Preparation, structural characterization and hypoglycemic activity of Pleurotus geesteranus stem polysaccharide

Abstract

In order to explore the potential molecular mechanism by which Pleurotus geesteranu s stem polysaccharide (PGSP) regulates glucose metabolism in insulin-resistant HepG2 cells. This study selected the stems of Pleurotus geesteranus ( P. geesteranus )as raw material for three main reasons: agricultural waste mushroom stalks are cheap, abundant and sustainable by-products of edible fungus cultivation; fungal polysaccharides have well-documented hypoglycemic and insulin-sensitizing bioactivities; and the hypoglycemic efficacy and molecular mechanism of polysaccharides extracted from P . geesteranus stems remain poorly characterized, requiring systematic elucidation. Crude polysaccharides were extracted via ultrasonic-assisted water extraction, followed by sevage deproteinization, dialysis, sequential purification using DEAE-52 cellulose anion-exchange chromatography and Superdex-200 size-exclusion chromatography to obtain purified PGSP. Multiple analytical techniques including ultraviolet-visible spectrophotometry, Fourier-transform infrared spectroscopy, ion chromatography and nuclear magnetic resonance spectroscopy were applied to characterize its chemical structure. Structural analyses revealed that PGSP possessed a pyranose backbone linked by α-glycosidic bonds, and was composed of fucose, arabinose, rhamnose, galactose, glucose, mannose and glucuronic acid at a molar ratio of 1.02: 42.02. Its weight-average molecular weight (Mw) and number-average molecular weight (Mn) were 66.39 kDa and 62.14 kDa, respectively, with a polydispersity index (Mw/Mn) of 1.07, confirming it as a homogeneous neutral polysaccharide fraction. The CCK-8 assay was adopted to evaluate cell viability, and a concentration gradient (40–640 µg/mL) was used to screen the effects of PGSP on cell proliferation and glucose consumption in both normal and insulin-resistant HepG2 cells. Cellular assays verified that PGSP exhibited no cytotoxicity across the tested concentration range and markedly facilitated HepG2 cell proliferation. Relative to untreated insulin-resistant cell models, PGSP at 320 µg/mL and 640 µg/mL remarkably elevated cellular glucose uptake capacity. Further immunofluorescence staining and western blot analyses using the optimal effective concentration (320 µg/mL) demonstrated that PGSP alleviated hepatic insulin resistance by upregulating IRS1 protein abundance, suppressing the expression of phosphorylated GSK-3β, and elevating the membrane translocation and total expression of GLUT4, collectively activating the IRS1/GSK-3β/GLUT4 insulin signaling cascade. This work systematically clarifies the structural features and hypoglycemic activity of PGSP, unveils its underlying molecular regulatory pathway against hepatic insulin resistance, and provides experimental evidence supporting the development of edible mushroom stem-derived polysaccharides as natural auxiliary hypoglycemic functional food ingredients.