Objective: To investigate the protective effect of Illicium verum extract on the vascularization of osteoporotic fracture in rats, and to elucidate its potential mechanism.
Methods: The osteoporotic fracture model was established in ovariectomized rats. Rats were infused with 0.05 ml/kg extract in the stomach every morning. Eighteen rats are then divided into control group, model group, and Illicium verum extract group with 6 rats in each group. To observe the therapeutic effect of Illicium verum extract on osteoporotic rats. Femoral bone mineral density and elastic segment end-point load were evaluated by dual-energy x-ray absorptiometry and three-point bending test. Hematoxylin-eosin staining was used to measure the number and area of callus blood vessels. The serum levels of VEGF and NO were detected by ELISA. Moreover, the expressions of NOX2, NOX4, NRF2, p-PI3K, CyclinD1, VEGF, HIF1α, and eNOS in HUVEC were detected by Western blot. CCK8 and wound healing assay were used to detect the proliferation and migration of HUVEC. Then, the ability of HUVEC to form blood vessels was detected by tube formation assay.
Results: Firstly, control group showed the normal pathomorphology and density of femoral bone, and model group showed significantly decreased bone density and consistent with bone microstructure degeneration, destruction, thinning, and fracture of bone trabecular structure vs control group, and illicium verum extract significantly increased femoral density and maximum load, increased the number and area of callus blood vessels and increased VEGF and NO levels in serum vs model group. Then, Illicium verum extract promoted the expression of NRF2, p-PI3K, CyclinD1, VEGF, HIF1α, and eNOS protein in HUVEC, inhibited the expression of NOX2 and NOX4, and enhanced the cell proliferation, migration, and angiogenesis. However, the effect was reversed by the overexpression of NRF2 and the treatment with LY294002.
Conclusion: Illicium verum extract protects the vascularization of the osteoporotic fracture model in rats.