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.