Abstract
Background: This study aimed to explore the synergistic hypoglycemic effect and mechanism of 1-deoxynojirimycin (DNJ) in mulberry leaves and theaflavins (TFs) in black tea. Methods: The synergistic inhibition of α-glucosidase and α-amylase by DNJ-TFs was evaluated using enzyme assays and the Chou-Talalay model. Insulin-resistant (IR) HepG2 cells and high-fat diet (HFD)-induced type 2 diabetes mellitus mice were treated with DNJ, TFs, or DNJ-TFs, determining the efficacy of drug combinations by measuring glycolipids and inflammatory factors. Network pharmacology and molecular docking were used to identify key target genes and signaling pathways, and CETSA experiments were used to verify the binding of drugs to targets. Key genes were further verified by immunofluorescence, Western blot, and Real-time PCR. Results: DNJ-TFs synergistically suppressed α-glucosidase (CI = 0.85) and α-amylase (CI = 0.76). In HepG2 cells, DNJ-TFs ameliorated palmitic acid-induced IR by promoting glucose uptake, attenuating lipid accumulation, and regulating glycolipid metabolism. In HFD mice, DNJ-TFs counteracted hyperglycemia, dyslipidemia, systemic inflammation and oxidative stress, elevated HOMA-IR, and hepatic steatosis. Network pharmacology integrated with experimental validation identified PTGS2 and MMP9 as key binding targets of DNJ and TFs. Furthermore, DNJ-TFs could inhibit the increase in liver TNFα protein and the decrease in p-AKT, p-GSKα, p-GSKβ, and GLUT2 protein caused by high fat, both in vivo and in vitro. Conclusions: DNJ and TFs exert synergistic glucose-lowering effects by targeting PTGS2/MMP9 and regulating the TNFα/AKT/GSK3/GLUT2 axis, providing a promising natural therapeutic strategy for diabetes management.