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Mechanism by Which Guben Zhuyu Decoction Repairs Impaired Osteogenesis in Glucocorticoid-Induced Osteonecrosis of the Femoral Head: Inhibition of the Mitochondrial Apoptotic Pathway by Targeting the PI3K/AKT Signaling Pathway

Year: 2026

Type: preprint

Abstract: Background: Steroid-induced osteonecrosis of the femoral head (SIONFH) is a highly prevalent disorder characterized by impaired osteogenesis caused by the long-term or high-dose use of corticosteroids. However, currently available effective therapeutic agents remain lacking. Guben Zhuyu Decoction (GBZY), a traditional Chinese medicine formula, has been shown clinically to effectively delay the progression of SIONFH, but its precise therapeutic mechanism remains poorly understood. Purpose: This study aimed to investigate the mechanism by which GBZY repairs impaired osteogenesis in SIONFH by targeting the PI3K/AKT signaling pathway to inhibit the mitochondrial apoptotic pathway. Methods: A rat model of SIONFH was established using lipopolysaccharide (LPS) and methylprednisolone (MPS) to simulate the pathological process in vivo. The therapeutic effects of GBZY on SIONFH were evaluated through behavioral assessments, imaging analysis, histopathological examination, and detection of osteogenic markers. An in vitro model of osteogenesis inhibition was established in MC3T3-E1 cells using dexamethasone. The ability of GBZY to promote osteogenic differentiation was assessed by EdU staining, ALP staining, mineralized nodule staining, and analysis of the expression of osteogenic differentiation markers. Key active components and potential signaling pathways of GBZY were identified by combining HPLC–MS/MS with network pharmacology and were subsequently validated by immunohistochemistry (IHC) and western blotting (WB). Indicators including reactive oxygen species, apoptosis, and mitochondrial membrane potential were measured to evaluate the effects of GBZY on the mitochondrial apoptotic pathway. Results: In vivo experiments showed that GBZY significantly improved gait, femoral head architecture, and bone microstructure in rats with SIONFH, while upregulating the expression of osteogenic differentiation markers, including ALP, OPN, OCN, BMP-2, and RUNX2. Cell experiments supported these in vivo observations by showing that GBZY enhanced MC3T3-E1 cell proliferation and restored osteogenic differentiation and mineral deposition under dexamethasone stress. HPLC–MS/MS and network pharmacology analyses revealed that tanshinone IIA, kaempferol, quercetin, β-sitosterol, and luteolin were the key active components of GBZY involved in the intervention of SIONFH. In vitro experiments showed that GBZY protected MC3T3-E1 cells from apoptosis triggered by the mitochondrial apoptotic pathway by reducing reactive oxygen species (ROS), increasing BCL-2 expression, decreasing BAX expression, and restoring membrane potential. Further studies confirmed that GBZY activated the PI3K/AKT pathway by increasing the phosphorylation levels of downstream proteins. Notably, the PI3K/AKT pathway inhibitor LY294002 completely reversed all of the abovementioned effects. Conclusion: This study demonstrated that GBZY promotes osteogenesis by activating the PI3K/AKT signaling pathway, thereby counteracting DEX-induced activation of the mitochondrial apoptotic pathway. These findings provide experimental evidence supporting GBZY as a potential therapeutic strategy for SIONFH.

Source: Research Square

DOI: 10.21203/rs.3.rs-10944480/v1