Yang Song,1– 3 Jing Hu,1– 3 Peng Yang,1– 3 Yuxing Zhang,1– 3 Zhaoyan Wu,1– 3 Siyu Chen,1– 3 Jun Zhang1– 3 

1Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University, Jinan, People’s Republic of China; 2Shandong Key Laboratory of Oral Tissue Regeneration, Jinan, People’s Republic of China; 3Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration, Jinan, People’s Republic of China

Correspondence: Jun Zhang, Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University, No. 44-1 Wenhua Road West, Jinan, Shandong, 250012, People’s Republic of China, Tel +86 13953109816, Fax +86 53188382923, Email zhangj@sdu.edu.cn

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.

Keywords: Astragaloside IV, periodontal ligament stem cells, osteogenic differentiation, PI3K/AKT/eNOS/NO