Zuoguiyin modulates M2 macrophage polarization to inhibit glycolysis in esophageal squamous cell carcinoma

Zuoguiyin (ZGY), a traditional Chinese medicine formula used to nourish Yin and replenish vital essence, possesses tonifying properties that are conceptually aligned with modern strategies aiming to modulate the immunosuppressive tumor microenvironment (TME). In esophageal squamous cell carcinoma (ESCC), M2 macrophages are pivotal components that drive tumor progression. Therefore, therapeutic strategies targeting M2 macrophages may offer a beneficial approach. This study aimed to explore whether ZGY suppresses ESCC by disrupting the pro-tumorigenic interactions between M2 macrophages and cancer cells, with a specific focus on the immunometabolic axis. A co-culture system of ESCC cells and macrophages was employed to investigate cellular interactions. An in vivo ESCC mouse model induced by a 4-nitroquinoline-1-oxide (4-NQO) was employed for validation. Proteomic analysis, Seahorse metabolic analysis, flow cytometry, Western blotting, molecular docking, and molecular dynamics simulations were conducted to elucidate the underlying mechanisms and identify active components. IL-4 secreted by ESCC cells bound to the IL-4 receptor on macrophages, activating the JAK/STAT6 signaling pathway and driving M2 polarization. These M2 macrophages, in turn, enhanced glycolytic metabolism in ESCC cells. ZGY treatment did not directly affect the cancer cells but effectively inhibited M2 polarization by blocking JAK2/STAT6 phosphorylation. This led to a reversal of the M2-driven enhancement of tumor glycolysis and a reduction in tumor growth in vivo . Isoferulic acid and norharman, key bioactive components of ZGY, were identified as stable binders of JAK2. This study elucidates the mechanism through which IL-4/JAK2/STAT6-mediated M2 macrophages promote glycolysis in ESCC and demonstrates that ZGY disrupts this pathway by targeting JAK2 in macrophages. These findings provide a mechanistic foundation for developing ZGY as a promising tumor microenvironment-modifying therapy for ESCC. • ESCC-derived IL-4 induces M2 polarization via the JAK/STAT6 pathway. • ZGY blocks the IL-4/JAK2/STAT6 signaling axis to suppress M2 polarization. • ZGY indirectly reverses tumor glycolysis by targeting M2 macrophages, not cancer cells. • Isoferulic acid and norharman in ZGY are identified as stable binders of JAK2.