Exploring the mechanism of Shuangyu Granule in regulating immune-inflammatory responses in influenza through UPLC-Orbitrap-MS/MS, GC-MS, and network target analysis

Elucidating the active compounds and anti-inflammatory mechanisms of Wubi Shanyao pill through random forest-based spectrum-effect relationship analysis and UPLC-MS/MS-based network pharmacology.

This study integrated the spectrum-effect relationship with UPLC-MS/MS-based network pharmacology to identify the active compounds and clarify the anti-inflammatory mechanism of Wubi Shanyao Pill (WSP). The chemical fingerprint of WSP was established using liquid chromatography-tandem mass spectrometry (LC-MS), and its anti-inflammatory properties were evaluated in lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-induced bone marrow macrophages (BMDM) by assessing four inflammatory factors. A random forest (RF) algorithm associated phytochemical components with anti-inflammatory activity, screening the top 10 potential active compounds. Network pharmacology was further utilized to predict the core targets and pathways, followed by molecular docking and in vitro validation. WSP significantly inhibited inflammatory factor production in BMDMs (P < 0.0001). Spectrum-effect relationship analysis screened 16 potential bioactive compounds, with echinacoside, acteoside, forsythoside I, schisantherin A, and schizandrin B ranked as the top five. Meanwhile, 106 compounds were identified from WSP, primarily lignans, triterpenoids, and phenylethanol glycosides. Network pharmacology predicted key anti-inflammatory targets based on these components including AKT1, PIK3R1 and PIK3CA, with the PI3K-Akt pathway identified as the key. Molecular docking confirmed stable binding of the top five compounds to core targets (binding energies < -5.0 kcal/mol). In vitro experiments confirmed that the predominant compound, echinacoside, markedly suppressed the phosphorylation of PI3K and AKT proteins (P < 0.05), thereby attenuating systemic inflammatory responses. This study reveals the anti-inflammatory mechanisms of WSP, demonstrating that its primary active ingredient, echinacoside, exerts anti-inflammatory effects by modulating the PI3K-Akt signaling pathway. Overall, the results suggest that WSP possesses promise as a therapeutic option for inflammatory disorders.

Integrative network pharmacology and transcriptomics analysis reveals the multi-target anti-allergic mechanism of ginsenoside Rh1

Zaoshi Huatan Xiefei formula improved severe pneumonia by regulating MAPK/NF-κB/VCAM1 signaling pathway mediated leukocyte transendothelial migration.

BACKGROUND Zaoshi Huatan Xiefei Formula (ZHXF) is a Chinese medicine prescription used in the treatment of severe pneumonia (SP). Although clinical studies have confirmed its efficacy in improving pneumonia, the specific bioactive components and their intricate regulatory pathwaysremain incompletely understood. PURPOSE This research aims to identify the active components of ZHXF, along with its potential targets and molecular mechanisms for treating SP. METHODS The main active ingredients of ZHXF and its blood chemical components were determined by UPLC-Q-Exactive Orbitrap MS. The core targets and biological immune regulatory mechanisms through which ZHXF improves SP were predicted using an integrated approach of network pharmacology, molecular docking, MD simulation, SPR, and proteomics. The protective effect of ZHXF on SP, along with its potential targets and mechanisms, was validated through experiments. RESULTS 130 ingredients of ZHXF were identified by UPLC-Q-Exactive Orbitrap MS, five were confirmed as its primary bioactive ingredients in the blood. Network pharmacology analysis showed that modulating the MAPK and NF-κB pathways is a key mechanism through which ZHXF improves SP. Molecular docking, MD simulations, and SPR indicated that the key components of ZHXF bind effectively to ERK1/2, JNK, and P38, thereby suppressing MAPK/NF-κB pathway expression. Proteomic analysis further revealed VCAM1 as the core target of ZHXF, indicating that ZHXF alleviates the impact of lung inflammation by regulating leukocyte transendothelial migration. In vitro study have confirmed the ZHXF inhibitory effect on leukocyte transendothelial migration. In vivo experiments have shown that ZHXF can significantly alleviate pulmonary inflammation symptoms in SP mice, and this protective effect on lung tissue was achieved through the inhibition of leukocyte transendothelial migration. CONCLUSION This study confirms that ZHXF improves lung inflammation by suppressing the activation of the MAPK/NF-κB pathway and regulating VCAM1 mediated leukocyte transendothelial migration. This study provides a novel perspective for the treatment of SP with ZHXF.

LC-MS-Guided Identification of Bioactive Constituents in Qingfei Jiedu Huatan Formula Reveals Ginsenoside Rb1 as an Endothelial Barrier-Protective Compound Targeting PAI-1 in Pneumonia.

