从自噬/溶酶体途径探讨组织蛋白酶D对特发性肺纤维化中Ⅱ型肺泡上皮损伤的分子机制及加减补肺汤的干预研究

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic lung disease. Current research suggests that IPF results in injury primarily to the alveolar epithelial type II cells (AEC II). Maintaining AEC II stability is very important to preventing or limiting the progression of IPF..Our previous research shows that the cathepsin D (Cat D) has a close relationship to idiopathic pulmonary fibrosis. However, it is not clear whether AEC II injury leads to cathepsin D activation, or if Cat D activation determines the development and direction of IPF. .The AEC II damage models used in this study are (1) a bloemycin (BLM) induced pulmonary fibrosis mice model, and (2) cells incubated with bloemycin. This study uses small interfering RNA (siRNA), western blotting, confocal laser, and real-time quantitative fluorescence PCR. In vivo and in vitro observations will examine the influence of lysosome membrane injury to Cat D activation and apoptosis of alveolar epithelial type II cells in the IPF models. This study will explore the Cat D signal transmission mechanisms in AEC II damage and in the progression of IPF; it will identify the potential target for Jiajianbufei decoction in the treatment of IPF; and it will elucidate the mechanisms of alveolar epithelium cells injury in IPF, and of traditional Chinese medicine in intervention. This will provide new directions for clinical protocols in the prevention and treatment of idiopathic pulmonary fibrosis.

特发性肺纤维化(IPF)是一种慢性、进行性恶化的肺间质疾病。肺泡Ⅱ型上皮细胞（AECⅡ）是IPF损伤的主要场所，维持AECⅡ的稳定对于肺纤维化的发生发展有重要的作用。目前认为，AECⅡ损伤可能是引起肺纤维化的关键因素。前期研究发现Cat D与IPF的发展有密切关系，但AECⅡ损伤是否导致Cat D活化并决定肺纤维化的发展方向尚不清楚。本研究拟通过博来霉素诱导IPF小鼠和AECⅡ损伤模型，运用RNA干扰技术、蛋白印迹、激光共聚焦和实时荧光定量PCR等方法，观察IPF过程中AECⅡ溶酶体膜的损伤对Cat D的活化、凋亡的影响。本研究将阐明Cat D在IPF中AECⅡ损伤的信号传导机制，揭示加减补肺汤抗IPF的潜在靶点，为深入探索IPF的发病机理和中药干预IPF的潜在靶点提供可靠的证据，以期为临床防治IPF提供新思路。

肺纤维化主要是肺组织受损后修复调节失控、重建异常所引起的病变，在纤维化发生发展过程中，大量的细胞因子表达异常、炎症细胞浸润，进而引起上皮细胞缺损、成纤维细胞增生和细胞外基质积聚，最终使成纤维细胞替代了行使正常功能的肺泡上皮细胞，导致了纤维化的发生。自噬对维持细胞及机体稳态具有重要意义，在纤维化的发生发展中自噬蛋白的激活受TGF-β1的影响。. 中医认为肺纤维化属“肺痹肺痿”的范畴，治疗应紧扣“补肺益肾、化瘀通络”的治则，在中医辨证与辨病相结合的理论指导下，根据气虚、痰浊、瘀血阻肺病机，以治病求本，标本同治为治则，以益气、活血化痰为治法。. 本研究主要探讨临床的经验方加减补肺汤抗肺纤维化的潜在靶点和可能作用机制，尝试阐明肺纤维化过程中组织蛋白酶D在自噬/溶酶体途径中的角色，为丰富中医药理论补肺健脾，活血化痰的组方提供实验依据。在研究中，建立了加减补肺汤的药物提取质控标准、复制了特发性肺纤维化小鼠模型和Ⅱ型肺泡上皮细胞损伤模型，通过整体动物实验、离体细胞模型，明确了加减补肺汤对实验性肺纤维化的药理学作用，其可能机制与自噬溶酶体途径相关因子的表达密切相关；并探讨了加减补肺汤联合甲强龙、地塞米松等激素对早期炎症治疗的协同作用，开展了以加减补肺汤中君药黄芪为主的有效部位治疗实验性IPF的系列探索；明确了加减补肺汤对实验性肺纤维化的药理学作用，其机制与自噬溶酶体途径相关因子的表达密切相关，丰富了中医药与激素联合治疗IPF的实验内涵，明确了黄芪甲苷Ⅳ和槲皮素具有激活细胞自噬抑制肺泡上皮细胞的EMT作用。