Abstract: BACKGROUND: Grape seed extract has been shown to be effective in inhibiting the growth of androgen-dependent tumors (e.g., breast cancer), and thus grape seed extract could theoretically inhibit epiphyseal closure induced by estrogen in late adolescence.
OBJECTIVE: To screen the effects of grape seed extract on apoptosis of growth plate chondrocytes and epiphyseal closure in rats. 
METHODS: (1) In vitro experiment: Growth plate chondrocytes from rat large tibia and femur at logarithmic growth stage were obtained and cultured in groups: normal control group, model control group (adding 17β-estradiol to induce apoptosis), positive control group (adding letrozole and 17β-estradiol), grape seed extract group (adding 17β-estradiol and 10 µg/mL grape seed extract), Caspase-9 inhibitor group (adding 17β-estradiol and Caspase-9 inhibitor), Caspase-9 agonist group (adding 17β-estradiol and Caspase-9 agonist). Cell apoptosis was detected by flow cytometry after 48 hours of culture. (2) In vivo experiment: Thirty 3-month-old Sprague-Dawley rats were randomly divided into model control group, positive control group and low-, medium- and high-dose groups, with five rats in each group. All rats were injected subcutaneously with 17β-estradiol (3 times per week) to establish epiphyseal closure models, followed by intragastric administration of letrozole in positive control group and 0.05, 0.2 and 0.8 g/kg grape seed extract in low-, medium- and high-dose groups, respectively, once a day until over 2/3 of the epiphyseal plate in the model control group was closed. The length of the tibia was then observed. Another 18 Sprague-Dawley rats were randomly divided into model control group, positive control group, and medium-dose group, with 6 rats in each group, treated as above for 1.5 continuous months. The expression of Caspase-9 protein in rat growth plate cartilage was detected by western blot.
RESULTS AND CONCLUSION: (1) In vitro experiment: 17β-estradiol could induce apoptosis in growth plate chondrocytes, and letrozole, grape seed extract, and caspase-9 inhibitors could all inhibit apoptosis in growth plate chondrocytes. (2) In vivo experiment: When more than 2/3 of the epiphyseal plate in the model control group was closed, the number of rats with epiphysis closure in the positive control and medium-dose groups was less than that in the model control group (P < 0.05), and the tibial length was longer than that in the model control group (P < 0.05), and the Caspase-9 protein expression in the tibial growth plate was lower than that in the model control group (P < 0.05). To conclude, the appropriate dose of grape seed extract can effectively inhibit the apoptosis of growth plate chondrocytes and delay epiphyseal closure, which has the potential to promote bone growth.

Key words: grape seed extract, growth plate chondrocyte, apoptosis, epiphyseal closure, Casepase-9