Preliminary structural characterization and potential glucose metabolism-regulating effects of Armillaria mellea mycelial polysaccharide in insulin-resistant HepG2 cells.

Armillaria mellea mycelial polysaccharide (AMMP) is a fungal bioactive macromolecule with potential metabolic regulatory activity; however, its preliminary structural characteristics and glucose metabolism-regulating effects in insulin-resistant hepatic cells remain inadequately clarified. In this study, AMMP was obtained by ultrasound-assisted enzymatic hydrolysis, purified through column chromatography, and preliminarily characterized using several physicochemical approaches. Its inhibitory effects on α-amylase and α-glucosidase were assessed as supporting in vitro evidence, and its regulatory activity in insulin-resistant HepG2 cells was further examined. The results showed that AMMP was a heteropolysaccharide fraction with a molecular weight of 15.6 kDa and was mainly composed of glucose (48.842%), galactosamine (14.647%), and mannose (13.564%). FT-IR and UV analyses revealed typical polysaccharide spectral characteristics, whereas one-dimensional 1H and 13C NMR spectra provided preliminary information on the general proton and carbon resonance regions. The relatively high proportion of galactosamine indicated the possible presence of amino sugar-containing components, although its exact origin and linkage pattern require further structural confirmation. AMMP exhibited moderate competitive and reversible inhibition of α-amylase and α-glucosidase, with weaker inhibitory potency than acarbose, and fluorescence quenching analysis suggested that the interactions were mainly mediated by van der Waals forces and hydrogen bonds. In insulin-resistant HepG2 cells, AMMP treatment (100-300 μg/mL) decreased glucose levels in the culture medium and cellular glycogen content, increased the activities of hexokinase, pyruvate kinase, and glucokinase, enhanced superoxide dismutase and glutathione peroxidase activities, and reduced malondialdehyde levels. In addition, AMMP regulated the mRNA expression of key genes in the PI3K/Akt signaling pathway. Transcriptomic analysis identified 32 DEGs under a stringent twofold-change criterion and 234 candidate DEGs under an exploratory threshold; among the latter, 35 remained statistically significant after Benjamini-Hochberg correction. GO enrichment analysis emphasized transcriptional regulation, metabolic and biosynthetic processes, oxidative stress, and metal ion-related responses, while PPI analysis identified IL6, JUN, and CXCL8 as the highest-degree candidate hub genes. KEGG analysis showed several exploratory pathway-level signals, although none remained significant after FDR correction. These findings provide preliminary in vitro evidence that AMMP may regulate glucose metabolism, oxidative stress, and insulin resistance-related molecular responses in insulin-resistant HepG2 cells, supporting further evaluation in suitable in vivo models.