Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

Aims: Induced membrane technique (IMT) is a novel two-stage reconstruction technology for large bone defects. As is widely known, promoting the vascularization of induced membrane tissue is of great importance in accelerating the mineralization process of bone graft. Naringin is a flavonoid compound with anti-inflammatory, anti-osteoporotic, and potential pro-angiogenic effects. The aim of this study is to explore the effect and mechanism of naringin on angiogenesis of induced membrane.

Methods: A total of 48 SD rats were randomly divided into a control group, low-dose group (L-Naringin), medium-dose group (M-Naringin), and high-dose group (H-Naringin). All rats underwent the establishment of an IMT model in the right femur. Four weeks after the first-stage surgery, which coincided with the completion of four weeks of treatment with different doses of naringin, the results of angiogenesis and cell proliferation within the induced membrane were evaluated. Eight weeks after the second-stage surgery, imaging and histological methods were employed to assess the healing of femoral bone defects and analyze the impact of induced membranes with varying vascular densities on the mineralization of bone grafts. The naringin-containing serum was collected from rats to cultivate endothelial progenitor cells (EPCs) derived from rat bone marrow in vitro. The effects of naringin on the proliferation, migration, angiogenesis, and transforming growth factor β (TGF-β)/SMAD signalling pathway of EPCs were assessed at corresponding stages. Meanwhile, the conditioned medium (CM) from EPCs was collected to treat osteoblasts, analyzing the effect of EPCs-derived angiogenic-osteogenic factors on osteoblast mineralization. Finally, naringin binding to TGF-β1 protein was studied by molecular docking, molecular dynamics simulation (MDS), and cellular thermal shift assay (CETSA).

Results: Compared with control and L-Naringin groups, the vascular number, cell proliferation rate, and TGF-β/SMAD pathway-related factors (TGF-β1, phosphorylated-SMAD (p-SMAD)2, and p-SMAD3) of the induced membrane were higher in the M-Naringin and H-Naringin groups. Meanwhile, the richer the blood vessels within the induced membrane, the stronger the mineralization capacity of the bone graft, and the faster the healing rate of the bone defect. In vitro, naringin-containing serum intervention notably increased cell viability, migration, tubular formation, secretion of angiogenic-osteogenic factors, and phosphorylation level of TGF-β/SMAD signalling pathway-related proteins in EPCs (p < 0.05 in the naringin group, vs the control group). Meanwhile, naringin-containing serum reversed the inhibitory effect of small interfering-TGF-β1 (si-TGF-β1) on the function of EPCs (p < 0.05 in the si-TGF-β1+ naringin group, vs the si-TGF-β1 group). The CM experiment demonstrated that angiogenic-osteogenic factors derived from EPCs could promote the formation of calcium nodules in osteoblasts. Additionally, results of molecular docking, myelodysplastic syndromes (MDS), and CETSA indicated that naringin could directly bind to the TGF-β1 protein and maintain its thermal stability.

Conclusion: Naringin could promote angiogenesis of the induced membrane and the mineralization of bone grafts in the later stage in a dose-dependent manner. This effect is partly related to naringin's targeted binding to TGF-β1 in EPCs, maintaining the stability of TGF-β1, and subsequently enhancing the activation and phosphorylation of the TGF-β1/SMAD signalling pathway.