Abstract: Our aim was to elucidate the effect and mechanism of puerarin on alleviating the osteogenic phenotypic transformation of vascular smooth muscle cells (VSMCs) in mice with type 2 diabetes mellitus (T2DM). A mouse model of T2DM was established using a high-fat diet combined with streptozotocin, and immunofluorescence staining was used to investigate the expression of osteogenic transformation proteins in the thoracic aorta of T2DM mice. Arterial calcification was detected by alizarin red S staining. VSMCs were harvested from the thoracic aorta and were induced to undergo osteogenic phenotype transformation and calcification by high glucose and high glucose calcifying medium. Western blot was used to detect the expression of osteogenic transformation marker proteins, azo coupling was used to detect alkaline phosphatase activity, and alizarin red S staining was used to detect the amount of calcium deposition. On this basis, network pharmacology and the Gene Expression Omnibus (GEO) database were used to explore the key targets of puerarin in regulating the osteogenic phenotype transformation of VSMCs induced by diabetes and high glucose. The results showed that puerarin ameliorated morphological alterations in the thoracic aorta of T2DM mice. Notably, it suppressed the expression of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2) in the aortic tissue, thereby attenuating vascular calcium deposition in this animal model. At the cellular level, puerarin at concentrations of 1 and 10 μmol/L significantly reduced the expression of RUNX2 and BMP2 in VSMCs, while promoting the expression of smooth muscle α22. Additionally, it inhibited cellular alkaline phosphatase activity and prevented calcium deposition induced by high glucose and calcification. Network pharmacology analysis showed that puerarin inhibits T2DM-induced osteogenic phenotype transformation of VSMCs via synergistic regulation of multiple targets. Further, using the GEO database, we selected nine key targets of puerarin in regulating the osteogenic transformation of VSMCs induced by high glucose. The key targets were screened using molecular docking, and it was found that the binding energy between puerarin and the solute carrier family 7 member 11 (SLC7A11) was the lowest (–40.735 kJ/mol). Cellular thermal shift assay showed the binding of puerarin to the target protein SLC7A11. Further analysis using molecular dynamics simulation elucidated the binding mode, key interacting amino acids, and binding stability between puerarin and SLC7A11. Cell-based experiments confirmed that puerarin promoted the protein expression of SLC7A11 and regulated SLC7A11-mediated glutathione content and reactive oxygen species (ROS) levels. Additionally, palmitoylation was involved in the regulation of SLC7A11 expression by puerarin. In conclusion, puerarin can inhibit T2DM-induced osteogenic phenotypic transformation and calcification of VSMCs, which is attributed to synergistic regulation of multiple targets. Modulation of SLC7A11 expression is one of the underlying mechanisms.

Key words: puerarin; aortic calcification in type 2 diabetic mellitus mice; osteogenic phenotype transformation of vascular smooth muscle cells; network pharmacology; solute carrier family 7 member 11