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This study aimed to observe the promoting effect of Jianpi Huogu Formula (JPHGF) on angiogenesis-osteogenesis coupling in rats with steroid-induced osteonecrosis of the femoral head (SONFH), and to explore its mechanism of action through the vascular endothelial growth factor (VEGF) -Notch-Noggin signaling pathway. A total of 120 Sprague-Dawley rats were randomly divided into a normal group, a model group, low-, medium-, and high-dose JPHGF groups (2.5, 5, and 10 g·kg-1), and a Jiangusheng Pills group (1.53 g·kg-1). Except for the normal group, all rats were injected with lipopolysaccharide (20 μg·kg-1) via the tail vein once daily on days 1 and 2, followed by intramuscular injection of methylprednisolone sodium succinate (40 mg·kg-1, gluteal muscle) once daily from days 3 to 5. The normal group received an equivalent volume of normal saline. Four weeks after model establishment, the rats were administered the corresponding drugs by gavage. The therapeutic effects of JPHGF on SONFH rats were evaluated by measuring mechanical pain threshold, cold pain sensitivity, hemorheological parameters, four coagulation indices, and by pathological and imaging analyses of the femoral head. The microvascular distribution of the femoral head was observed by ink perfusion and Microfil angiography. Immunofluorescence was used to detect the expression of platelet endothelial cell adhesion molecule-1 (CD31), endothelial mucin (Emcn), and the osteoblast-specific transcription factor Osterix, as well as the co-expression of CD31 and Noggin proteins in the femoral head. Immunohistochemistry was employed to detect the expression of type Ⅰ collagen (collagen Ⅰ) and Notch-1 proteins, while Western blot was used to measure the expression levels of VEGF and Noggin proteins in the femoral head. The results showed that JPHGF significantly increased the mechanical pain threshold and reduced the number of foot lifts in response to cold stimulation in SONFH rats. It markedly increased trabecular bone area, reduced the number of adipocytes and the percentage of empty lacunae, improved imaging manifestations of femoral head necrosis, and increased the vascular area in ink perfusion and the vessel volume in Microfil perfusion within the medullary cavity of the femoral head. JPHGF also improved hemorheological parameters and coagulation function, upregulated the expression of CD31, Emcn, and Osterix proteins, enhanced the co-expression of CD31 and Noggin proteins, and promoted the expression of collagen Ⅰ, Notch-1, VEGF, and Noggin proteins. These findings indicate that JPHGF promotes angiogenesis-osteogenesis coupling by activating the VEGF-Notch-Noggin signaling pathway, thereby effectively treating SONFH in rats. This study elucidates the pharmacological connotation of JPHGF from the perspective of "angiogenesis-osteogenesis coupling" and provides an experimental basis for its clinical application.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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