Research on the Role and Mechanism of Rehmannioside D in Regulating Fracture Healing

Rehmannioside D (RD), a characteristic iridoid glycoside from Rehmannia glutinosa, possesses antioxidant and bone metabolism-regulating activity. Existing studies have confirmed its protective effects on cartilage injury, but its specific molecular mechanism in fracture repair remains unclear. This study aimed to explore the regulatory function and underlying signaling pathway of RD on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and mouse femoral fracture healing.

This study combined network pharmacology prediction with in vitro cell experiments and in vivo mouse fracture model validation. First, potential targets of RD and fracture-related disease were retrieved from the PubChem, GeneCards, and OMIM databases; PPI networks and KEGG enrichment analyses were performed to identify core signaling pathways. in vitro, CCK-8, Alizarin Red S (ARS) staining, immunofluorescence, and Western blot were used to detect the proliferation and osteogenic differentiation of C57BL/6J mouse BMSCs treated with gradient concentrations of RD (0-160 μg/mL). in vivo, a stable mouse femoral transverse fracture model was established; mice were divided into the model group, low-dose RD (0.05 mg/kg/d), and high-dose RD (0.1 mg/kg/d) groups via intragastric administration for 3 weeks. Micro-CT, HE/Safranin O-Fast Green staining, and immunohistochemistry (IHC) were applied to evaluate callus remodeling and the expression of osteogenic markers RUNX2, BMP2, OPG, and OPN. PI3K inhibitor Alpelisib was used for reverse verification of core pathways.

In vitro assays showed that RD (0-160 μg/mL) exhibited no cytotoxicity to BMSCs; 40 μg/mL and 80 μg/mL RD significantly enhanced mineralized nodule formation (P < 0.05). Western blot and immunofluorescence verified that RD dose-dependently upregulated phosphorylated PI3K and AKT protein expression. After PI3K inhibition, the osteogenic promotion effect of RD was markedly abolished. in vivo Micro-CT results demonstrated that RD significantly reduced BV/TV and Tb.N of fracture callus (P < 0.05), accelerating callus remodeling. Histological staining revealed that RD facilitated endochondral ossification, reduced residual immature chondrocytes, and promoted mature bone formation. uIHC results confirmed that RD was significantly elevated at the fracture site, with increased expression of BMP2, OPN, OPG, and RUNX2 (P < 0.05). Network pharmacology predicted PI3K/AKT as the core pathway mediating RD's bone-protective effects.

RD exerts dual regulatory effects on fracture repair by facilitating BMSC osteogenic differentiation and accelerating callus remodeling. Mechanistically, RD activates the PI3K/AKT signaling cascade to upregulate key osteogenic transcription factors and matrix proteins, thereby boosting endochondral ossification during fracture healing. The present study complements the pharmacological mechanism of iridoid glycosides in bone regeneration, providing experimental evidence for the application of Rehmannia-derived active ingredients in orthopedic fracture treatment. Limitations of this study include a lack of long-term animal observation and clinical sample validation, which require further exploration.

RD promotes BMSC proliferation and osteogenic differentiation via activating the PI3K/AKT signaling pathway, accelerates callus remodeling and endochondral ossification, and ultimately improves femoral fracture healing in mice. RD is a promising natural active candidate for fracture repair intervention.