抑制生长板软骨细胞凋亡药物筛选及白藜芦醇延迟大鼠骨骺闭合的作用

Screening of drugs for inhibiting growth plate chondrocyte apoptosis and the effect of resveratrol on delaying epiphyseal closure in rats

背景:骨骺闭合长骨停止生长,骨骺闭合的直接诱因是生长板软骨细胞的凋亡。有报道芳香化酶抑制剂可以抑制雌激素的生成,而骨骼生长的停止是由于骨骺闭合引起的,因此推断芳香化酶抑制剂可以抑制生长板软骨细胞凋亡,延迟骨骺闭合。目的:比较几种芳香化酶抑制剂(白藜芦醇、鹰嘴豆素A、亚麻木酚素)抑制大鼠生长板软骨细胞凋亡的效果,并对白藜芦醇延迟大鼠骨骺闭合促进长骨生长的作用进行验证。方法:解剖大鼠胫骨及股骨生长板软骨,二步酶消化法体外分离获得生长板软骨细胞并进行培养,分别利用倒置相差显微镜及Ⅱ型胶原免疫荧光实验对软骨细胞进行形态学观察和鉴定。然后用白细胞介素1β诱发生长板软骨细胞凋亡,分别加入含有10,20,40,60μmol/L的白藜芦醇培养液,5,10,20,30μmol/L的鹰嘴豆素A培养液,1,10,20,40μmol/L的亚麻木酚素培养液进行培养,比较各个浓度的药物对生长板软骨细胞凋亡的影响。将筛选出效果最好的药物白藜芦醇给予大鼠灌胃,观察其对大鼠胫骨平台骨骺闭合及对长骨生长的影响。结果与结论:①适宜浓度的白藜芦醇、鹰嘴豆素A、亚麻木酚素均可以不同程度地抑制生长板软骨细胞凋亡,最佳抑制细胞凋亡的药物浓度分别为40,10,20μmol/L,其中40μmol/L白藜芦醇抑制生长板软骨细胞凋亡的效果最好;②而用40 mg/kg白藜芦醇给予大鼠灌胃处理,发现可以延迟大鼠胫骨平台的骨骺闭合促进长骨生长,为骨骼生长争取更长时间从而促进发育期骨骼增长。

BACKGROUND:Epiphyseal closure indicates the long bones stop growing,which is caused by the apoptosis of growth plate chondrocytes.It has been reported that aromatase inhibitors can inhibit the production of estrogen,and the cessation of bone growth is caused by epiphyseal closure.Therefore,it has been inferred that aromatase inhibitors can inhibit growth plate chondrocyte apoptosis and delay epiphyseal closure.OBJECTIVE:To compare the effects of several aromatase inhibitors(resveratrol,biochanin A,and flax lignans)on inhibiting the apoptosis of rat growth plate chondrocyte and to verify the effect of resveratrol on delaying epiphyseal closure and promoting the growth of long bones in rats.METHODS:Rat tibial and femoral growth plate cartilage samples were dissected,and growth plate chondrocytes were isolated and cultured in vitro by two-step enzyme digestion.Chondrocytes were morphologically observed and identified by inverted phase contrast microscope and type II collagen immunofluorescence experiments.Then interleukin 1βwas used to induce apoptosis of growth plate chondrocytes.Cells were cultured with culture medium containing 10,20,40,and 60μmol/L resveratrol,with culture medium containing 5,10,20,30μmol/L biochanin,or with culture medium containing 1,10,20,40μmol/L flax lignans to compare the effects of different concentrations of drugs on the apoptosis of growth plate chondrocytes.Resveratrol which had the best effect was administered to rats by gavage to investigate its effect on the epiphyseal closure of rat tibial plateau and the growth of long bones.RESULTS AND CONCLUSION:Appropriate concentrations of resveratrol,biochanin A,and flax lignans could inhibit the apoptosis of growth plate chondrocytes to varying degrees.The best drug concentrations for inhibiting cell apoptosis were 40,10,and 20μmol/L in sequence,and 40μmol/L resveratrol had the best effect on inhibiting the apoptosis of growth plate chondrocytes.Administration of 40 mg/kg resveratrol by gavage could delay epiphyseal closure of tibial plateau and promote the growth of long bones in rats.Therefore,resveratrol can promote bone growth during developmental period by prolonging the time of bone growth.