ABSTRACT
Objective To investigate the effect of excessive caffeine intake on fetal cartilage ossification in female rats during pregnancy and its mechanism. Methods From gestational day(GD) 9 to GD 20, the pregnant Wistar rats in caffeine exposure group were intragastrically administered 120 mg/kg day caffeine, and the control group was administered the same volume of distilled water. The pregnant mice were sacrificed at day 20, and the body length of the fetal mice was measured. The distal femur of fetal rats was isolated, the length of distal femur cartilage was measured, and primary chondrocytes were prepared. The cells were treated with caffeine (0.1, 1 and 10 μmol/L), insulin-like growth factor 1 (IGF-1, 100 (μg/L) and extracellular regulated protein kinases(ERK) inhibitor (10 μmol/L), respectively. Then the cells were harvested for apoptosis, gene and protein analysis. Results Compared with the control group, the body length and femur length of the fetuses in the caffeine exposed group decreased significantly (P< 0.05), and the serum corticosterone levels increased significantly (P<0.05). Immunohistochemical analysis showed that the expressions of IGF-1, proliferating cell nuclear antigen (PCNA) and sex determining region Y box protein 9 (SOX9) in mast chondrocyte area of caffeine exposed group were significantly lower than those of control group (P<0.05). In vitro, caffeine treatment reduced the expression of IGF-1, PCNA, SOX9 mRNA and p-ERK protein in primary chondrocytes in a concentration-dependent manner, while exogenous IGF-1 could reversed these changes induced by caffeine, and the effect of exogenous IGF-1 was reduced by ERK inhibitors (all P<0.05). Conclusion Prenatal caffeine exposure leads to shortening of the long bones of the fetus and prolongation of the hypertrophy by inhibiting the proliferation of chondrocytes. The IGF-l/MAPK/ERK signaling pathway in chondrocytes may be partially involved in the adverse effects of caffeine on chondrocyte proliferation.