Chromobox protein homolog 7 (CBX7) deficiency inhibits osteoblast ferroptosis by activating the Nrf2 function in type 2 diabetic osteoporosis.

Background: Ferroptosis occurs in osteoblasts in a diabetic environment, which impairs osteoblast number and function, promotes osteoblast death, destroys bone homeostasis, and eventually contributes to type 2 diabetic osteoporosis (T2DOP). Chromobox protein homolog 7 (CBX7) deficiency plays a positive role in bone formation and skeletal development. Besides, CBX7 interference has been reported to protect against disease development by inhibiting ferroptosis. This study focuses on determining whether CBX7 is involved in the progression of T2DOP by regulating osteoblast ferroptosis and explore the underlying mechanism.

Methods: The bone tissues of 28 patients with T2DOP (the research group) and severe bone trauma (the control group) were collected, and CBX7 expression in bone tissues was detected through RT-qPCR and western blotting. Mouse pre-osteoblast MC3T3-E1 cells were treated with high glucose (HG; 25mM) for 72h to establish an in vitro model of T2DOP. The effects of CBX7 knockdown or overexpression on ferroptosis in HG-treated MC3T3-E1 cells were assessed by examining the levels of intracellular Fe²⁺, lipid peroxidation, ROS, MDA, 4‑HNE, GSH, and ferroptosis regulatory proteins SLC7A11 and GPX4. The osteogenic differentiation of HG-treated MC3T3-E1 cells after knocking down or overexpressing CBX7 was assessed by performing ALP and ARS staining and measuring the levels of osteogenesis markers (ALP, RUNX2, and OCN). Whether CBX7 knockdown affects osteoblast differentiation and ferroptosis by regulating the Nrf2 pathway was validated by using the Nrf2 inhibitor ML385. Furthermore, Sprague Dawley (SD) rats were fed a high-fat diet and injected with streptozotocin to induce type 2 diabetes (T2DM), followed by the induction of T2DOP for 2 months and subsequently the determination of the anti-osteoporotic, anti-ferroptotic, and pro-osteogenic effects of CBX7 deficiency in vivo.

Results: CBX7 expression was markedly increased in the bone tissues of T2DOP patients compared to control patients. HG stimulation enhanced CBX7 expression, intracellular Fe²⁺, lipid peroxidation, ROS, MDA, and 4‑HNE levels but attenuated GSH, SLC7A11, and GPX4 levels in MC3T3-E1 cells. These effects of HG were reversed by CBX7 knockdown but were intensified by CBX7 overexpression. CBX7 silencing antagonized while CBX7 overexpression enhanced the inhibitory effects of HG treatment on the osteogenic differentiation of MC3T3-E1 cells. CBX7 knockdown promoted Nrf2 expression and nuclear translocation in HG-treated MC3T3-E1 cells and CBX7 showed potential protein interaction with Nrf2. The inhibition of CBX7 depletion on HG-induced ferroptosis in MC3T3-E1 cells and its promotion on the osteogenic differentiation of MC3T3-E1 cells were abrogated by ML385. Additionally, CBX7 silencing ameliorated bone mass loss and bone microstructure destruction, reduced MDA and iron ion levels, and elevated ALP, OCN, Nrf2, and GPX4 in T2DOP rats.

Conclusion: Our study demonstrates that CBX7 expression is increased during T2DOP and that CBX7 knockdown promotes osteogenesis and ameliorates T2DOP progression by inhibiting osteoblast ferroptosis through activating the Nrf2/SLC7A11/GPX4 pathway. These findings suggest that CBX7 is a promising novel therapeutic target for T2DOP.