Pulmonary fibrosis (PF) is a chronic progressive lung disease characterized by the impaired gas transfer, restricted ventilation patterns and respiratory failure (Liu et al., 2022). The pathogenesis of PF is usually accompanied with the deposit of extracellular matrix (ECM), proliferation of fibroblasts, the deposition of collagen and complex inflammatory processes, with predominantly damage of alveolar epithelial cells II and abnormal remodeling of respiratory architecture. Until now, the therapeutic options is limited (Henderson et al., 2020). However, the precise molecular mechanisms underpinning these reactions remain to be elucidated and required further attention.

Abnormal alterations of the nonessential amino acids metabolism, including glycine, glutamine and arginine, can drive the profibrotic cellular and promote the secretion of the collagen in PF (Liu et al., 2022). Arginine (L-Arg) is altered in lung fibrosis, and precipitates the susceptible to abnormal synthesis of collagen and airway remodeling disorders for the patients of PF (Oliveira-Paula et al., 2016). Furtherly, L-Arg is used as the substrate of the cytoplasmic Arginase 1 (ARG1) and mitochondrial Arginase 2 (ARG2) enzyme for the production of L-Ornithine (L-OT), L-Proline and glutamate, and nitric oxide synthase (NOS) enzyme for the generation of nitric oxide (NO) (Jiang et al., 2023). L-Arg is a key precursor of NO, which abnormally increases in fibrotic environment, and lead to the injury of alveolar epithelial cell, inflammation and oxidative stress, and in turn trigger the formation of PF (Oliveira-Paula et al., 2016; Wang et al., 2023b). The current study discovered that the high levels of NO may be due to the induction of NOS2, which will contribute to the development of fibrosis, and also participates in host defense and inflammatory response (Wang et al., 2023b). In another study, L-Arg degradation to L-Proline via L-OT, and L-Proline biosynthesis driving ECM production in PF (Guzy and Redente, 2021). Abnormal arginine metabolism is involved in the occurrence of PF, and regulation of arginine metabolism is beneficial to alleviate PF.

Traditional Chinese medicine (TCM) plays an important role in the treatment of PF due to multi-target, multi-pathway and little adverse reaction (Shao et al., 2022). PF was defined as “lung impediment” or “lung wilting” in Synopsis of the Golden Chamber, and its syndrome types were lung dryness, Qi asthenia and phlegm-stasis syndrome (Zhu et al., 2021). Pueraria lobata (Willd.) Ohwi (the plants' name were checked at http://www.worldfloraonline.org on January 24, 2024) has been recorded in Shen Nong's herbal classic, which is the earliest authoritative monograph on pharmacy in China. According to the documentation in bencao xinbian, P. lobata could relieve lung dryness and antiasthmatic. Based on TCM theory and data from previous literature studies, P. lobata is uasually used in treating upper respiratory tract infection, fever and pneumonia (Chang et al., 2012; Shree et al., 2022). Additionally, the active ingredients of P. lobata are mainly flavonoids, coumarin, terpenoids, steroids (Wong et al., 2011), and the major bioactive ingredient of P. lobata is puerarin, which can be used in treating PF by combination with other compounds or drugs (Jiang et al., 2022; Zimmermann and Rothenberg, 2006). At present, P. lobata has been considered to be effective in the treatment of renal fibrosis, hepatic fibrosis and cardiac fibrosis (Liu et al., 2016). A study demonstrated that botanical isoflavone puerarin in P. lobata could attenuate NO-mediated neurotoxicity and diminish NO production via modulating the enzymes in the L-Arg-NO pathway in the fibrosis diseases (Zhao et al., 2016). However, whether arginine metabolism mediate typical inflammation response in the treatment of P. lobata on PF still needs to be explained.

In this study, we prepared aqueous extracts of P. lobata, which could be delivered directly by gastric irrigation to treat PF in rats. The anti-pulmonary fibrosis effect of P. lobata was determined using bleomycin-induced (BLM-induced) PF rats model. Firstly, untargeted metabolomics analysis approach revealed metabolite variations of before and administration of P. lobata in PF rats. Then, we predicted the targets and pathway of P. lobata in the treatment of PF based on network pharmacology analysis. Molecular docking simulation was performed between P. lobata and possible targets screened by network pharmacology. Finally, the above targets were verified.

In the present study, we explored whether P. lobata exerted a potential mechanism for the treatment of PF. We also found P. lobata alleviated PF by regulating arginine metabolism based on reducing NOS2 and L-OT singal molecular. Thus, interference with NOS2 and L-OT expression may provide a novel therapeutic option for patients with PF.