Ticlopidine induces craniofacial cartilage developmental toxicity in zebrafish via downregulation of BMP signaling

Ticlopidine is a clinically used P2Y12 receptor antagonist for thrombotic disorders. The P2Y12 receptor is widely expressed across tissues, including chondrocytes, where its activation protects cartilage. However, its developmental toxicity on craniofacial structures remains unknown. In this study, zebrafish embryos were exposed to ticlopidine (1.2, 1.5, and 1.8 mg/L) from 10 to 96 hours post-fertilization, demonstrating concentration-dependent craniofacial cartilage developmental toxicity. Phenotypic analysis revealed disorganized pharyngeal arch structure, aberrant chondrocyte arrangement, and reduced craniofacial bone mineralization following ticlopidine exposure. Molecularly, ticlopidine exposure disrupted the normal expression of genes associated with cartilage development ( col2a1a, pax9 ), cranial neural crest cell development ( fgf8a, zic2a ), osteogenic mineralization ( bglap, sparc, runx2b, rankl ), and apoptosis ( p53, caspase3, caspase9, bax, bcl2 ). Mechanistically, ticlopidine suppressed the expression of BMP signaling pathway-related genes and increased apoptosis in craniofacial pharyngeal arch chondrocytes. Notably, SJ000291942 administration significantly rescued the cartilage malformation and associated apoptosis, but not the bone mineralization defect, indicating that BMP signaling primarily mediates the cartilage phenotype while mineralization defects likely involve additional, non-BMP mechanisms. In conclusion, ticlopidine might disrupt craniofacial development in zebrafish by downregulation of the BMP signaling pathway, thus leading to defective pharyngeal arch cartilage formation. Therefore, this study provides important evidence for ticlopidine-induced craniofacial cartilage developmental toxicity through BMP signaling pathway inhibition in zebrafish, offering valuable insights for clinical safety assessment and therapeutic applications.