Abstract: Background　Idiopathic pulmonary fibrosis（IPF） has a complex pathogenesis，limited treatment options and a poor prognosis. Therefore，it is crucial to identify the safe and effective therapeutic targets. Objective　To investigate the regulatory effect of Nrp1 on the activation of mouse pulmonary fibroblasts（MPFs） through PI3K/AKT signaling pathway and the therapeutic effect and safety of recombinant Nrp1 protein on pulmonary fibrosis（PF） in mice. Methods　GSE150910，GSE218997，GSE173523，GSE47460，and GSE32537 were downloaded from the GEO database to analyze the expression of Nrp1 in lung tissues of PF mouse models and IPF patients，as well as the correlation between Nrp1 expression and pulmonary function parameters in IPF patients. A total of 45 IPF patients diagnosed in the Department of Respiratory and Critical Care Medicine and 29 healthy controls who underwent physical examinations in the health management center in General Hospital of Ningxia Medical University from 2022 to 2024 were selected. Cell-free RNA（cfRNA） sequencing was used to analyze the expression levels of Nrp1 in the plasma of IPF patients and healthy controls and its diagnostic efficacy. MPFs were divided into the control group，transforming growth factor β1（TGF-β1）group，Nrp1 overexpression group and TGF-β1+Nrp1 overexpression group.TGF-β1 was used to induce the activation of MPFs，and the effects of Nrp1 overexpression on the PI3K/AKT pathway and the expression of α-SMA，Vim and Fn were detected. Thirty male C57BL/6J mice were randomly divided into the control group，bleomycin（BLM） group and BLM+Nrp1 group，with 10 mice in each group. After establishing the mouse PF model，the BLM+Nrp1 group was intraperitoneally injected with 100 μg·kg-1·d-1 of recombinant Nrp1 protein for 20 consecutive days. HE and Masson staining were used to observe the pathological changes of mouse lung tissues，and liver and kidney function indicators were evaluated. Western blot and immunohistochemistry were used to detect the expression of α-SMA，Vim and Fn in lung tissues. ELISA was used to measure the levels of Nrp1 in plasma and bronchoalveolar lavage fluid（BALF）. Results　Compared with normal lung tissues，The expression of Nrp1 was decreased in the lung tissues of PF mouse and IPF patients（P<0.05），and the expression of Nrp1 was positively correlated with FEV1%，FVC% and DLCO%. The expression of Nrp1 mRNA in plasma of IPF patients was significantly lower than that of healthy controls（P<0.05），and the area under the receiver operating characteristic curve of plasma Nrp1 for diagnosing IPF was 0.754（95%CI=0.634-0.874）. Compared with the TGF-β1 group，the ratios of p-PI3K/PI3K and p-AKT/AKT in the TGF-β1+Nrp1 overexpression group were decreased，and the expression of α-SMA，Vim and Fn was decreased（P<0.05）. After intraperitoneal injection of recombinant Nrp1 protein，the pathological features of PF in mice were improved. There were no statistically significant differences in the levels of alanine aminotransferase（ALT），aspartate aminotransferase（AST），alkaline phosphatase（ALP），blood urea nitrogen（BUN）and creatinine（CREA）in plasma among the control group，BLM group and BLM+Nrp1 group（P>0.05）. Compared with the BLM group，the expression of α-SMA and Fn in the lung tissues of the BLM + Nrp1 group was decreased，and the levels of Nrp1 in plasma and BALF were increased（P<0.05）. Conclusion　The expression of Nrp1 is decreased in the lung tissues，plasma and BALF of IPF patients. Nrp1 inhibits the activation of mouse pulmonary fibroblasts by negatively regulating the PI3K/AKT signaling pathway. Intraperitoneal injection of recombinant Nrp1 protein can alleviate pulmonary fibrosis in mice and has systemic safety.

Key words: Pulmonary fibrosis, Neuropilin-1, Fibroblast activation, Recombinant Nrp1 protein

摘要： 背景　特发性肺纤维化（IPF）发病机制复杂、治疗手段有限、预后差，因此寻找安全有效治疗靶点至关重要。目的　探究神经纤毛蛋白1（Nrp1）通过磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）信号对小鼠肺成纤维细胞（MPFs）活化的调控作用，以及重组蛋白Nrp1对小鼠肺纤维化（PF）的治疗作用及安全性。方法　从GEO数据库下载数据集GSE150910、GSE218997、GSE173523、GSE47460和GSE32537，分析PF小鼠模型和IPF患者肺组织中Nrp1表达以及Nrp1与IPF患者肺功能指标的相关性。选取2022—2024年在宁夏医科大学总医院呼吸与危重症医学科确诊的IPF患者45例，以及同期在本院健康管理中心体检的健康对照者29例，采用游离RNA（cfRNA）测序分析IPF患者和健康对照者血浆Nrp1表达水平及其诊断效能。将MPFs分为对照组、转化生长因子β1（TGF-β1）组、Nrp1过表达组和TGF-β1+Nrp1过表达组，TGF-β1诱导MPFs活化，过表达Nrp1检测其对PI3K/AKT通路和α-SMA、Vim和Fn表达的影响。将30只雄性C57BL/6J小鼠随机分为对照组、博来霉素（BLM）组、BLM+Nrp1组，每组10只。构建小鼠PF模型后，BLM+Nrp1组连续20 d腹腔注射100 μg·kg-1·d-1的重组蛋白Nrp1。HE和Masson染色观察小鼠肺组织病理变化，评估小鼠肝肾功能指标，Western blotting和免疫组化检测肺组织中α-平滑肌肌动蛋白(α-SMA）、波形蛋白Vimentin（Vim）和纤连蛋白Fibronection（Fn）表达，酶联免疫吸附试验（ELISA）测定血浆和支气管肺泡灌洗液（BALF）中Nrp1水平。结果　数据集分析结果显示，与正常肺组织相比，PF小鼠和IPF患者肺组织中Nrp1表达降低（P <0.05），Nrp1表达与第一秒用力呼气量占预计值百分比（FEV1%）、用力肺活量占预计值百分比（FVC%）、一氧化碳弥散量占预计值百分比（DLCO%）呈正相关。IPF患者血浆Nrp1 mRNA表达水平低于健康对照者（P <0.05），血浆Nrp1诊断IPF的受试者工作特征曲线下面积为0.754（95%CI=0.634~0.874）。与TGF-β1组相比，TGF-β1+Nrp1过表达组中p-PI3K/PI3K、p-AKT/AKT降低，α-SMA、Vim和Fn表达降低（P <0.05）。腹腔注射重组蛋白Nrp1后，PF小鼠肺部病理特征改善，对照组、BLM组、BLM+Nrp1组血浆丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、碱性磷酸酶（ALP）、血尿素氮（BUN）和肌酐（CREA）水平比较，差异均无统计学意义（P >0.05）。与BLM组比较，BLM+Nrp1组小鼠肺组织中α-SMA和Fn表达降低，血浆和BALF中Nrp1水平增加（P <0.05）。结论　IPF患者肺组织、血浆及BALF中Nrp1表达降低。Nrp1通过负向调控PI3K/AKT信号通路抑制小鼠肺成纤维细胞活化。腹腔注射重组Nrp1可以缓解小鼠肺纤维化，具有系统安全性。

关键词: 肺纤维化, 神经纤毛蛋白 1, 肺成纤维细胞活化, 重组蛋白 Nrp1