Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study.

PURPOSE: Periodontal ligament stem cells (PDLSCs) play a critical role in alveolar bone regeneration and orthodontics. Astragaloside IV (AS-IV) is the chief ingredient of Astragalus, which has been shown to promote osteogenesis. The study aimed to detect the impact of AS-IV on osteogenic differentiation of PDLSCs and to investigate the role of the PI3K/AKT/eNOS/NO pathway in this process. METHODS: PDLSCs were isolated from clinically healthy premolars that were extracted for orthodontic purposes from patients aged 14-20 years. The isolated cells were then cultured in vitro and characterized by flow cytometry. After treating the cells with different doses of AS-IV, LY294002 (PI3K inhibitor), and L-NAME (eNOS inhibitor), alkaline phosphatase (ALP) staining, alizarin red staining, qRT-PCR, Western blotting, nitric oxide (NO) assay and immunofluorescence staining were utilized to ascertain the expression level of related factors and the validity of PI3K/AKT/eNOS/NO pathway. Divided sixteen male Wistar rats into the control and AS-IV groups, and the orthodontic tooth movement model was created for 14 days. Micro-computed tomography scan, hematoxylin and eosin staining and immunohistochemical staining were conducted to investigate relevant indicators. RESULTS: PDLSCs expressed high levels of surface antigens CD44 and CD90 while negatively expressing CD34 and CD45. AS-IV at each experimental concentration did not inhibit the proliferation of hPDLSCs, and 20 μM AS-IV could significantly enhance ALP activity, mineral deposition, and ALP, runt-related transcription factor 2 (RUNX-2), collagen I (COL-1) expression. After adding inhibitors LY294002 and L-NAME, the effect of AS-IV was inhibited. In vivo, AS-IV increased bone volume/total volume (BV/TV), trabecular thickness (Tb. Th), and the expression of ALP, COL-1 and eNOS on the tension side in rats. CONCLUSION: AS-IV can promote the osteogenic differentiation of PDLSCs, and PI3K/AKT/eNOS/NO was involved. Meanwhile, AS-IV exhibits positive effects on tension-side osteogenesis during tooth movement in rats.