This study found that acute SD quickly broke the balance of bone remodeling, the bone formation markers decreased and bone resorption markers increased significantly. Interestingly, the damage will be repaired gradually after sleep recovery. At the same time, we further explored the effect of SD on the expression of mRNA in bone tissues.
Our study found that the bone formation marker P1NP decreased significantly within the first 24 hours of sleep deprivation, while bone resorption marker β-CTX increased significantly. A few previous studies with small samples focused on the changes of BTMs after SD or sleep restriction, while the results of them were not sure. Staab et al.[10] conducted a 72-hour sleep restriction (4:30 − 6:30 sleep every night) in 10 healthy young men and monitored the changes of BTMs. Bone alkaline phosphatase (BALP) decreased after 24 hours SD, while tartrate resistant acid phosphatase (TRAP) and β-CTX, markers of serum bone resorption, increased after 48 hours and 72 hours of intervention. The percentage of BALP/TRAP decreased significantly after 48 and 72 hours of sleep restriction, showing that short-term SD disrupted the balance of bone turnover status and tilted towards bone resorption. In a small sample cohort study, repeated sleep restriction for 3 weeks led to uncoupling of bone turnover, in which bone formation markers decreased rapidly and significantly in the early stage, although there was no significant change in bone resorption markers in the later stage[6, 11, 12].
In this study, we further found that some genes related to bone resorption, formation and differentiation were up-regulated after SD, such as Tnfrsf11a, Ctsk, spp1, pth1r. And a variety of signal pathways were up-regulated, including bone resorption and bone remodeling. Via KEGG enrichment analysis, oxidative phosphorylation pathway and Alzheimer's disease pathway were up-regulated, while thyroid hormone signal pathway, parathyroid hormone synthesis, secretion pathway and insulin signal pathway were significantly down-regulated. The differences on gene expression were most focused on the pituitary and hippocampus of the brain in previous study, but there is lack of research on gene expression in bone tissue at present.
Our RNA-seq data further provided evidence to show that bone remodeling signal pathway was up-regulated and the expression of mRNA related to osteoclast markers were also up-regulated, which is confirmed by qPCR meanwhile. CTSK, as specific highly expressed in osteoclast, degrades the N-terminal peptide and C-terminal peptide of Col I protein to produce N-terminal peptide cross-linking of type I collagen (NTX) and C-terminal peptide cross-linking of type I collagen (CTX), which are considered the most sensitive and specific biochemical indexes of bone resorption and degradation of bone organic matrix[17–19]. CTSK also can regulate the apoptosis of osteoclasts, therefore then the level of it affects the number and function of osteoclasts[19, 20]. The mRNA RANK, encoded by Tnfrsf11a, is a key factor regulating osteoclast differentiation and bone resorption, by binding to RANKL. it promotes the expression of downstream transcription factors, thus affecting the expression of bone resorption related enzymes such as TRAP and bone resorption specific factor CTSK, which are important in the signal transduction process leading to osteoclast differentiation[21, 22].
In our study, the results also showed that the expression of osteogenesis-related mRNAs were also up-regulated. The process of bone remodeling is a dynamic process between osteoblasts and bone resorption osteoclasts. Osteoblasts form new bone and regulate the activity of osteoclasts by releasing RANKL and OPG. Mature osteoclasts dissolve bone matrix by secreting CTSK and TRAP to remove old bone. Osteogenesis is coupled with osteoclast activity to maintain balance. Runx2 is a vital transcription factor in the early and middle stages of osteoblast differentiation. The osteocalcin (OCN), a marker of late stages of osteoblast differentiation. The precursor protein of osteopontin (OPN), encoded by Spp1, also known as a non-collagen protein and one of the phenotypes of osteoblasts. The up-regulated expression of these mRNAs reflects the enhancement of bone remodeling in bone tissues.
Our study found that the expressions of RANKL and OPG mRNA in SD rats were both down-regulated, but the ratio of RANKL/OPG increased, and the expression of TRAP mRNA and CTSK mRNA increased, which suggested that osteoclast was more active. Due to the coupling of bone formation and resorption, the activated osteoclast may increase the osteogenic activity to some extent, resulting in compensation for bone loss, which may partly explain the reason of osteogenesis-related genes expressed increased meanwhile.
Although there are more and more scholars realizing the importance of healthy sleep habit, little attention has been paid to the effect of short-term SD on bone metabolism. In this study, 6-week-old Wistar male rats were subjected to continuous 72-hour sleep deprivation by modified multi-platform method. The differences between SD rats and NC rats of serum BTMs were compared, and the changes of BTMs during the intervention of SD were monitored. It was found that acute SD quickly broke the balance of bone remodeling, abnormal bone turnover, and up-regulated the expression of mRNA related to bone resorption and bone remodeling in bone tissues.
In the previous studies, only a few small sample size cohort studies (n ≤ 20) focused on this phenomenon. Because of human, the participants have more interference factors, such as activity factors, age and sex, which can not meet the needs of the model of simple sleep deprivation. While this study also has some limitations, whether the results of this study can be deduced to the real world of human beings also needs to be further explored by clinical studies with a large sample size in the future.