| Objective To investigate the bone metabolism effects of caffeine. Methods At 6h after fertilization, zebrafish embryos were treated with 100, 200, and 400μmol/L of caffeine, and controls were cultured with E3, followed by histological observation, alcian blue staining，alizarin red staining，single-cell RNA sequencing, GO（Gene Ontology）and KEGG（Kyoto Encyclopedia of Genes and Genomes）enrichment analyses. The statistical method was one-way analysis of variance. Results The cranio-caudal length, inter-chondral distance, inter-joint interval, bone mineralization, calcium levels in high caffeine treatment groups [(14.53±1.91)μm, (11.08±0.97)μm, (19.79±2.22)μm, (10742.80±3244.60), (1759.40±88.50)ug/g] significantly reduced as compared with those in the control group [(19.4±1.61)μm, (14.99±1.19)μm, (24.46±2.59)μm, (16831.20±3055.80), (2272.10±100.10)ug/g, P<0.05]. Single-cell RNA sequencing revealed that caffeine would significantly influence osteoblast differentiation and function by regulating specific gene expression. GO and KEGG enrichment analyses showed that high-concentration caffeine treatment would affect zebrafish osteoblast function by regulating multiple key biological processes and signaling pathways, such as oxidative phosphorylation, MAPK signaling pathway and calcium signaling pathway. Conclusion It is inferred that high caffeine has a significant negative impact on bone metabolism, potentially interfering with normal bone development and metabolism through multiple mechanisms. |