NAD+/Nrf2 signaling promotes osteogenesis by regulating oxidative level of BMSCs under mechanical stress.

Abstract

Background: Mechanical stress triggers an increase in cellular reactive oxygen species (ROS), which is associated with the impairment of osteogenesis. During orthodontic treatment, bone marrow mesenchymal stem cells (BMSCs) experience mechanical stress, yet the oxidative profile and redox regulatory mechanisms under such stress, especially involving Nicotinamide adenine dinucleotide (NAD⁺), are not well understood, necessitating further research into their roles in orthodontic therapies.

Methods: The Tension System was established to detect ROS changes in BMSCs under cyclic stretch stress, with H₂O₂ simulating uncontrolled ROS. Flow cytometry and fluorescence staining measured ROS, while an NAD⁺/NADH assay kit assessed NAD⁺ levels. qRT-PCR and Western blotting analyzed expression of NAD⁺ synthesis and consume enzymes. Osteogenic potential was evaluated by qRT-PCR, Western blotting, and Alkaline phosphatase (ALP) staining. Loss-of-function and supplementation assays explored role of NAD⁺ in oxidative stress and Nrf2 regulation, with localization assessed by immunofluorescence and Western blotting. In vivo osteogenic effects were confirmed using an orthodontic tooth movement (OTM) model, with osteogenesis assessed by immunohistochemistry and microCT for OTM measurements.

Results: Cyclic stretch stress increased ROS in BMSCs over 24 h and boosted osteogenic differentiation. However, increased ROS from H₂O₂ hindered this process. Notably, NAD⁺ levels rose with cyclic stretch, and experiments showed it supported osteogenesis by controlling ROS level in BMSCs. Furthermore, NAD⁺ regulated BMSC ROS via Nrf2 nuclear translocation. Rat models indicated that NMN supplementation enhanced osteogenic and osteoclastic markers and accelerated tooth movement, while FK866 inhibited this effect.

Conclusions: We identified that NAD⁺/Nrf2 signaling regulated oxidative level and thus promoted osteogenic commitment of BMSCs under cyclic stretch stress. Targeting NAD⁺ metabolism or administrating exogenous supplementation to promote bone rebuilding could be a prospective therapy to accelerate OTM.