## 4 Discussion  
The bronchial epithelial barrier plays a pivotal role in maintaining respiratory homeostasis by preventing the penetration of inhaled allergens and pathogens while regulating  
Figure 6: The model of allergen-induced airway epithelium barrier dysfunction. ZO-1 immunofluorescence staining reveals allergen-induced widespread epithelial barrier disruption compared to a normal bronchial epithelium. Allergens like HDMs can directly cleave epithelial junctions or activate pattern recognition receptors (PRRs) such as PAR-2, C-type lectins (CLR) and purinergic receptors. Activation of these receptors leads to the degradation and/or mislocalization of junctional proteins. When epithelial repair is impaired, sustained loss of E-cadherin activates \(\beta\)-catenin pathways, further promoting epithelial-to-mesenchymal transition, goblet cell hyperplasia and ciliated cell loss, which are hallmarks of asthma-related epithelial changes. Created with BioRender.com. immune responses. Disruption of this barrier is a hallmark of asthma pathogenesis, contributing to inflammation, airway hyperresponsiveness and remodeling [5, 37, 44]. Enzymes in allergens act as key enhancers of allergic reactions [45]. Studies confirm that protease activity in allergens, including those from pollen and HDMs, disrupts epithelial integrity and activates innate immune responses [46]. HDMs degrade TJs by cleaving occludin and causing intracellular ZO-1 proteolysis, leading to epithelial barrier breakdown [47]. Allergen-derived proteases also activate protease-activated receptor-2, triggering the immune response via thymic stromal lymphopoetin (TSLP) production. Additionally, proteases in sensitized patients may increase epithelial permeability by disrupting adhesion proteins like occludin and claudin-1 [48]. In this study, we investigated the individual and combined effects of RW and HDM extracts on bronchial epithelial barrier integrity using a physiologically relevant air-liquid interface model of normal human bronchial epithelial cells. The use of an ALI culture model of NHBE cells is supported by numerous studies demonstrating the physiological relevance of this system due to the formation of functional TJs and the production of mucus [49, 50, 51, 52, 53]. Similarly, a study by [PERSON] et al. (2020) used ALI cultures to investigate the effects of HDMs on epithelial barrier function and found results consistent with those obtained in animal models [10, 54]. These studies validate our experimental approach and support the reliability of our results. The use of HDM and ragweed allergens is of significant interest due to the rising prevalence of sensitization and the increasing incidence of severe allergic forms of disease. The concentrations employed in this study were in line with those used in previous research, which has demonstrated significant biological effects on respiratory epithelial cells [55]. Moreover, a range of concentrations was utilized to explore the detrimental effects of various allergen exposures on the epithelial barrier. Notably, the 200 ug/mL concentration yielded the most robust response in TEER measurements, allowing for a comprehensive evaluation of the cellular response to these allergens. This aspect is important for understanding their potential damaging effects on bronchial epithelial cell cultures. Lower concentrations may not have induced a sufficiently strong response to reveal substantial disruption of the epithelial barrier, as displayed by ZO-1 architectural alterations demonstrated in this study, particularly in a controlled laboratory setting. Although the TEER assay is widely used, the TEER readings are highly dependent on the electrode positions and careful handling of the electrodes is required while introducing them into the well under test to avoid any disturbance to the cells. The uniformity of the current density generated by the electrodes across the cell layer has a significant effect on the TEER measurements [18, 34, 56].