大豆苷元和葛根素对去卵巢大鼠骨代谢生化指标的影响

Effect of daidzein and puerarin on biochemical markers of bone metabolism in the ovariectomized rats

目的:植物雌激素对骨改善的作用已经得诸多实验验证,通过观察大豆苷元和葛根素对去卵巢模型大鼠骨代谢的影响,探讨生化指标与骨钙代谢的关系。方法:2003-06/2004-10在南京师范大学金陵女子学院营养学实验室和东南大学公共卫生学院动物实验室进行动物实验,将60只3月龄清洁级SD大鼠建立去卵巢模型,随机分为青年组、假手术组、去卵巢模型对照组、雌激素对照组、大豆苷元组和葛根素组共6组,每组10只。无菌条件下,对模型对照组、雌激素对照组、大豆苷元组、葛根素组大鼠进行骨代谢去卵巢模型的制备,假手术组不切除卵巢,只切除少许脂肪。术后青霉素抗感染3d,自由饮水、摄食,5d后进行喂养试验。青年组未去卵巢,饲养条件同其他组。每日清晨以10μg/kg体质量剂量己烯雌酚溶液给予雌激素对照组灌胃;以100mg/kg体质量剂量大豆苷元犤纯度为98%的结晶体(安徽农业大学实验室提供)犦给予大豆苷元组大鼠灌胃;以20mg/kg体质量剂量葛根素给予葛根素组大鼠灌胃;模型对照组去卵巢后、假手术组手术后灌胃蒸馏水;青年组不予灌胃。将青年组大鼠3月龄时处死,将其余大鼠置于无菌室动物房中饲养3个月,6月龄时处死,股动脉放血收集血样,分离血清,4000r/min,离心15min,-20℃保存待测。利用Olympuls全自动生化分析仪,偶氮胂Ⅲ法测定血清钙;钼蓝比色法测定血清磷,碱性磷酸酶试剂盒测定血清碱性磷酸酶;采用放免试剂盒,SN-695B型智能放免仪γ测量仪计数大鼠血清雌激素和骨钙素。结果:动物实验60只,在饲养过程中,由于动物去卵巢手术或灌胃操作不当死亡6只,共54只动物进入结果分析。①大豆苷元组、雌激素对照组、葛根素组钙磷比明显高于去卵巢模型组(1.18±0.1,1.3±0.16,1.13±0.13,0.87±0.2,P<0.01),且钙磷比均保持在1.1～1.2左右的较佳状态。②大豆苷元和葛根素组的碱性磷酸酶相对于假手术组明显增高犤(87.5±20.5),(90±10.13),(47.5±22.17)U/L;P<0.05犦;③大豆苷元组和葛根素组的骨钙素相对于假手术组显著增高犤(8.39±0.84),(6.69±2.34),(3.42±0.93)ng/L;P<0.05,0.01犦。结论:去卵巢大鼠属于高转换型骨代谢,补充大豆苷元与葛根素使骨转换持续活跃,可能通过与雌激素受体结合抑制骨的重吸收达到骨改善;雌激素和植物雌激素对骨钙改善的影响在一定程度上受雌激素受体活性的介导。

AIM： The improvement of bone by the vegetable estrogen has been substantiated by many experiments. This paper aims to observe the effect of daidzein and puerarin on biochemical indexes of the bone and calcium metabolism in ovariectomized rats, and discuss the relationship between them. METHODS： The experiment was completed at Trophological Laboratory of Jinling Female College of Nanjing Normal University and Animal Laboratory of Public Health College of Southeast University from june 2003 to October 2004. Totally 60 SD rats, three-month old, clean grade, were divided into young group, sham operation group, ovarieetomized model group, estrin control group, daidzein group and puerarin group with 10 in each group. Under sterile stage, rats in model control group, estrin control group, daidzein group and puerarin group were performed with bone metabolism to establish ovariectomized model, and rats in sham operation group were cut off a few fat but not ovaries. After operation, benzylpenicillin was used against infection for 3 days, rats drank and ate freely, and feeding test was performed 5 days later. Rats in young group were not cut off ovaries under the same feeding environment as other groups. Each morming, 10 μg/kg diethylstilbestrol of body mass was perfused into rats in estrin control group; 100 mg/kg daidzein （98% crystal, provided by Laboratory of Anhui Agriculture University） perfused into rats in daidzein group; 20 mg/kg puerarin was perfused into rats in puerarin group; distilled water was perfused into rats in model control gruup after ovariectomized and in sham operation group after opcratior,; and rats in young group were not perfused. Rats in young group were sacrificed at 3-month old, and rats in other groups were fed in sterilizing room till 6-month old. Blood was collected from bone artery and serum was centrifugalizated at 4 000 r/minute for 15 minutes and maintained at -20 %;. Serum calcium was assayed with Olympuls automatic biochemistry analyzer and azo-arsine Ⅲ method; serum phosphorus was assayed with molybdenum blue chromatometry; serum alkaline phosphatase （ALP） was assayed with alkaline phosphatase kit; and serum estrin and osteoealcin were assayed with radioimmunoguided kit and SN-695B intelligent radioimmunoguided 7 measurement device. RESULTS： Among 60 rats, 6 rats died because of ovafiectomized operation or perfusion, so 54 rats entered the final analysis. ① Ratio between calcium and phosphorum was higher in daidzein group, estrin control group and puerarin group than that in ovarieetomized model group （1.18±0.1, 1.3±0.16, 1.13±0.13, 0.87±0.2, P 〈 0.01）, and the ratio was maintained about 1.1-1.2. ② Alkaline phosphatase in daldzein group and puerafin group was increased as compared with that in sham operation group [（87.5±20.5）, （90±10.13）, （47.5±22.17） U/L; P〈 0.05]. ③ Osteocalcin in daidzein group and puerarin group was increased as compared with that in sham operation group [（8.39±0.84）, （6.69±2.34）, （3.42±0.93） ng/L; P 〈 0.05, 0.01]. CONCLUSION： The ovariectomized rats belong to high bane turnover model. Daidzein and puerarin keep the high bone turnover and attribute to its estrogen action through inhabiting the resorption of bone of calcium. The effect of the estrogen and vegetable estrogen on improvement of hone and calcium to some extent depends on estrogen receptor.