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

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 fluorescence staining, and flow cytometry. Mitochondrial membrane potential and energy metabolism were evaluated by JC-1 fluorescence staining and ATP content detection. Mitochondrial superoxide levels were measured using MitoSOX fluorescence probe and flow cytometry. Intracellular total reactive oxygen species and glutathione content were also measured to assess oxidative stress status. Additionally, Western blot and qRT-PCR were used to detect the expression of apoptosis-related proteins (Bax, Bcl-2) and autophagy markers (LC3B, p62), flow cytometry was used to analyze apoptosis rate, and autophagy flux was observed via mRFP-GFP-LC3 adenovirus transfection combined with confocal microscopy. RESULTS AND CONCLUSION: Compared with the control group, the dexamethasone group showed significantly reduced osteogenic differentiation capacity of MC3T3-E1 cells, abnormal mitochondrial structure (swelling, cristae disruption), decreased mitochondrial membrane potential, reduced ATP synthesis, increased mitochondrial superoxide and total reactive oxygen species levels, and increased glutathione consumption (P < 0.05). The dexamethasone group showed significantly upregulated pro-apoptotic protein Bax (P < 0.01), significantly downregulated anti-apoptotic protein Bcl-2 (P < 0.01), increased LC3B-II/I ratio (P < 0.01), and decreased p62 levels (P < 0.01); dexamethasone treatment significantly increased the apoptosis rate (P < 0.01). mRFP-GFP-LC3 adenovirus tracing revealed increased formation of autophagosomes and autolysosomes. These results indicate that dexamethasone induces mitochondrial dysfunction and oxidative stress, synergistically regulating apoptosis and autophagy in MC3T3-E1 cells, thereby impairing bone formation and repair function. This mechanism may be a key pathological basis for the pathogenesis of steroid-induced osteonecrosis of the femoral head.

1. Introduction

Steroid-induced osteonecrosis of the femoral head is a non-traumatic osteonecrosis caused by long-term or high-dose glucocorticoid use, leading to abnormal bone metabolism, lipid metabolism imbalance, blood microcirculation disorders, trabecular bone fracture, bone tissue necrosis and collapse, and hip joint dysfunction. With the widespread use of glucocorticoids in rheumatic immune diseases, organ transplantation, and hematological diseases, the incidence of steroid-induced osteonecrosis of the femoral head has been increasing annually, posing a major clinical challenge. Studies have shown that the incidence of osteonecrosis is positively correlated with the dose and duration of glucocorticoid use. For example, a daily dose of 40 mg for more than 3 months results in a 5%-40% incidence of osteonecrosis, and each 10 mg increase in daily dose raises the incidence by 4%. Steroid-induced osteonecrosis of the femoral head is characterized by bilateral symmetry, extensive necrosis, and high disability rate. Approximately 80% of patients develop femoral head collapse within two to three years after diagnosis, ultimately requiring total hip arthroplasty, imposing a heavy medical burden on patients and society. Given the irreplaceable role of glucocorticoids in clinical treatment, in-depth research on the pathogenesis of steroid-induced osteonecrosis of the femoral head is of great theoretical significance for its prevention and treatment.

The pathogenesis of steroid-induced osteonecrosis of the femoral head is complex and not fully elucidated. Current research suggests that glucocorticoids disrupt the homeostasis of the femoral head microenvironment through multiple pathways, among which osteoblast dysfunction and mitochondrial damage are considered key pathological mechanisms. Glucocorticoids significantly inhibit the proliferation and differentiation of osteoblasts, involving dysregulation of multiple signaling pathways. They can also hinder osteogenic differentiation by downregulating key osteogenic transcription factors such as Runt-related transcription factor 2 (Runx2) and Osterix. Mitochondria, as the energy metabolism center and apoptosis regulatory hub of cells, play a critical role in the pathogenesis of steroid-induced osteonecrosis of the femoral head. This study aims to investigate the effects of dexamethasone on mitochondrial function in osteoblasts and its regulatory roles in apoptosis and autophagy, providing new insights into the pathogenesis of this disease.