Leptocaramine from Hypecoum erectum inhibits inflammatory responses via CSF2-NF-κB signaling pathway
Hailian Yuan1, Hongyan Wang2, Cheng Wang3
1Key Laboratory of Immune Microenvironment and Inflammatory Disease Research in universities of Shandong province, School of Basic Medical Sciences, Shandong Second Medical University, Weifang 261053, PR China; School of Traditional Chinese Medicine, Faculty of Medicine, Yangzhou University, Yangzhou, 225009, PR China.
Background:
Inflammation is a critical defense mechanism whose dysregulation underpins numerous diseases. Natural products, particularly isoquinoline alkaloids, are valuable sources for anti-inflammatory drug discovery due to their structural diversity and multi-target potential. Hypecoum erectum (H. erectum), a traditional medicinal plant, is rich in isoquinoline alkaloids, but its active anti-inflammatory constituents and their mechanisms remain unclear.
Methods:
Four isoquinoline alkaloids were isolated from H. erectum. Their anti-inflammatory activities and cytotoxicities were screened in LPS-stimulated RAW264.7 macrophages. The most promising candidate, leptocaramine, was further evaluated for its broad-spectrum efficacy across multiple in vitro models (using LPS, PGN and HSV-1 stimuli) and cell types (RAW264.7, mouse peritoneal macrophages and THP-1 cells). RNA-sequencing (RNA-seq), bioinformatics analysis and qPCR were employed to investigate the transcriptomic changes and mechanism of action. Immunoblotting and target prediction were used to elucidate the signaling pathways involved.
Results:
Among the four alkaloids, leptocaramine exhibited the most potent anti-inflammatory activity with minimal cytotoxicity. It demonstrated broad-spectrum efficacy by significantly inhibiting pro-inflammatory mediator production across all tested in vitro models. RNA-seq analysis revealed that leptocaramine profoundly suppressed the LPS-induced inflammatory transcriptome. Functional enrichment highlighted a marked downregulation of genes in immune and cytokine-related pathways, with colony-stimulating factor 2 (CSF2/GM-CSF) being a key suppressed target. Mechanistically, leptocaramine specifically inhibited the NF-κB signaling pathway without affecting MAPK, and molecular docking predicted IKKβ as its direct binding target. Furthermore, leptocaramine's anti-inflammatory effect was compromised by exogenous CSF2, indicating the disruption of a CSF2-mediated positive feedback loop.
Conclusion:
This study identifies leptocaramine as a novel broad-spectrum anti-inflammatory isoquinoline alkaloid from H. erectum. Its primary mechanism involves suppressing the NF-κB pathway-potentially via direct IKKβ inhibition-and disrupting the CSF2-driven inflammatory amplification loop. Leptocaramine emerges as a promising lead compound for the development of new anti-inflammatory therapeutics.
