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Phenobarbital is an antiepileptic drug that is widely used to treat epilepsy in a clinical setting. However, a long term of phenobarbital
administration in pregnant women may produce side effects on embryonic skeletogenesis. In this study, we aim to investigate the
mechanism by which phenobarbital treatment induces developmental defects in long bones. We first observed that phenobarbital
treatment decreased chondrogenesis and inhibited the proliferation of chondrocytes in chick embryos. Phenobarbital treatment
also suppressed mineralization in both in vivo and in vitro long bone models. Next, we established that phenobarbital treatment
delayed blood vessel invasion in a cartilage template, and this finding was supported by the down-regulation of vascular endothelial
growth factor in the hypertrophic zone following phenobarbital treatment. Phenobarbital treatment inhibited tube formation and the
migration of human umbilical vein endothelial cells. In addition, it impaired angiogenesis in chick yolk sac membrane model and
chorioallantoic membrane model. In summary, phenobarbital exposure led to shortened lengths of long bones during embryogenesis,
which might result from inhibiting mesenchyme differentiation, chondrocyte proliferation, and delaying mineralization by impairing
vascular invasion.