To investigate the effect and mechanism of Huogu injection (HG) on steroid-induced osteonecrosis of the femoral head (SONFH), we established a SONFH model in rabbits using horse serum and dexamethasone (DEX) and applied HG locally at the hip joint. We evaluated the therapeutic efficacy at 4 weeks using scanning electron microscopy (SEM), micro-CT, and qualitative histology including H&E, Masson’s trichrome, ALP, and TUNEL staining. In vitro, we induced osteogenic differentiation of bone marrow stromal cells (BMSCs) and performed analysis on days 14 and 21 of cell differentiation. The findings, in vivo, including SEM, micro-CT, and H&E staining, showed that HG significantly maintained bone quality and trabecular number. ALP staining indicated that HG promoted the proliferation of bone cells. Moreover, the results of Masson’s trichrome staining demonstrated the essential role of HG in collagen synthesis. Additionally, TUNEL staining revealed that HG reduced apoptosis. ALP and ARS staining in vitro confirmed that HG enhanced osteogenic differentiation and mineralization, consistent with the WB and qRT-PCR analysis. Furthermore, Annexin V-FITC/PI staining verified that HG inhibited osteoblast apoptosis, in agreement with the WB and qRT-PCR analyses. Furthermore, combined with the UPLC analysis, we found that naringin enhanced the osteogenic differentiation and accelerated the deposition of calcium phosphate. Salvianolic acid B protected osteoblasts derived from BMSCs against GCs-mediated apoptosis. Thus, this study not only reveals the mechanism of HG in promoting osteogenesis and anti-apoptosis of osteoblasts but also identifies the active-related components in HG, by which we provide the evidence for the application of HG in SONFH.

为了探讨活骨注射液（HG）对激素性股骨头坏死（SONFH）的作用和机制，我们使用马血清和地塞米松（DEX）建立家兔SONFH模型，并将HG局部应用于髋关节。我们使用扫描电子显微镜 (SEM)、显微 CT 和定性组织学（包括 H&E、Masson 三色、ALP 和 TUNEL 染色）评估了 4 周时的治疗效果。在体外，我们诱导骨髓基质细胞 (BMSC) 成骨分化，并在细胞分化的第 14 天和第 21 天进行分析。体内研究结果（包括 SEM、显微 CT 和 H&E 染色）表明 HG 显着维持了骨质量和小梁数量。ALP染色表明HG促进骨细胞增殖。此外，Masson三色染色的结果证明了HG在胶原蛋白合成中的重要作用。此外，TUNEL 染色显示 HG 减少细胞凋亡。体外 ALP 和 ARS 染色证实 HG 增强成骨分化和矿化，与 WB 和 qRT-PCR 分析一致。此外，Annexin V-FITC/PI 染色证实 HG 抑制成骨细胞凋亡，与 WB 和 qRT-PCR 分析一致。此外，结合UPLC分析，我们发现柚皮苷增强成骨分化并加速磷酸钙的沉积。丹酚酸 B 可以保护 BMSC 来源的成骨细胞免受 GC 介导的细胞凋亡。因此，本研究不仅揭示了HG促进成骨和抗成骨细胞凋亡的机制，而且鉴定了HG中的活性相关成分，为HG在SONFH中的应用提供了证据。