Maladjustment of β-CGRP/α-CGRP Regulation of AQP5 Promotes Alveolar Epithelial Cell Apoptosis to Pulmonary Fibrosis

Background: To explore the triggering mechanism of interstitial lung disease (ILD) by establishing the effects of immunogenic and neurogenic calcitonin gene-related peptide (CGRP) imbalance on the regulation of AQP5 expression and the repair responses to promote alveolar epithelial cell (AEC) apoptosis in pulmonary fibrosis. Methods: Newly diagnosed ILD patients (n=60) were enrolled, whoseserological levels of β-CGRP, α-CGRP, AQP5, RAMP1, and RCP were detected by ELISA. Th1 and Th2 cytokines and CD4+ and CD8+ cells were measured by flow cytometry method (FCM). In vivo, bleomycin (BLM) was set for modeling pulmonary fibrosis. The CALCA-HET model was set as a chronic pulmonary fibrosis model. Hematoxylin-eosin (H&E), immunohistochemistry, Masson's trichrome staining, and TUNEL staining was performed to assess the role of apoptosis in the injured lung. The concentrations of cytokines were determined by cytokine antibody arrays. Results: Abnormal activation of serological CD4+T lymphocytes and predominant Th2 response was established in patients with ILD. Moreover, the ratio of β-CGRP/α-CGRP was positively correlated with the increased level of AQP5 in patients with ILD. In vivo, a significant increase of AQP5 and β-CGRP at the chronic stage of pulmonary fibrosis induced by bleomycin in mice model, whereas the expression of AQP5 protein was the generally decreased in the acute alveolitis phase. Moreover, the levels of AQP5 and α-CGRP in the CALCA-HET rats were elevated. The high ratio βCGRP/α-CGRP enhanced the expression of AQP5, inhibited TGFβ1/PSmad1/Smad4 pathway, and upregulated NRF2 signal, whereas the apoptosis of alveolar epithelial cells was significantly reduced in late-stage pulmonary fibrosis. Conclusions: The high ratio of β-CGRP/α-CGRP induced predominant Th2 response and promoted AEC apoptosis through inactivation of the TGFβ1/smad1 signaling pathway and upregulation of the Nrf2 signaling in the chronic stage of pulmonary fibrosis. Funding Statement: This work was financially supported by the National Natural Science Foundation of China (no.81800070 to Xiaoting Lv and no.81871293, no.81571613 to Qicai Liu), the Joint Fund for Program of Science innovation of Fujian Province (no.2016Y9011 to Feng Gao), the Sail Foundation of Fujian Medical University (no.2016QH063 to Xiaoting Lv). Declaration of Interests: The authors have no competing interests to declare. Ethics Approval Statement: This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Fujian Medical University (no. [2016]156). Consent for participation and publication the case reports was obtained. The animal experiment was approved by the Experimentation Ethics Committee on Animal Rights Protection of Fujian Medical University Animal Experiment Center (Fujian, China). During the experiments, the animals were treated in compliance with the “Guiding Opinions on the Ethical Treatments of Laboratory Animals” published by the Ministry of Science and Technology in 2006. All procedures strictly followed the National Institutes of Health guidelines for the care and use of laboratory animals.