Study on the response of Punicalagin to AGEs - Induced inflammation based on lipidomics.

BACKGROUND: Punicalagin (PUN), the main bioactive in pomegranate peel polyphenols, plays a key therapeutic effect against diverse ailments, especially inflammatory diseases. Advanced Glycation End Products (AGEs) can disrupt lipid metabolism, trigger inflammation, and are closely linked to metabolic inflammatory diseases. Based on PUN's known protective effects, this study uses lipidomic analysis to examine its influence on AGEs-induced metabolic inflammation, providing a basis for anti-inflammatory drug research. PURPOSE: To develop the medicinal value of PUN by studying its effects on AGEs-induced inflammation and lipid metabolism disorders. METHODS: RAW264.7 macrophages were first stimulated with AGEs (200 μg/mL), followed by treatment with PUN (25-100 μg/mL). Next, TNF-α and IL-1β levels were measured by ELISA. Western blotting was then performed to assess the expression of PPARγ and key proteins in the NF-κB and JNK signaling pathways, nuclear translocation of NF-κB p65 was visualized via immunofluorescence. By using the antagonist GW9662, the upstream and downstream relationship between PPARγ and NF-κB, as well as JNK, was demonstrated. Finally, non-targeted lipidomics using UPLC-Q/TOF-MS, combined with PCA and OPLS-DA multivariate analysis, identified differential lipid metabolites. RESULTS: PUN significantly suppressed AGEs-induced secretion of TNF-α and IL-1β in a concentration-dependent manner. Mechanistically, PUN upregulated PPARγ expression, inhibited the phosphorylation of IKKα/β, IκB-α, and NF-κB, reduced p-JNK, c-Jun, and c-Fos levels, and blocked nuclear translocation of p65. The use of the PPARγ antagonist GW9662 led to a re-upregulation of the phosphorylation levels of the NF-κB and JNK pathways downstream of PPARγ. The original inhibitory effect of PUN was blocked. This confirmed that the anti-inflammatory mechanism of PUN depends on the activation of PPARγ. Lipidomics analysis revealed that PUN reversed 31 dysregulated lipid metabolites induced by AGEs, with glycerophospholipid metabolism identified as the most significantly enriched pathway. CONCLUSION: This study demonstrates that PUN alleviates AGEs-induced inflammation by restoring glycerophospholipid metabolism homeostasis, upregulating PPARγ expression, and subsequently suppressing NF-κB and JNK signaling pathways.