The negative impact of caffeine on zebrafish bone metabolism: Investigation of cartilage development, bone mineralization, and single-cell sequencing of osteoblasts

This study aims to investigate the effects of caffeine on bone metabolism in zebrafish, specifically its impact on cartilage development, bone mineralization, osteoclast activity.Zebrafish larvae at 72 hours post-fertilization (hpf) were exposed to varying concentrations of caffeine. Cartilage development was assessed through Alcian blue staining, while bone mineralization was evaluated using Alizarin red staining. Osteoclast activity was examined via TRAP staining. Single-cell RNA sequencing was performed to analyze gene expression profiles in osteoblasts and other related cell types. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to identify key biological processes and pathways affected by caffeine.Caffeine exposure significantly inhibited normal cartilage development. High caffeine concentrations markedly reduced bone mineralization, with some samples showing a lack of otolith mineralization. TRAP staining revealed decreased osteoclast number and activity, suggesting impaired bone resorption. Single-cell RNA sequencing identified significant alterations in the gene expression profiles of osteoblasts, with key changes observed in calcium signaling pathways. GO and KEGG analyses further highlighted affected biological processes, including those related to bone development and remodeling. This study demonstrates that caffeine has a detrimental impact on bone metabolism in zebrafish, disrupting cartilage development, bone mineralization, and osteoclast function. These findings suggest potential risks to bone health, emphasizing the need for further investigation into caffeine’s effects on bone metabolism and its implications for human health.