Huogu injection protects against SONFH by promoting osteogenic differentiation of BMSCs and preventing osteoblast apoptosis

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.