肺泡上皮细胞来源外泌体调节自噬在肺纤维化形成中的作用机制

Pulmonary fibrosis (PF) is the end-stage change of various interstitial lung diseases with the complicated pathogenesis and the poor prognosis. Our previous study confirmed that autophagy inhibition promoted the progress of pulmonary fibrosis, but the regulation of autophagy in pulmonary fibrosis formation mechanism is still not fully clear, especially for the key target cells in the pulmonary fibrosis of alveolar epithelial cells (AEc) and lung fibroblasts (LF) communication.Exosome can mediate the intercelluar material transfer and communication. In the early stage, exosome has been extracted from the culture of alveolar epithelial cells, but it is not clear what component, how to regulate the proliferation and transformation of LF and the formation of pulmonary fibrosis through autophagy. This project will establish the AEc and LF co-cultivation system to observe the characteristic of proliferation, differentiation and apoptosis of LF. AEc-derived exosomes are harvested and added to LF, cell differentiation, proliferation and apoptosis were tested. AEc-derived exosomes dealing with LF are co-cultured with AEc to observe the AEc differentiation.The autophagy associated miRNA and target protein and signaling pathway in AEC Exo were detected by high-throughput sequencing, and the function of the special miRNA and target protein was verified. The model of bleomycin induced pulmonary fibrosis were established, and the level of miRNA and target protein in alveolar lavage fluid were detected. Pulmonary fibrosis and autophagy were detected through interfering with the miRNA and target protein expression. This project will further clarify the mechanism of autophagy in pulmonary fibrosis and lay a solid foundation for the screening of drug therapy for pulmonary fibrosis.

肺纤维化(PF)是各种间质性肺疾病的终末期改变，发病机制复杂，预后差。我们前期研究证实了自噬抑制促进了肺纤维化的进展，但肺纤维化形成中自噬的调控机制，尤其是关键的肺泡上皮细胞(AEc)和肺成纤维细胞(LF)之间相互作用机制仍不清楚。外泌体可以介导细胞间的物质转运和信息传递，前期已经从肺泡上皮细胞培养中提取外泌体，但何种成分、如何调节自噬影响LF的增殖转化及在肺纤维化形成的作用并不清楚。本项目通过提取AEc来源的外泌体（Exo）及各成分，建立AEc与LF共培养体系观察对LF增殖、分化、凋亡和自噬的影响。高通量测序检测AEc-Exo中自噬表达相关miRNA、靶向蛋白及通路预测，并验证miRNA和靶向蛋白功能。构建肺纤维化模型，检测肺泡灌洗液中外泌体miRNA及靶向蛋白，干预miRNA及靶蛋白表达后对肺纤维化及自噬的影响。本课题的完成将进一步阐明自噬在肺纤维化中的作用机制。

特发性肺纤维化（IPF）是一种慢性的、进行性的肺部疾病，其发病率逐年增长。肺泡上皮细胞来源的外泌体（AEc-Exo）被证实影响肺纤维化的进展。使用TGF-β来构建细胞纤维化模型，用博来霉素（BLM）构建小鼠肺纤维化模型，用AEc-Exo进行治疗，使用miR-450b-5p抑制剂干扰治疗。体外实验表明AEc-Exo通过激活自噬，抑制EMT和PI3K/Akt信号通路减弱TGF-β诱导的肺纤维化，抑制miR-450b-5p减弱AEc-Exo对TGF-β诱导的肺纤维化的治疗作用。通过基因靶向验证HDAC7是miR-450b-5p的靶基因，且同时抑制miR-450b-5p和敲降HDAC7后，部分恢复了AEc-Exo对TGF-β诱导的肺纤维化的治疗作用。体内实验表明，BLM诱导小鼠肺纤维化，抑制自噬，促进EMT和PI3K/Akt信号通路，添加AEc-Exo后减弱了BLM的作用，进一步抑制miR-450b-5p后，又部分恢复了BLM的作用。我们的研究证明了AEc-Exo通过miR-450b-5p/HDAC7分子轴激活自噬显著抑制肺纤维化发展，为肺纤维化的临床治疗提供了新的思路。.吸烟被认为是特发性肺纤维化(IPF)发病率的重要危险因素，香烟烟雾对肺泡上皮细胞有多种损伤作用，可以通过诱导肺泡上皮细胞的外泌体的释放，IPF相关的外泌体Lnc LINC01133 对IPF进展的影响尚不清楚。利用香烟烟雾提取物 (cigarette smoke extract，CSE)诱导IPF小鼠模型。利用5%CSE处理人肺上皮细胞HLF-a后，将其与人肺成纤维细胞MRC-5建立共培养模型。结果发现CSE通过诱导的肺上皮细胞外泌体 LINC01133促进IPF的进展。CSE诱导的肺上皮细胞分泌外泌体 LINC01133抑制了肺成纤维细胞的细胞自噬水平，促进了肺成纤维细胞过度活化和脂质代谢物积累，加速了胶原纤维沉积，进而促进了IPF进展。机制为：LINC01133靶向结合SRSF6，通过抑制FOXA1，下调UCP1的表达，进而对肺成纤维细胞自噬，异常激活和脂质代谢物发挥作用的。研究证明了CSE诱导的肺上皮细胞外泌体 LINC01133通过SRSF6介导FOXA1抑制MRC-5细胞自噬，促进细胞活化和脂肪酸累积，加快了IPF进展，这将为IPF患者在临床治疗过程中提供了新的见解。