Background Pathological scarring is driven by persistent crosstalk between profibrotic cytokine signaling and extracellular matrix remodeling. Transforming growth factor-β1 (TGF-β1)/Smad2/3 signaling broadly activates fibroblasts, whereas bone morphogenetic protein 2 (BMP2)/Smad1/5/9 signaling reinforces matrix-producing fibroblast states. Concurrent modulation of these pathways may therefore provide a more comprehensive therapeutic strategy. Purpose To determine whether hypericin (HYP), prioritized through receptor-based virtual screening, attenuates pathological scarring while concurrently modulating TGF-β1 and BMP2 signaling. Methods HYP was prioritized through receptor-based virtual screening involving transforming growth factor beta receptor 1 and bone morphogenetic protein receptor type 1A and evaluated in human skin fibroblasts using low-dose viability assays, single-ligand and TGF-β1/BMP2 co-stimulation models, reference inhibitors, cellular thermal shift assays, quantitative polymerase chain reaction, immunoblotting, immunofluorescence, proliferation and migration assays, and integrated transcriptomic analysis. HYP-loaded micelles were characterized and assessed in a rabbit ear hypertrophic scar model, whereas non-micellar HYP was evaluated in a human keloid patient-derived xenograft model. Results Virtual screening prioritized HYP as a candidate compound with predicted interactions with transforming growth factor beta receptor 1 and bone morphogenetic protein receptor type 1A. HYP at 1 and 2 μM did not significantly affect fibroblast viability, whereas viability remained above 90% at 4 μM. Cellular thermal shift assays demonstrated enhanced thermal stabilization of both receptor proteins in HYP-treated cells, supporting cellular target engagement. HYP suppressed TGF-β1/Smad2/3- and BMP2/Smad1/5/9-associated fibrotic responses. Under simultaneous TGF-β1 and BMP2 stimulation, HYP attenuated both signaling branches, whereas SB-431542 and LDN-193189 predominantly inhibited their respective pathways. Integrated transcriptomic analysis identified 96 shared HYP-suppressed genes and convergent downregulation of extracellular matrix, collagen, focal adhesion, integrin, ossification, and chondrogenesis programs. HYP-loaded micelles exhibited a mean particle size of 148.1 ± 15.1 nm, a polydispersity index of 0.219 ± 0.015, an encapsulation efficiency of 72.18 ± 4.05%, and a drug loading of 11.65 ± 1.28%. Local HYP treatment reduced scar elevation, collagen deposition, xenograft volume, and pathway activation in vivo. Conclusion HYP coordinately suppresses TGF-β1/Smad2/3 and BMP2/Smad1/5/9 signaling and exerts consistent antifibrotic effects across cellular, rabbit ear, and human keloid xenograft models. These findings provide a preclinical rationale for further investigation of HYP as a locally delivered antifibrotic therapy with concurrent TGF-β1- and BMP2-pathway modulatory activity.
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.
Background Traditional Chinese medicine polysaccharides (TCMPs) have diverse pharmacological activities, yet their mechanisms are often difficult to interpret because direct polysaccharide–target recognition must be distinguished from downstream signaling and microbiota-mediated effects. Purpose This review evaluates current strategies for TCMP target discovery and validation and develops a structure-informed framework for interpreting target claims. Study Design A critical narrative review focused on purified or fraction-defined TCMPs, target-discovery workflows, validation methods, and representative evidence patterns. Methods A targeted literature search integrated affinity capture, chemical probes, omics-assisted prioritization, biophysical assays, NMR, computational methods, and structure-target-activity analysis. Evidence was organized into a six-level hierarchy from phenotypic activity to in vivo target-causal validation. Results Direct-binding evidence has been reported for structurally characterized TCMPs interacting with lectins, immune receptors, adhesion-related proteins, enzymes, and other disease-relevant proteins, with target-dependent functional evidence available in a subset of cases, whereas in vivo target-causal validation remains limited. Other findings are better interpreted as proximal functional nodes or microbiota–metabolite–host axes. Across studies, target confidence depends on ligand definition, assay format, specificity controls, orthogonal binding validation, and functional perturbation. The review links the six-level hierarchy with four classes of ligand definition and emerging structure-target-activity relationships. Conclusion A structure-first workflow that couples carbohydrate characterization with direct-binding measurements, functional perturbation, and in vivo validation can move TCMP research from pathway association toward testable ligand–target mechanisms and more comparable target evidence.
Background Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with a dismal prognosis, mainly due to cisplatin resistance and severe adverse effects of conventional therapies. Wogonin, a natural flavonoid, exhibits anticancer activity in several cancers, but its efficacy and potential molecular mechanisms in ESCC remain unclear. Purpose Our study aimed to investigate the anticancer effect of wogonin in ESCC and elucidate its molecular mechanism, with a particular focus on ferroptosis regulation and synergy with cisplatin. Methods A series of experiments were conducted, including cell functional assays, key gene knockdown, RNA-seq, bioinformatics analyses, western blotting, qPCR, molecular docking, surface plasmon resonance (SPR), and in vivo xenograft models and ex vivo tumor explant assays. Results Wogonin could dose-dependently inhibit ESCC cell proliferation and cisplatin (DDP) resistance by inducing ferroptosis. Mechanistically, wogonin directly interacts with GATA-binding protein 1 (GATA1) to stabilize the protein and enhance its nuclear translocation. Nuclear-accumulated GATA1 upregulates the transcription of lysophosphatidylcholine acyltransferase 3 (LPCAT3) to exacerbate lipid peroxidation and elevates transferrin receptor 1 (TFRC) abundance to trigger intracellular iron overload. Wogonin synergizes with cisplatin to enhance anticancer efficacy via distinct ferroptotic pathways. In vivo, wogonin suppressed tumor growth without affecting mouse body weight and increased the expression of ferroptosis markers in tumor tissues. Conclusions This study is the first to demonstrate that wogonin induces ferroptosis in ESCC by regulating GATA1. This highlights the potential of wogonin as a treatment candidate for ESCC, particularly in overcoming cisplatin resistance, and provides a novel synergistic strategy with cisplatin.
