Tang HaoXu, Yuan Lingli, Xu ZhiYuan, Jiang GuiFen, Liang YingJie, Li Ce, Ding PengLin, Qian MinLong
Department, Bengbu Medical University, Anhui, China.
Department of the Second, Affiliated Hospital of Bengbu Medical University, Anhui, China.
Sci Rep. 2025 Aug 9;15(1):29205. doi: 10.1038/s41598-025-15018-4.
Glucocorticoid-induced osteoblast dysfunction is the primary cause of steroid-induced osteonecrosis of the femoral head (SONFH). However, the specific underlying biological mechanisms of glucocorticoids' effect on osteoblasts remain undetermined. Recently, the role of hypoxia-inducible factor 1-alpha (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway in modulating bone formation has been studied. This study aimed to investigate the association and mechanism of the HIF-1α/VEGF signaling pathway in glucocorticoid-induced osteogenesis suppression in MC3T3-E1 cells. This study performed CCK8 and live/dead staining assays by stimulating MC3T3-E1 cells with varying dexamethasone (DEX) doses to elucidate its influence on cell proliferation and activity. Furthermore, Western blotting was carried out to investigate the expression of HIF-1α, runt-related transcription factor 2 (RUNX2), VEGF, osteopontin (OPN), and alkaline phosphatase (ALP) proteins to identify the optimal DEX concentration for simulating steroid-induced osteonecrosis cell models. Moreover, the osteogenic differentiation of cells was assessed by transfecting them with control or HIF-1α overexpression lentiviral vectors. Similarly, in vivo, hematoxylin and eosin staining, immunohistochemical staining, and micro-computed tomography were performed to validate in vitro results in the SONFH rat model. In vitro analyses revealed that a 10 M concentration of DEX significantly suppressed cell viability and osteogenesis by decreasing HIF-1α and VEGF levels. Furthermore, HIF-1α upregulation increased osteogenic activity and VEGF expression in MC3T3-E1 cells. However, the HIF-1α antagonist 3-(5'-hydroxymethyl-2'-furyl) -1-benzylindazole (YC-1) indicated opposite effects in DEX-treated MC3T3-E1 cells. Moreover, SONFH femoral heads had reduced bone density, bone tissue content, and femoral head integrity, as well as increased bone cell lacunae, while decreased HIF-1α, OPN, VEGF, and ALP levels in bone tissue compared to normal rats. This study indicated that DEX suppresses osteoblast differentiation via the HIF-1α/VEGF pathway, thus promoting SONFH.
糖皮质激素诱导的成骨细胞功能障碍是类固醇诱导的股骨头坏死（SONFH）的主要原因。然而，糖皮质激素对成骨细胞作用的具体潜在生物学机制仍未明确。最近，缺氧诱导因子1α（HIF-1α）/血管内皮生长因子（VEGF）信号通路在调节骨形成中的作用已得到研究。本研究旨在探讨HIF-1α/VEGF信号通路在糖皮质激素诱导的MC3T3-E1细胞成骨抑制中的关联及机制。本研究通过用不同剂量的地塞米松（DEX）刺激MC3T3-E1细胞进行CCK8和活/死染色试验，以阐明其对细胞增殖和活性的影响。此外，进行蛋白质免疫印迹法以研究HIF-1α、 runt相关转录因子2（RUNX2）、VEGF、骨桥蛋白（OPN）和碱性磷酸酶（ALP）蛋白的表达，以确定模拟类固醇诱导的骨坏死细胞模型的最佳DEX浓度。此外，通过用对照或HIF-1α过表达慢病毒载体转染细胞来评估细胞的成骨分化。同样，在体内，进行苏木精和伊红染色、免疫组织化学染色和微计算机断层扫描，以在SONFH大鼠模型中验证体外结果。体外分析显示，10 μM浓度的DEX通过降低HIF-1α和VEGF水平显著抑制细胞活力和成骨。此外，HIF-1α上调增加了MC3T3-E1细胞的成骨活性和VEGF表达。然而，HIF-1α拮抗剂3-（5'-羟甲基-2'-呋喃基）-1-苄基吲唑（YC-1）在DEX处理的MC3T3-E1细胞中显示出相反的作用。此外，与正常大鼠相比，SONFH股骨头的骨密度、骨组织含量和股骨头完整性降低，骨细胞腔增加，而骨组织中HIF-1α、OPN、VEGF和ALP水平降低。本研究表明，DEX通过HIF-1α/VEGF途径抑制成骨细胞分化，从而促进SONFH。
