Jingfang granule alleviates cold-damp lung injury by suppressing NF-κB/HIF-1α-associated macrophage glycolysis
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Zhao Sun1, Xiaozhe Jiang1, Chang Liu1
1State Key Laboratory of Integration and Innovation of Classical formula and Modern Chinese Medicines, National Chinmedomics Research Center, Metabolomics Laboratory, Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China.
Background:
Jingfang Granule (JFG) is clinically used for exterior cold-damp disorders, but its pulmonary effects and associated inflammatory responses remain incompletely understood.
Purpose:
To evaluate the protective effects of JFG against cold-damp syndrome-like lung injury and its effects on macrophage glycolytic inflammatory activation.
Study Design:
A combined in vivo, multi-omics and in vitro mechanistic study was conducted.
Methods:
Rats were exposed to low-temperature/high-humidity conditions and treated with JFG. Lung pathology, inflammatory responses, targeted serum energy metabolomics, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), transcriptomics and proteomics were evaluated. UPLC fingerprinting and UPLC-Q-TOF-MS/MS-based serum pharmacochemistry were used to characterize JFG and its absorbed constituents. LPS-stimulated RAW264.7 macrophages were treated with JFG-medicated serum. DMOG rescue, HIF-1α knockdown, BAY 11-7082-mediated NF-κB inhibition, fluorometric ECAR assay and representative-compound validation were performed to strengthen mechanistic interpretation.
Results:
JFG improved cold-damp-related phenotypes, reduced pulmonary edema, mucus hypersecretion, inflammatory cytokine levels and CD68/iNOS-positive macrophage accumulation. Metabolomics and MALDI-MSI showed that JFG corrected glycolysis- and TCA cycle-related disturbances. Integrated multi-omics highlighted the NF-κB/HIF-1α-HK2/LDHA pathway as a major JFG-regulated axis. In LPS-stimulated macrophages, JFG-medicated serum reduced glycolysis-associated markers and CD86-positive M1-like activation. DMOG partially reversed these effects, whereas HIF-1α knockdown reduced HK2, LDHA, iNOS expression and extracellular acidification, weakening the additional inhibitory effect of JFG-medicated serum. BAY 11-7082 further supported the involvement of NF-κB in HIF-1α-associated glycolytic activation. Prim-O-glucosylcimifugin (POG), nobiletin and quercetin partially reproduced the inhibitory effects of JFG-medicated serum on pathway-associated proteins in vitro.
Conclusion:
JFG attenuated cold-damp syndrome-like lung injury in rats, accompanied by improvements in pulmonary inflammation and glycolysis-related metabolic disturbances. Complementary experiments in LPS-stimulated macrophages supported the involvement of NF-κB/HIF-1α-associated glycolytic activation in the anti-inflammatory effects of JFG-medicated serum, while serum pharmacochemistry provided evidence of absorbed constituents.
