Mechanism of glucocorticoid-induced mitochondrial dysfunction in osteoblasts in steroid-induced osteonecrosis of the femoral head

doi:10.12307/2026.872

Abstract

Abstract: BACKGROUND: The pathogenesis of steroid-induced osteonecrosis of the femoral head remains unclear; however, it is closely associated with mitochondrial damage in osteoblasts.
OBJECTIVE: To explore the impact of dexamethasone on mitochondrial dysfunction in osteoblasts following steroid-induced osteonecrosis of the femoral head and to analyze its regulatory roles in osteoblast apoptosis and autophagy.
METHODS: MC3T3-E1 cells were cultured in vitro and divided into control group (no treatment) and dexamethasone group (1 μmol/L dexamethasone treatment for 24 hours). Osteoblast differentiation capacity was assessed by alizarin red staining and qRT-PCR. Mitochondrial morphology was examined using transmission electron microscopy, MitoTracker Red fluorescent staining, and flow cytometry. Mitochondrial membrane potential and energy metabolism were evaluated via JC-1 fluorescent staining and ATP content measurement. Mitochondrial superoxide levels were quantified using MitoSOX™ Red fluorescent probe combined and flow cytometry. Intracellular total reactive oxygen species and glutathione levels were simultaneously measured to evaluate oxidative stress status. Additionally, western blot and qRT-PCR assays were performed to examine Bax/Bcl-2 (apoptosis) and LC3B/p62 (autophagy) expression. Flow cytometric analysis was performed to assess apoptotic rates. Autophagic flux was assessed using mRFP-GFP-LC3 adenovirus transfection followed by confocal microscopy analysis.
RESULTS AND CONCLUSION: (1) Compared with the control group, dexamethasone-treated MC3T3-E1 cells exhibited significantly impaired osteogenic differentiation capacity (P < 0.05), accompanied by mitochondrial structural abnormalities (including swelling and cristae disruption), decreased membrane potential, reduced glutathione synthesis, elevated mitochondrial superoxide and total reactive oxygen species levels, as well as increased glutathione depletion. (2) Western blot analysis revealed that dexamethasone treatment significantly upregulated Bax (P < 0.01) while downregulating Bcl-2 (P < 0.01), concurrently increasing the LC3B-II/I ratio (P < 0.01) and decreasing p62 levels (P < 0.01). Flow cytometry analysis further confirmed that dexamethasone treatment significantly increased the apoptotic rate (P < 0.01). (3) mRFP-GFP-LC3 adenovirus tracer detection demonstrated a marked enhancement in the formation of both autophagosomes and autolysosomes. To conclude, dexamethasone regulates the apoptosis and autophagy processes of MC3T3-E1 cells in a coordinated manner by inducing mitochondrial dysfunction and oxidative stress response, and thereby impairs bone formation and repair functions. This mechanism may be the key pathological basis for the pathogenesis of steroid-induced osteonecrosis of the femoral head.

Key words: steroid-induced osteonecrosis of the femoral head, mitochondria, mitochondrial dysfunction, dexamethasone, apoptosis, autophagy

CLC Number:

Ma Runqiu, Yang Huixia, Li Xuer, Bai Zhigang, Li Guizhong, Hao Yinju, Ma Shengchao, Jiang Yideng. Mechanism of glucocorticoid-induced mitochondrial dysfunction in osteoblasts in steroid-induced osteonecrosis of the femoral head[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(34): 8845-8851.

Figures/Tables 6

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