Nrf2 epigenetic derepression induced by running exercise protects against osteoporosis

Osteoporosis (OP), characterized by low bone mineral density (BMD) and fragility fractures, is a significant health concern, particularly among postmenopausal women. Bone remodeling, a balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation, is crucial for bone health. In OP, osteoclastogenesis exceeds osteoblastogenesis, leading to reduced BMD and impaired trabecular microstructure. The pathogenesis of OP involves various factors, including aging, hormonal imbalances, and altered gene transcription. Epigenetic modifications, such as DNA methylation, play a significant role in regulating osteoclastogenesis and osteoblastogenesis. DNA methylation, catalyzed by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b), silences gene transcription by adding methyl groups to cytosine bases in CpG islands within gene promoters. Conversely, DNA demethylation reactivates gene transcription. Emerging evidence suggests that physical exercise can modulate DNA methylation, influencing various physiological processes. Exercise is a well-established method for improving bone health and preventing OP, but the underlying epigenetic mechanisms remain largely unclear. Oxidative stress also contributes to OP pathogenesis, with weak oxidative stress responses and low circulating antioxidants associated with lower BMD. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a crucial transcription factor in cellular defense against oxidative stress and bone homeostasis. Nrf2 transactivates multiple antioxidant enzymes upon stress stimulation. Nrf2 deficiency is linked to increased susceptibility to OP, while Nrf2 activation has protective effects. The Nrf2 promoter contains CpG islands, and Nrf2 downregulation due to altered DNA methylation has been observed in various age-related diseases. This study investigated whether Nrf2 epigenetic repression contributes to OP development and if running exercise protects against OP by restoring Nrf2 expression.

Existing literature demonstrates the significance of epigenetic mechanisms in bone biology and osteoporosis. Studies have identified numerous genes with altered DNA methylation patterns in osteoporotic patients. DNA methylation is a reversible process, and DNA demethylating drugs have shown promise in animal studies, although their long-term use can have side effects. Several studies have highlighted the beneficial effects of physical exercise on DNA methylation in various conditions. Exercise, being easily accessible and side-effect free, shows great promise for maintaining bone health and protecting against osteoporosis. However, the specific epigenetic mechanisms through which exercise confers its benefits remain largely uninvestigated. The role of Nrf2 in bone homeostasis and its potential connection to oxidative stress and osteoporosis are also established in previous research, emphasizing the need to explore Nrf2's epigenetic regulation in the context of exercise and osteoporosis.

The study employed an ovariectomized (Ovx) mouse model of osteoporosis, a widely used model for studying postmenopausal osteoporosis. Female Nrf2 knockout (*Nfe2l2^-/-*) mice and wild-type (WT) littermates underwent Ovx surgery. Femoral bone microstructure was assessed using micro-computed tomography (µCT) to quantify BMD, BV/TV, Tb.N, Tb.Th, and Tb.Sp. Nrf2 and collagen 1 protein levels were analyzed using western blotting. Methylation status of the Nrf2 promoter was assessed by methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP). Levels of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b) were determined by western blotting and immunohistochemistry (IHC). To investigate the effects of running exercise (RE), half of the sham and Ovx mice were subjected to daily 1-hour treadmill running for four weeks after a one-week recovery period. The same parameters as mentioned above were measured in these mice post-intervention. Human bone samples from osteoporotic and non-osteoporotic patients were also analyzed for Nrf2, Dnmts, and Nrf2 promoter methylation. Antioxidant enzyme activities (catalase, SOD, GPX) were measured using commercial kits. Statistical analysis was performed using ANOVA and Tukey's post hoc test.

Nrf2 knockout mice showed significantly worse BMD reduction after Ovx compared to WT mice, demonstrating Nrf2's crucial role in bone protection. Osteoporotic femurs from both patients and Ovx mice exhibited significantly reduced Nrf2 levels, accompanied by increased Dnmt1, Dnmt3a, and Dnmt3b levels and Nrf2 promoter hypermethylation. Running exercise significantly reduced Nrf2 promoter methylation in Ovx mice, reversed the increased Dnmt levels, and restored Nrf2 expression. RE also normalized the expression of osteoblast and osteoclast markers, along with antioxidant enzymes (catalase, SOD2, GPX). The osteoprotective effects of RE were significantly diminished in Nrf2 knockout mice, confirming Nrf2's essential role in mediating RE's benefits. RE effectively mitigated the suppression of antioxidant enzymes downstream of Nrf2, suggesting that restoration of Nrf2's activity contributes to the reduction of oxidative stress and improved bone health. The three-way ANOVA indicated a significant effect of genotype (P1 < 0.00001), RE intervention (P2 = 0.00221), and their interaction (P3 = 0.02765) on BMD. In summary, Nrf2 repression due to Dnmt elevation and promoter hypermethylation is a crucial epigenetic feature of OP pathogenesis, while Nrf2 derepression is essential for RE's anti-osteoporotic effects.

This study provides strong evidence linking Nrf2 epigenetic repression to osteoporosis development and demonstrating the critical role of Nrf2 in mediating the osteoprotective effects of running exercise. The findings suggest that aberrant Dnmt activity and subsequent Nrf2 promoter hypermethylation contribute significantly to the pathogenesis of osteoporosis. Running exercise effectively counteracts this epigenetic dysregulation, resulting in Nrf2 recovery, reduced oxidative stress, improved bone microstructure, and enhanced bone mineral density. The study underscores the importance of considering epigenetic mechanisms in understanding the pathogenesis and treatment of osteoporosis. The results provide a molecular basis for the well-known benefits of physical exercise in protecting against osteoporosis, emphasizing the potential for exercise-based interventions to mitigate epigenetic alterations and improve bone health.

This study demonstrates that Nrf2 repression due to aberrant Dnmt elevations and Nrf2 promoter hypermethylation is a significant epigenetic feature of osteoporosis pathogenesis. Running exercise effectively reverses these epigenetic abnormalities, leading to Nrf2 restoration and subsequent improvements in bone health. The study highlights the potential of exercise as a therapeutic strategy for mitigating epigenetic alterations in osteoporosis. Future research should explore the broader impact of different exercise modalities and their epigenetic effects on various bone cell types, as well as investigate potential synergistic effects with other therapeutic interventions.

The study primarily utilized a mouse model of osteoporosis, which might not fully capture the complexity of human osteoporosis. The sample size for human studies was relatively small, potentially limiting the generalizability of the findings. While the study focused on running exercise, further investigation is needed to determine if other types of physical activity exert similar epigenetic effects. The precise mechanisms by which running exercise modulates Dnmt activity and Nrf2 promoter methylation require further investigation.