-1 t=5.42, P=0.001), respectively. WB results showed that the expression of TFF3 in non-eosinophilic chronic sinusitis (Non-eCRS) group and eCRS group was higher than that in the control group (t=3.62, P=0.036; t=5.93, P<0.001). The expression of occludin, claudin-1 and ZO-1 in eCRS group was lower than that in the control group (occludin t=5.14, P=0.002; claudin-1 t=6.35, P<0.001; ZO-1 t=6.64, P<0.001), respectively. The RT-qPCR results showed that compared with the control group, the levels of TFF1 and TFF3 mRNA were increased in the nasal mucosal epithelium of the Non-eCRS and eCRS groups (TFF1 t=3.98, P=0.046, t=4.89, P=0.002; TFF3 t=3.50, P=0.044, t=6.78, P<0.001). There was no statistically significant difference in TFF2 mRNA levels between the Non-eCRS and eCRS groups (t=1.34, P=0.061; t=3.37, P=0.055). Compared with the control group, Non-eCRS and eCRS groups showed a decrease in the mRNA levels of occludin, claudin-1 and ZO-1 (occludin t=4.27, P=0.011, t=5.61, P=0.007; claudin-1 t=3.62, P=0.036, t=6.80, P<0.001; ZO-1 t=3.47, P=0.047, t=7.86, P<0.001). The mRNA levels of TFF3 and TJs in eCRS nasal mucosa tissue showed a moderate positive correlation (occludin r=0.661, claudin-1 r=0.614, ZO-1 r=0.548, all P<0.001); TFF1 showed a low degree of positive correlation with the expression of occludin, claudin-1 and ZO-1 (occludin r=0.467, P=0.040; claudin-1 r=0.362, P=0.012; ZO-1 r=0.425, P=0.025). The establishment of cell models showed that compared with normal HNECs, the mRNA expression of TFF3 was most significantly increased at a concentration of 50 ng/ml stimulated by IL-13 (t=3.72, P=0.013); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.18, P=0.031; claudin-1 t=3.86, P=0.010; ZO-1 t=5.16, P=0.002). The expression of TFF3 mRNA increased most significantly after 15 hours of IL-13 stimulation (t=3.14, P=0.034); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.97, P=0.010; claudin-1 t=4.78, P=0.004; ZO-1 t=5.16, P=0.004). TJs damage model could be established by treating HNECs with 50 ng/ml IL-13 for 15 hours. Intervention experiments showed that compared with the IL-13 group, the IL-13+TFF3 group showed an increase in TJs mRNA expression (occludin t=6.10, P=0.009; claudin-1 t=5.90, P=0.013; ZO-1 t=9.44, P=0.007). Compared with the IL-13 group, the expression of TJs protein in the IL-13+TFF3 group increased (occludin t=3.23, P=0.013; claudin-1 t=9.40, P=0.017; ZO-1 t=2.23, P=0.032); The expression of TJs protein decreased in the IL-13+TFF3+LY294002 group (occludin t=4.73, claudin-1 t=8.77, ZO-1 t=3.51, all P<0.001). Compared with the IL-13+TFF3 group, the IL-3+TFF3+LY294002 group showed a decrease in PI3K and p-Akt/Akt protein expression (PI3K t=13.29, p-Akt/Akt t=10.30, all P<0.001). The increased mRNA and protein expression of occludin, claudin-1 and ZO-1 induced by TFF3 were also inhibited by LY294002. Conclusion: TFF3 can up-regulate the expression of occludin, claudin-1, and ZO-1 through PI3K/Akt pathway, and has a certain protective effect on the nasal mucosal epithelial barrier, providing a new idea for treating eCRS.