Abstract
Maternal cigarette smoking during pregnancy is a known high-risk factor for having a child with a cleft lip and/or palate (CLP), a common congenital malformation. Nicotine is the major teratogen component of cigarettes and e-cigarettes, and nicotine plays an important role in the development of CLP. However, the mechanism underlying nicotine's effect on CLP remains unclear. Here, we aimed to determine the role and molecular mechanisms of nicotine-induced autophagy, an important process involved in regulating the cellular stress response in mouse embryonic palatal cells (MEPCs). First, we found that nicotine promoted MEPCs proliferation and inhibited their apoptosis from 0 to 12 h. After 12 h, the proliferation was inhibited, and apoptosis was promoted. The migration of MEPCs was also inhibited by nicotine. Simultaneously, long-term nicotine stimulation inhibited the osteogenic differentiation of MEPCs. We then found that nicotine significantly increased autophagy flux in MEPCs at 12 h by increasing the expression of microtubule-associated protein light chain 3 (LC3) and reducing P62 expression levels. After nicotine exposure, intracellular reactive oxygen species (ROS) and extracellular signal-regulated kinase-1/2 (ERK1/2) expression significantly increased, and the expression of ERK1/2 was reversed by the ROS scavenging agent N-acetylcysteine (NAC). Moreover, the autophagy induced by nicotine was reversed by SCH772984, a specific inhibitor of ERK1/2, and the autophagy inhibitor chloroquine (CQ). These results suggest that in the early stage of nicotine exposure, MEPCs may trigger autophagy through the ROS/ERK1/2 signaling pathway to avoid cell damage caused by nicotine.