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

Abstract

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.