Effects Of Sleep Deprivation On Bone Metabolism In Growing Rats And Its Mechanis

Chronic sleep deprivation impaired bone formation in growing ratsBackground:Chronic sleep deprivation(CSD)is increasingly common in adolescents.For them it is a critical period for growth and development.Previous clinical cross-sectional surveys have found sleep deprivation impair bone health,but there are no unified consensus.This study aimed to assess the effects of CSD on bone metabolism in growing rats and the likely underlying mechanism.Methods:Twenty 5-week-old male Wistar rats and randomly divided into the CSD and normal control(NC)groups after one-week acclimatization.After a 6-week intervention of sleep deprivation(8:00-18:00),the left distal femurs of both groups were harvested for micro-computed tomography scans and histological analysis.Meanwhile,the right femur were measured the mRNA and protein expression via RNA sequencing(RNA-seq)and western blot.Serum bone turnover markers were evaluated at 0,2,4,and 6 weeks.Result:(1)CSD impaired the bone growth and development of growing rats,especially inhibited the process of bone formation.① From the micro-CT results,in CSD group,the bone microstructure deteriorated and the trabecular parameters BV/TV and Tb.N decreased significantly.②The analysis of bone histomorphology by HE staining showed that in the CSD group,the growth plate of the metaphysis of the femur was abnormal,the cells in the proliferative area of chondrocytes decreased,the hypertrophic area of chondrocytes widened,the cancellous bone in the calcified area of cartilage decreased,the structure of trabecular bone was poor,the arrangement was disordered,the trabecular space was enlarged,and the osteoblasts decreased,which suggested that the osteogenesis of growing rats was decreased and the formation and mineralization of new bone were damaged.③In CSD group,the serum bone formation marker(P1NP)decreased gradually and finally lost coupling with the bone resorption marker(β-CTX),even after adjusting for confounding factors such as body weight,the difference was still significant.Our results suggest that the status of bone turnover decreased significantly after 6 weeks of CSD intervention.(2)This study found that CSD inhibited the expression of genes and proteins related to osteoblast differentiation,thus impairing bone formation and development.①The results of immunohistochemical analysis showed that the expression of osteogenic associated protein Collal and OCN in femur tissue of CSD group decreased significantly.②The results of RT-qPCR also showed that the expression of osteogenesis related mRNA,such as igf1,bglap,runx2,Collal and pthlr,were significantly down-regulated by several times comparing NC group.③GO enrichment analysis showed that osteoblast differentiation signal pathway and skeletal development signal pathway were significantly down-regulated in CSD group.The functional enrichment of GSEA showed that most of the genes in these two pathways were significantly down-regulated in CSD rats.(3)CSD inhibited the PI3K/AKT signal pathway in bone tissue,and the phosphorylation levels of PI3K and AKT decrease,which may partly explain the damage of osteogenic differentiation and bone formation.Differentially expressed genes were detected,and KEGG functional enrichment analyses revealed that the PI3K/AKT pathway was significantly down-regulated in the CSD group,which was further verified by the protein expression levels.Conclusion:These results suggest that CSD can significantly impaired bone health,and it may exert these effects in part by suppressing bone formation and osteoblast differentiation,and inactivating the PI3K/AKT signaling pathway.Adolescence is a critical period for accumulating bone mass and an important therapeutic window for preventing bone loss;Good sleep is essential for accumulating bone mass and preventing osteoporosis in later life.Bone turnover markers and transcriptomes changes induced by acute sleep deprivationObjective:Acute sleep deprivation(SD)means several hours to dozens of hours of partial or all sleep loss,common in shift workers or people with special conditions.Previous studies found that overnight sleep deprivation can affect gene transcription in hippocampal and cardiovascular endothelial tissues,but the effect of acute sleep deprivation on mRNA expression in bone tissue has not been studied.The effects of SD on bone metabolism are currently not clear.To explore the effect of acute sleep deprivation on the bone turnover status and the changes in bone tissue gene expression in rats by RNA sequencing.Methods:Wistar rats,5 weeks age,randomly divided into 3 groups:normal controls(NC)group,SD group,SD and recovery(SD+R)group after one-week acclimatization.Acute sleep deprivation model was established using a modified multi-level bench method.Bone turnover markers(P1NP,β-CTX)were measured.The normal control(NC)and SD rats were used for RNA sequencing,and the genes with differential expression were screened and the KEGG functional annotation analysis and GO enrichment analysis.Differential expression genes were screened and the KEGG functional annotation analysis and GO enrichment analysis.Results:① The serum P1NP levels were significantly decreased after the 72 hours of SD intervention(P<0.05)and serum β-CTX levels were increased(p<0.05).②There were 4441 differential genes(padj value<0.05),of which 1985 genes were up-regulated and 2456 genes were down-regulated.Some genes associated with osteogenesis and osteoclast differentiation such as Tnfrsf11a,Ctsk,spp1,and pth1r were significantly up-regulated.KEGG enrichment analysis suggested oxidative phosphorylation pathway and Alzheimer’s disease pathway,and thyroid hormone signaling,PTH synthesis,secretion and insulin signaling.Conclusions:(1)This study found that acute sleep deprivation rapidly breaks the balance of bone turnover,and and increased related mRNA expression.(2)Sleep is essential for maintaining bone turnover status and is an important factor for bone.