Effect of Illicium henryi extract on angiogenesis in rats with osteoporotic fractures
Shi Guirong1, Ren Bowen2, Zhang Zhongbo2, Wang Lisha2, Zhang Qiwei2, Shi Dongliang2
1Department of Traditional Chinese Medicine, Clinical School of Shangqiu Medical College, Shangqiu 476000, Henan Province, China; 2First Department of Orthopedics, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou 450000, Henan Province, China
Abstract: BACKGROUND: The extract of Illicium henryi is a Chinese herbal preparation with anti-inflammatory and analgesic properties and invigorating blood circulation and stasis. Its ethanol extract has markedly curative effect on rat soft tissue injury. However, there is yet no report on whether Illicium henryi has therapeutic effect on osteoporotic fractures.
OBJECTIVE: To investigate the protective effect of Illicium henryi extract on angiogenesis in osteoporotic fracture rats and to clarify its possible mechanism.
METHODS: (1) In vivo experiments: A rat model of osteoporotic fracture was prepared using castration method and closed fracture method. A total of 36 healthy female Sprague-Dawley rats were selected, 30 of which were randomized into sham, model, and treatment groups, with 10 rabbits in each group, and the remaining 6 were included in the control group. Each rat in the treatment group was given 0.05 mL/kg Illicium henryi extract after modeling. The dual-energy X-ray and three-point bending mechanics methods were used to detect femoral bone mineral density and the end-point load of the femoral elastic segment. Hematoxylin-eosin staining was used to measure the number and area of blood vessels in bone calluses. ELISA was used to detect serum levels of vascular endothelial growth factor, basic fibroblast growth factor, platelet-derived growth factor, and nitric oxide. (2) In vitro experiments: Human microvascular endothelial cells were given drug intervention. Treated cells were divided into control group, Illicium henryi extract (50, 100, or 200 mg/L) groups, Vector+dimethyl sulfoxide group, NRF1 overexpression group, LY294002 (10 μmol/L) treatment group, and NRF1 overexpression+LY294002 treatment group. MTT, Transwell, and angiogenesis assays were used to detect the proliferation, migration rate and angiogenesis of human microvascular endothelial cells, respectively. Western blot assay was used to detect the levels of NRF1, PI3K, p-PI3K, AKT, and p-AKT proteins in human microvascular endothelial cells. ELISA was used to detect the levels of vascular endothelial growth factor, basic fibroblast growth factor, platelet-derived growth factor, and nitric oxide in human microvascular endothelial cells.
RESULTS AND CONCLUSION: (1) Illicium henryi extract significantly improved the bone mineral density and maximum load of the femur in osteoporotic rats (P < 0.05), and increased the number and area of blood vessels in the callus tissue, and the serum levels of angiogenesis-related factors (P < 0.05). (2) Illicium henryi extract promoted the expression of NRF1, p-PI3K and p-AKT proteins in human microvascular endothelial cells (P < 0.05), and increased the proliferation, migration, angiogenesis, and angiogenesis-related factor expressions of human microvascular endothelial cells (P < 0.05). Overexpression of NRF1 had the same effects, but LY294002 treatment reversed the effects of NRF1 overexpression. (3) To conclude, Illicium henryi extract can promote angiogenesis in osteoporotic fracture rats by increasing the expression of NRF1 protein and activating the PI3K/AKT signaling pathway.
Key words: Illicium henryi, osteoporosis, fracture, human microvascular endothelial cell, NRF1, the PI3K/AKT pathway