Punicalagin Alleviates Acute Liver Injury via Dual STAT1 / NF ‐ κB Inhibition and STAT3 Activation to Orchestrate M1‐to‐M2 Macrophage Polarization

Acute liver injury (ALI) is characterized by excessive inflammation and macrophage polarization, with the M1-to-M2 phenotypic shift emerging as a critical regulatory node. Punicalagin (PUN), a polyphenolic compound derived from pomegranate, has demonstrated hepatoprotective effects in preclinical models; however, its molecular mechanisms underlying macrophage polarization and inflammatory signaling remain unclear. This study aims to investigate the role of PUN in modulating macrophage polarization and inflammatory pathways in chemical- and drug-induced ALI. CCL₄- and APAP-induced ALI mouse models were employed to assess the hepatoprotective effects of PUN through liver enzyme analysis, histology, and immune phenotyping. In vitro, LPS-stimulated RAW264.7 macrophages were treated with PUN, and mechanisms were examined through western blotting, qRT-PCR, flow cytometry, and ELISA. PUN (12.5 mg kg^-1) significantly reduced serum levels of ALT and AST, as well as the necrotic area in both ALI models. It reduced hepatic infiltration of monocyte-derived macrophages (MDMs) and lowered the expression of M1 markers (CD86, iNOS), while simultaneously increasing the expression of M2 markers (CD206, Arg-1). Mechanistically, PUN inhibited the phosphorylation of NF-κB and STAT1, while promoting the activation of STAT3, which resulted in a reduction of TNF-α and IL-6 levels (approximately 62%-71%), alongside an increase in IL-10 and TGF-β, both in vivo and in vitro. Similar reprogramming from M1 to M2 and cytokine shifts were observed in LPS-challenged RAW264.7 cells. PUN alleviates ALI by promoting M1-to-M2 macrophage polarization through the dual inhibition of NF-κB/STAT1 and the activation of STAT3. These findings underscore PUN as a potential therapeutic agent for ALI by reprogramming the hepatic immune microenvironment.