Integrative analysis and in vitro assessment of the effects of α-mangostin on high-glucose-induced inflammatory and oxidative stress responses in RAW264.7 macrophages.

BACKGROUND: Persistent macrophage-mediated inflammation under hyperglycemic conditions is an important feature of the inflammatory microenvironment associated with diabetic wounds. α-Mangostin (α-MG), a natural xanthone derived from Garcinia mangostana, has been reported to exert anti-inflammatory activity in various experimental models. However, its effects on high-glucose-induced macrophage inflammation and oxidative stress remain insufficiently characterized.

OBJECTIVE: This study aimed to investigate the effects of α-MG on inflammatory and oxidative responses in high-glucose-stimulated macrophages and to explore its potential molecular mechanisms using an integrative strategy combining network pharmacology, molecular docking, molecular dynamics simulation, and in vitro assessment.

METHODS: Potential targets of α-MG were collected and intersected with diabetic wound-associated genes to identify candidate targets. Protein-protein interaction analysis and functional enrichment analyses were performed to identify hub targets and related biological pathways. Molecular docking was used to evaluate the binding affinity between α-MG and key targets, followed by a 110 ns MD simulation of the α-MG-TNF complex and a 50 ns supplementary MD simulation of the α-MG-PTGS2 complex. In vitro experiments were performed using high-glucose-stimulated RAW264.7 macrophages, with mannitol included as an osmotic control. The effects of α-MG on inflammatory mediators, oxidative stress markers, PTGS2/COX-2 expression, and NF-κB activation were further evaluated.

RESULTS: A total of 24 overlapping targets between α-MG and diabetic wound-associated genes were identified. Enrichment analyses suggested that these targets were mainly associated with inflammatory and immune-related biological processes, with TNF and IL-17 signaling pathways identified as computationally predicted and potentially relevant pathways. Molecular docking showed favorable binding interactions between α-MG and several hub targets, including PTGS2, AKT1, TNF, and PARP1. The 110 ns molecular dynamics simulation further suggested that the α-MG-TNF complex remained relatively stable during the simulation period. A supplementary 50 ns MD simulation also supported the conformational stability of the α-MG-PTGS2 complex. In high-glucose-stimulated RAW264.7 macrophages, α-MG significantly reduced the mRNA expression and secretion of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. α-MG also decreased PTGS2/COX-2 expression and inhibited NF-κB p65 phosphorylation. In addition, α-MG alleviated high-glucose-induced oxidative stress, as indicated by increased GSH content and SOD activity, together with reduced MDA levels. The inclusion of the mannitol control helped distinguish glucose-associated inflammatory and oxidative responses from osmotic stress-related effects.

CONCLUSION: α-MG attenuated high-glucose-induced inflammatory and oxidative responses in RAW264.7 macrophages. The integrative computational analysis suggested that computationally predicted TNF- and IL-17-related pathways may be involved, whereas the in vitro experiments provided supportive evidence only for changes in inflammatory cytokines, PTGS2/COX-2, oxidative stress markers, and NF-κB activation. These findings provide preliminary experimental and computational evidence for the anti-inflammatory and antioxidant potential of α-MG under diabetic-like macrophage inflammatory conditions. However, direct pathway-specific validation and functional diabetic wound-related models are required before drawing conclusions regarding TNF/IL-17 pathway regulation or therapeutic efficacy in diabetic wound healing.