Integrating network pharmacology and proteomics to elucidate the mechanism of Allium macrostemon total saponins in alleviating high-altitude acute lung injury.

BACKGROUND: High-altitude acute lung injury (HALI) is a severe respiratory disorder occurring after exposure to high-altitude hypoxic environments. Its pathological features primarily include pulmonary edema, inflammatory response, oxidative stress, and apoptosis, which may progress to high-altitude pulmonary edema in severe cases, posing a life-threatening risk. Allium macrostemon Bunge total saponins (AMBS), one of the main active components of A. macrostemon, have been investigated; however, their specific role and molecular mechanisms in HALI remain unclear. PURPOSE: This study employed network pharmacology and proteomics approaches to investigate the protective effects and molecular mechanisms of AMBS against HALI. METHODS: Hypoxia models in mice were established to evaluate the anti-hypoxic activity and dose-response relationship of AMBS. HALI rat and mouse models were constructed using a simulated high-altitude hypobaric hypoxia chamber at 6500 m altitude. Parameters measured included lung index, lung wet/dry weight ratio, hematological parameters, inflammatory factors, oxidative stress markers, and apoptosis levels. Pathological changes in lung tissue were observed via hematoxylin and eosin (HE) staining. In vitro, a cobalt chloride (CoCl2)-induced hypoxic injury model in human pulmonary microvascular endothelial cells (HPMECs) and a 1% O2 hypoxia model were established to assess cell viability, lactate (LACT) and lactate dehydrogenase (LDH) release, ROS levels, and mitochondrial membrane potential. Meanwhile, the contents of malate and oxaloacetate, as well as the activity and expression level of malate dehydrogenase (MDH), were measured in both in vivo and in vitro models. The mechanisms underlying AMBS-mediated improvement of HALI were elucidated using integrated network pharmacology and proteomics analysis, combined with surface plasmon resonance (SPR), RT-qPCR, Western blot, immunofluorescence, immunohistochemistry, and co-immunoprecipitation (Co-IP). RESULTS: AMBS significantly prolonged the survival time of hypoxic mice, reduced the lung index and lung wet/dry weight ratio in HALI rats, improved blood oxygen saturation and partial pressure of oxygen, corrected acid-base balance disturbances, and alleviated lung tissue pathological injury. Furthermore, AMBS decreased the levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), cysteine-aspartic acid protease 3 (Caspase-3), and Bcl-2-associated X protein (Bax), while increasing glutathione peroxidase (GSH-Px), catalase (CAT), B-cell lymphoma 2 (Bcl-2), and superoxide dismutase (SOD) activities. In vitro experiments demonstrated that AMBS attenuated CoCl2- and 1% O2-induced hypoxic injury in HPMECs by inhibiting LACT and LDH release, reducing ROS accumulation, and restoring mitochondrial membrane potential. In both HALI rats and HPMECs hypoxia models, hypoxia significantly decreased malate content while markedly increasing MDH activity and oxaloacetate content, and AMBS intervention reversed these metabolic abnormalities. Integrated network pharmacology and proteomics analysis revealed that AMBS alleviates HALI primarily through inhibition of the Janus kinase 2/signal transducer and activator of transcription 3/malate dehydrogenase 2 (JAK2/STAT3/MDH2) pathway. SPR confirmed that Macrostemonoside I, an active component of AMBS, directly binds to JAK2 and STAT3 proteins. WP1066 (a JAK2 inhibitor) produced protective effects consistent with AMBS, whereas STAT3 overexpression enhanced the protein-protein interaction between STAT3 and MDH2, antagonized the regulatory effects of AMBS on MDH2 expression and malate-oxaloacetate metabolic balance, and subsequently abolished the cytoprotective effects of AMBS. CONCLUSION: AMBS likely exerts its protective effects against HALI by downregulating MDH2 expression through inhibition of the JAK2/STAT3 signaling pathway.