脑啡肽酶表达下调激活肺成纤维细胞在特发性肺纤维化中的作用及机制

Idiopathic pulmonary fibrosis (IPF) is a fibrotic interstitial pneumonia. Although the pathogenesis of it is not fully elucidated, it is generally believed that fibroblast activation produces a large number of extracellular matrix inducing pulmonary fibrosis which play a key role in the development of IPF. Our previous study using proteomics, Western blot and Immunohistochemistry revealed that the expression of neprilysin (NEP) in IPF lung tissue was decreased; collagen1α and α-SMA increased after NEP knocked down in human embryonic lung fibroblasts (HELF). In activated HELF induced by TGF-β1, the expression of collagen1α and α-SMA were elevated, whereas expression of NEP was decreased. It is speculated that NEP loss might participate in IPF by regulating activation of pulmonary fibroblasts. This project will use IPF clinical specimens, NEP knocked down and overexpressed pulmonary fibroblasts, NEP knocked out mice to clarify that loss of NEP could participate in IPF by activating pulmonary fibroblasts, and to explore molecular mechanisms of NEP loss regulates pulmonary fibroblasts activation through TGF-β1/Smads pathway. The results will reveal the role and mechanisms of NEP loss might promote pulmonary fibroblast activation in IPF, and provide important experimental basis for IPF intervention.

特发性肺纤维化（IPF）是一种纤维化性间质性肺炎，其发病机制还不十分清楚，但普遍认为成纤维细胞激活产生大量细胞外基质使肺纤维化在其中发挥关键作用。我们前期用蛋白质组学、Western blot及免疫组化发现IPF肺组织中脑啡肽酶（NEP）表达降低；在人胚肺成纤维细胞（HELF）中敲减NEP后，collagen1α和α-SMA表达增加；用TGF-β1激活HELF后，NEP表达降低且collagen1α和α-SMA表达增加。推测，NEP可能通过调控肺成纤维细胞激活参与IPF发生发展。本项目拟用IPF临床标本、NEP敲减和过表达的HELF细胞模型和NEP-/-小鼠肺纤维化模型，阐明NEP表达下调促进肺成纤维细胞激活参与IPF；围绕TGF-β1/Smads信号通路探讨NEP表达下调影响IPF的分子机制。本研究将揭示NEP表达下调促进肺成纤维细胞激活在IPF中的作用和机制，并为干预提供重要实验依据。

特发性肺纤维化（IPF）是一种纤维化性间质性肺炎，发病率逐年增加，预后差，目前缺乏有效的治疗手段，急需研发新的治疗药物。但是IPF发病机制还不十分清楚，但普遍认为成纤维细胞激活产生大量细胞外基质使肺纤维化，在其中发挥关键作用。本项目利用蛋白质组学、Western blot及免疫组化发现IPF肺组织中脑啡肽酶（NEP）表达降低。进一步研究了NEP通过调控肺成纤维细胞激活参与IPF发生发展。围绕TGF-β1/Smads信号通路探讨NEP影响IPF的分子机制。在肺纤维化小鼠模型中，证实了NEP抑制剂可阻止肺纤维化形成。本项目发现，用TGF-β激活人胚肺成纤维细胞（HELF）后，NEP表达增加且collagen1α和α-SMA表达增加。在HELF中敲减NEP后，collagen1α和α-SMA表达降低，Smads信号通路磷酸化降低；而过表达NEP后，collagen1α和α-SMA表达增加，且Smads信号通路磷酸化活性增加。用NEP抑制剂AHU377预处理细胞，抑制NEP表达，可减少collagen1α和α-SMA表达。进一步在小鼠肺纤维化动物模型中验证，腹腔内注射NEP抑制剂AHU377，可明显减轻肺纤维化发生。说明抑制NEP表达，对肺纤维化有保护作用。本项目阐明了NEP通过激活肺成纤维细胞，增加细胞外基质产生而参与了IPF发生发展。进一步阐释了NEP基于对TGF-β1/Smads信号通路的调节作用，进一步激活肺成纤维细胞。体外实验发现，NEP抑制剂或从基因层面敲减NEP，均可抑制成纤维细胞的激活。动物模型证实了，NEP抑制剂AHU377可抑制小鼠肺纤维化的发生。本项目研究结果提示，NEP可能是IPF的治疗靶点，为IPF临床新药研发奠定了的实验室基础。