Liquiritigenin ameliorates circadian rhythm-mediated osteoblast differentiation through Rorα and Nr1d1 signaling pathways

The decrease of bone mineral density in zebrafish larvae caused by continuous light is closely related to circadian rhythm gene disorder. • LG improved the circadian rhythm gene disorder of zebrafish larvae caused by continuous light. • LG alleviated the decrease of bone mineral density in zebrafish larvae caused by continuous light. • LG reversed the inhibition of osteogenic differentiation of MC3T3-E1 cells caused by circadian rhythm gene disorder. • Circadian rhythm genes play a key role in LG intervention in osteogenic differentiation. Circadian rhythm disorders can destroy the balance of bone metabolism and hence closely related to bone metabolic diseases. To further explore the relationship between circadian rhythm and bone metabolism, the sleep deprivation model of zebrafish induced by continuous light and the MC3T3-EI cell model induced by Nr1d1-specific agonist STL1267 (STL1267) were used to study the intervention mechanism of liquiritigenin on bone metabolism imbalance mediated by circadian rhythm. Molecular docking, behavioral studies, calcein staining, alizarin red staining, alkaline phosphatase and osteocalcin activity, qRT-PCR, Western blotting, and other methods were adopted to screen and study the effects of licorice flavonoids on circadian rhythm disorder and its induced decrease of bone mineral density in zebrafish, osteogenic differentiation, and mineralization inhibition of MC3T3-EI cells. The results showed that liquiritigenin significantly alleviated the expression disorders of zebrafish circadian rhythm genes brain and muscle ARNT-like 1 (Bmal1), circadian locomotor output cycles kaput (Clock), period (Per), cryptochrome (Cry), retinoic acid receptor-related orphan receptor α (Rorα), and nuclear receptor subfamily 1 group D member (Nr1d1) induced by continuous light and caused zebrafish behavioral defects, decreased alkaline phosphatase and osteocalcin activity, and bone mineral density as well. Similarly, liquiritigenin effectively improved the expression of circadian rhythm-related genes at mRNA and protein levels in MC3T3-EI cells induced by STL1267 and enhanced cell viability, alkaline phosphatase and osteocalcin activity, and bone matrix mineralization. Therefore, this work reveals the key role of circadian rhythm in bone metabolism homeostasis, and proves that liquiritigenin can effectively alleviate the imbalance of bone homeostasis caused by circadian rhythm disorders, providing research basis for the application of natural small molecule components to improve circadian rhythm disorders and bone metabolism imbalance. Download: Download high-res image (338KB) Download: Download full-size image