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

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

This study explored the triggering mechanism of interstitial lung disease (ILD). We established the effects of immunogenic and neurogenic calcitonin gene-related peptide (CGRP) imbalance on the regulation of aquaporin 5 (AQP5) expression and the repair responses that promote the transition from alveolar epithelial cell (AEC) apoptosis to pulmonary fibrosis. Newly diagnosed ILD patients (n = 60) were enrolled, whose serological levels of beta-CGRP, alpha-CGRP, AQP5, receptor activity modifying protein 1, and receptor component protein were detected by ELISA. Th1 and Th2 cytokines and CD4(+)and CD8(+)cells were measured by flow cytometry method.In vivo, bleomycin (BLM) was set for modeling pulmonary fibrosis. ACALCA-HET model was set as a chronic pulmonary fibrosis model. Hematoxylin-eosin, immunohistochemistry, and Masson's trichrome staining were performed to assess the role of apoptosis in the injured lung. The concentrations of cytokines were determined by cytokine antibody arrays. Abnormal activation of serological CD4(+)T lymphocytes and predominant Th2 response was established in the patients with ILD. Moreover, the ratio of beta-CGRP/alpha-CGRP positively correlated with the increased level of AQP5 in patients with ILD.In vivo, a significant increase of AQP5 and beta-CGRP at the chronic stage of pulmonary fibrosis was induced by BLM in the mice model, whereas the expression of AQP5 protein was generally lower in the acute alveolitis phase. Moreover, the levels of AQP5 and alpha-CGRP in theCALCA-HET rats were lower than those of the normal saline group. The high ratio beta-CGRP/alpha-CGRP enhanced the expression of AQP5, inhibited transforming growth factor-beta 1 (TGF beta 1)/P-Smad1/Smad4 pathway, and upregulated the NRF2 signal, whereas the apoptosis of AECs was significantly reduced in late-stage pulmonary fibrosis. The imbalance of beta-CGRP/alpha-CGRP may be associated with the predominance of Th2 response and participate in the process of AEC apoptosis in lung fibrosis. The high ratio of beta-CGRP/alpha-CGRP may elevate the level of AQP5 through inactivation of the TGF-beta 1/smad1 signaling pathway and upregulation of the Nrf2 signaling in the chronic stage of pulmonary fibrosis.

本研究探讨了间质性肺疾病（ILD）的触发机制。我们确定了免疫源性和神经源性降钙素基因相关肽（CGRP）失衡对水通道蛋白5（AQP5）表达的调节作用，以及促进从肺泡上皮细胞（AEC）凋亡向肺纤维化转变的修复反应。纳入新诊断的ILD患者（n = 60），通过酶联免疫吸附测定（ELISA）检测其血清中β - CGRP、α - CGRP、AQP5、受体活性修饰蛋白1和受体组分蛋白的水平。通过流式细胞术检测Th1和Th2细胞因子以及CD4(+)和CD8(+)细胞。在体内，使用博来霉素（BLM）建立肺纤维化模型，设置ACALCA - HET模型作为慢性肺纤维化模型。进行苏木精 - 伊红染色、免疫组织化学染色和马松三色染色以评估凋亡在受损肺中的作用。通过细胞因子抗体阵列测定细胞因子的浓度。ILD患者存在血清CD4(+)T淋巴细胞异常激活和以Th2反应为主的情况。此外，ILD患者中β - CGRP/α - CGRP的比值与AQP5水平升高呈正相关。在体内，小鼠模型中BLM在肺纤维化慢性期诱导AQP5和β - CGRP显著增加，而在急性肺泡炎期AQP5蛋白的表达普遍较低。此外，CALCA - HET大鼠中AQP5和α - CGRP的水平低于生理盐水组。高比值的β - CGRP/α - CGRP增强了AQP5的表达，抑制了转化生长因子 - β1（TGF - β1）/P - Smad1/Smad4通路，并上调了NRF2信号，而在肺纤维化晚期AECs的凋亡显著减少。β - CGRP/α - CGRP的失衡可能与Th2反应占优势有关，并参与肺纤维化中AEC凋亡的过程。在肺纤维化慢性期，高比值的β - CGRP/α - CGRP可能通过使TGF - β1/smad1信号通路失活和上调Nrf2信号来提高AQP5的水平。