Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 25 6 2026 09 02 908 918 Jie Deng Department of Otorhinolaryngology Head and Neck Surgery, Air Force Medical Center, PLA, Beijing, China Hongyi Liu Department of Otorhinolaryngology Head and Neck Surgery, Air Force Medical Center, PLA, Beijing, China Yiran Zang Department of Otorhinolaryngology Head and Neck Surgery, Air Force Medical Center, PLA, Beijing, China Zhihan Wei Department of Otorhinolaryngology Head and Neck Surgery, Air Force Medical Center, PLA, Beijing, China Xue Yue Department of Otorhinolaryngology Head and Neck Surgery, Air Force Medical Center, PLA, Beijing, China Zhanguo Jin Department of Otorhinolaryngology Head and Neck Surgery, Air Force Medical Center, PLA, Beijing, China 2025 07 31 2025 10 11 The research intended to elucidate the synergistic effects of eosinophils (Eos) and mast cells (MCs) on human nasal epithelial cells (HNEpCs) in the context of allergic rhinitis (AR), focusing on inflammation, tight junction protein expression, and DNA damage. Cell proliferation capacity was measured using the Cell Counting Kit-8 (CCK-8) method, apoptosis was examined via the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and inflammatory cytokine levels were assayed via enzyme-linked immunosorbent assay. Western blotting evaluated the protein abundance of tight junction proteins (ZO-1, Occludin) and CD40L/CD40. Immunofluorescence was used to detect phosphorylated histone H2AX (γH2AX) (DNA damage), as well as subcellular ZO-1/Occludin distribution. Co-immunoprecipitation (Co-IP) was used to analyze the CD40L-CD40 interaction between Eos and MCs. Eos and MCs significantly reduced HNEpC viability and enhanced apoptosis, with the most pronounced effects in the AR+Eos+MC group. Inflammatory cytokine levels were markedly elevated in the Eos+MC and AR+Eos+MC groups, with the highest concentrations observed in the AR+Eos+MC group. Western blot and immunofluorescence analyses showed decreased expression of ZO-1 and Occludin in treatment groups compared to Control, along with a shift in their localization from the cell membrane to the cytoplasm. γH2AX expression, indicating DNA damage, was significantly elevated, with the highest levels observed in the AR+Eos+MC group. Co-IP analysis confirmed enhanced CD40L-CD40 interaction involving Eos and MCs within the Eos+MC and AR+Eos+MC groups. Eosinophils and MCs synergistically promote inflammation, disrupt the nasal epithelial barrier, and exacerbate DNA damage. The CD40L-CD40 pathway serves an essential function in their interaction, providing a potential therapeutic target for AR. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/4534 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/4534/2318