INTRODUCTION Qingfei Jiedu Huatan Formula (QJHF) is a traditional Chinese medicine formula developed for severe pneumonia with the syndrome of "phlegm heat obstructing the lung." PURPOSE This study aimed to investigate the protective effects and underlying mechanisms of QJHF on pulmonary vascular endothelial barrier dysfunction in severe pneumonia. MATERIALS AND METHODS Mouse models of pneumonia/lung injury were established by intratracheal instillation of Klebsiella pneumoniae or LPS to evaluate the protective effects of QJHF. A novel strategy integrating LC-MS-based serum pharmacochemistry and single-cell RNA sequencing was performed to elucidate the underlying mechanisms of QJHF. An LPS-induced HUVEC endothelial barrier injury model was used to screen active compounds and investigate their mechanisms of action. RESULTS QJHF markedly alleviated lung injury and inflammatory responses in mice challenged with Klebsiella pneumoniae or LPS. Specifically, QJHF reduced inflammatory cell infiltration, particularly neutrophil accumulation, decreased the levels of IL-1β, IL-6, and TNF-α, and suppressed pulmonary myeloperoxidase activity. Single-cell transcriptomic analysis revealed that QJHF mainly modulated inflammatory responses and vascular endothelial function. Moreover, QJHF decreased the lung index, lung wet/dry weight ratio, total protein concentration, and Evans blue extravasation, while restoring VE-cadherin expression, indicating preservation of pulmonary endothelial barrier integrity. Integrated serum component profiling and network pharmacology analyses identified 10 major absorbed constituents that may underlie the endothelial barrier-protective effects of QJHF. Among these constituents, ginsenoside Rb1 (G-Rb1) was identified as a key bioactive component. G-Rb1 upregulated VE-cadherin expression and protected endothelial barrier integrity both in vitro and in vivo and further ameliorated LPS-induced lung tissue injury and inflammatory responses in mice. Target identification further implicated plasminogen activator inhibitor-1 (PAI-1) as a putative molecular target of G-Rb1, which was subsequently validated by molecular dynamics simulations, cellular thermal shift assays, and surface plasmon resonance analysis. Importantly, PAI-1 overexpression attenuated the protective effects of G-Rb1 on endothelial barrier function. CONCLUSION QJHF ameliorates inflammatory lung injury in pneumonia mice by preserving the pulmonary vascular endothelial barrier. This effect is at least partly mediated by G-Rb1, which targets and suppresses PAI-1, thereby restoring VE-cadherin-mediated endothelial junction integrity.

Exploration of Compounds Identified from Amomum cardamomum Seed Extract: LC-MS Profiling, Network Pharmacology, and Computational Analyses for Innovative Multi-Targeted Breast Cancer Therapy

Background Breast cancer remains one of the leading causes of cancer-related mortality worldwide, and the emergence of drug resistance, systemic toxicity, and limited efficacy of current therapies highlight the need for safer and more effective treatment. Natural products have emerged as promising sources of multi-target anticancer agents. A. cardamomum has demonstrated preliminary anticancer potential, yet the bioactive constituents and their molecular mechanisms in breast cancer remain poorly elucidated. Methods This study integrated in silico approaches to investigate the therapeutic potential of A. cardamomum seed extract against breast cancer. LC–MS analysis identified phytochemical compounds, followed by network pharmacology to determine their potential targets and molecular pathways. Pharmacokinetic and toxicity predictions were assessed through ADMET and Lipinski’s rule of five analyses to evaluate drug-likeness and safety. Molecular docking and molecular dynamics (MD) simulations were conducted to evaluate binding affinity and structural stability of compounds with key oncogenic proteins. Results LC-MS profiling identified 22 distinct compounds in A. cardamomum seeds. ADMET and Lipinski analyses demonstrated that most compounds possessed high gastrointestinal absorption, favorable oral bioavailability, and low toxicity risk. Network pharmacology highlighting SRC, TNF-α, Caspase-3, and EGFR as central nodes in the protein-protein interaction network. Molecular docking identified compounds C17 and C20 as the most promising bioactives, showing strong binding affinities and interactions similar to control ligands. MD simulations confirmed their stable complexes, indicating conformational stability and robust ligand–protein interactions. Conclusion This study highlights the promising multi-target anticancer potential of A. cardamomum seeds. Compounds C17 and C20 were identified as lead candidates with strong and stable interactions with key breast cancer-related proteins and favorable pharmacokinetic properties. These results suggest that A. cardamomum could serve as a potential source for developing new plant-based therapies against breast cancer. Further in vitro and in vivo investigations are warranted to validate their efficacy and safety.

Unraveling the therapeutic mechanisms of Huanglian Jiedu Tang in bacterial meningitis

This study aims to comprehensively elucidate the therapeutic mechanisms of Huanglian Jiedu Tang in bacterial meningitis via an integrated strategy combining network pharmacology, molecular docking, and experimental validation. Network pharmacology was utilized to screen for bioactive constituents, pivotal targets, and associated signaling pathways of HLJDT. The potent candidate Obacunone and its target MAPK14 were prioritized for verification. An in vitro bacterial meningitis cell model was established, followed by therapeutic interventions using Obacunone, the MAPK14 inhibitor VX-702, or their combination. Therapeutic effects were assessed using the CCK-8 assay, sodium ion permeability assays, TUNEL staining, RT-PCR, Western blot, and ELISA. Bioinformatics analysis of the GEO dataset GSE40586 identified 495 BM-related genes, 30 of which intersected with HLJDT targets. GO and KEGG enrichment analyses highlighted biological processes including antibiotic and glucocorticoid responses, as well as transcriptional misregulation and HIF-1 signaling pathways. Molecular docking simulations revealed that Obacunone possessed superior binding affinity to MAPK14. In the BM cell model, treatment with Obacunone or VX-702 alone significantly improved cell viability, attenuated the increased sodium ion permeability, inhibited apoptosis, and suppressed inflammatory cytokine levels. Notably, combination therapy exhibited the most potent synergistic effects. In a bacterial meningitis co-culture model, combined treatment with Obacunone and VX-702, as well as MAPK14 silencing, significantly restored the viability and migratory capacity of HMC3 cells while effectively attenuating early apoptosis. This study delineated the pharmacological underpinnings of HLJDT in BM treatment, identifying its active components and targets. Experimental validation confirmed the synergistic action of Obacunone and the MAPK14 inhibitor, providing a robust scientific foundation for future drug development and clinical strategies.