Nrp1 Negatively Regulates PI3K/AKT Pathway to Inhibit the Activation of Lung Fibroblasts and Its Role in Mouse Pulmonary Fibrosis postprint

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.