The purpose of this study was to prepare phosphorylated Athyrium multidentatum (Doll.) Ching polysaccharide (PPS) and investigate its protective effect on vascular endothelial cells (VECs) in vitro and in vivo and the underlying mechanisms. Sodium tripolyphosphate (STPP) and sodium trimetaphosphate (STMP) were used as phosphorylation reagents and PPS was characterized by Fourier transform infrared (FT-IR), 13 C nuclear magnetic resonance ( 13 C NMR) and 31 P nuclear magnetic resonance ( 31 P NMR) spectra. Chemical analysis demonstrated that PPS was composed of mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose with a molar ratio of 11.36:0.42:4.03:1.12:1.81:0.26:33.25:24.12:6.85:14.46:2.32 and a molecular weight of 28,837?Da. Results from in vitro and in vivo assays revealed that PPS protected human umbilical vein endothelial cells (HUVECs) against H 2 O 2 -induced oxidative injury and attenuated D-galactose-induced VECs damage in mice. RNA sequencing (RNA-seq) analysis identified 18 differentially expressed genes (DEGs) between D-galactose-treated and PPS-pretreated mice abdominal aorta. A deep analysis of these DEGs disclosed that PPS regulated the expression of genes involved in the functions of vascular endothelium repairment, cell growth and proliferation, cell survival and apoptosis, inflammation, angiogenesis and antioxidant, indicating that these biological processes might play crucial roles in the protective actions of PPS on VECs.
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