葛根素体外影响小鼠胚胎长骨雌激素受体β表达的作用

Effect of puerarin on the expression of estrogen receptor beta in long bones of mouse embryoes in vitro

目的:以体外培养小鼠胚胎长骨为靶器官,观察葛根素对骺板雌激素受体亚型雌激素受体β分布及含量的影响,并与内源雌激素比较,分析葛根素对骨可能的作用机制。方法:实验于2003-05/2004-05在中国人民武装警察部队医学院药物化学教研室药物活性筛选室进行。以小鼠胚胎长骨为靶器官,以雌二醇1×10-6mol/L处理为阳性对照组,同时设空白对照组、葛根素干预组分为1×10-6,1×10-5,1×10-4mol/L3个浓度组。①小鼠怀孕16d时取出胚胎,剔除雌性胎鼠前肢肌肉和软组织,剥离出尺骨,做为实验靶器官置于BGJb培养基中,葛根素和雌二醇作用48h后,测量长骨总长和骨干长。每组培养长骨8根,重复3次。②长骨固定、脱钙后作石蜡切片,进行免疫组织化学染色,光镜下观察阳性细胞定位,图像分析系统下测定免疫反应阳性细胞数和阳性细胞面积。结果:雌性胎鼠长骨全部进入结果分析,无脱失。①长骨经葛根素和经雌二醇处理48h后,葛根素1×10-4,1×10-5mol/L组及阳性对照组骨总长及骨干长与空白对照组相比,差异有非常显著性意义[(3.97±0.12),(1.51±0.07);(3.79±0.06),(1.40±0.03);(4.05±0.1),(1.62±0.05);(3.43±0.05,1.24±0.04)mm;P<0.01],葛根素10-6mol/L组仅骨总长与空白对照组相比差异有显著性意义[(3.56±0.06)mm,P<0.05];葛根素1×10-4mol/L的作用与阳性对照组差异无统计学意义(P>0.05),1×10-5,1×10-6mol/L组作用弱于阳性对照组(P<0.01)。②空白对照组骺板静止区和肥大区雌激素受体β蛋白几乎不表达,仅增殖区有较弱表达。经葛根素和1×10-6mol/L雌二醇作用后,增殖区表达明显增强,肥大区和静止区软骨细胞也都出现雌激素受体β蛋白的阳性表达。葛根素1×10-6,1×10-5,1×10-4mol/L组阳性细胞数和阳性细胞面积均小于阳性对照组[29.8±2.9,43.5±5.1,63.3±5.8,83.3±6.9,(819.78±164.53),(1175.76±188.73),(1585.15±198.93),(2036.12±384.23)μm2,P<0.01～0.05],作用明显弱于雌二醇,并表现出剂量依赖的趋势。结论:葛根素在体外可促进软骨内成骨。同雌激素一样,葛根素也能够上调骺板雌激素受体β的表达。提示葛根素对软骨内成骨的促进作用可能与其上调雌激素受体β在骺板的表达有关。

AIM： To regard the cultured long bone of mouse fetuse as target organ in vitro, and study the effects of puerarin on the distribution and the content of estrogen receptor beta （ERbeta） protein product in fetal mouse epiphyseal growth plate and compare the differences between the effects of puerarin and of endoestrogen for postulating the mechanism by which puerarin exerts its effect on bone. METHODS： The experiment was conducted at the Drug Active Screening Room, Department of Medicinal Chemistry, Medical College of Chinese People＇s Armed Police Forces from May 2003 to May 2004. Regarding long bone of mouse fetuse as target organ, and 1×10^-6 mol/L estradiol as positive control group, at the same time established blank control group, 1×10^-6, 1×10^-5, 1×10^-4 mol/L puerarin groups. ①The fetuses of pregnant mice were removed on day 16 of gestation, the long bone of forelimbs of female fetal mice were free of muscle and soft tissue and cultured for 48 hours in BGJb medium treated respectively with estradiol and purearin. After cultured for 48 hours, the long bones were harvested; the bones length and the length of their diaphyses were measured. Eight long bones were cultured in each group, for duplicated 3 times, ②After fixing long bones and decalcification, paraffin section was made, and then performed immunohistochemical staining. Positive cells location was observed under light microscope. The number and area of receptor-positive cells were determined with an image analytic system. RESULTS： Long bones of female foetal mice were involved in the result analysis, no drop-out. ①After being treated by puerarin or estradiol for 48 hours, the bones length and the length of their diaphyses in the 1×10^-4 and 1×10^-5 mol/L puerarin groups and positive control group had significantly difference with the blank control group [（3.97±0.12）, （1.51±0.07）; （ 3.79 ±0.06 ）, （ 1.40 ±0.03 ） ： （4.05 ±0.1 ）, （ 1.62 ±0.05 ） ; （ 3.43 ±0.05,1.24 ±0.04） mm;P 〈 0.01]. There was significant difference in the bones length between the 10^-6 mol/L puerarin group and blank control group [（3.56 ±0.06） mm,P 〈 0.05]. There was no statistical significance of 1×10^-4 mol/L puerarin as compared with the positive control group （P 〉 0.05 ）. The effect was weak in the 1×10^-5 and 1×10^-6 mol/L puerarin groups than in the positive control group （P 〈 0.01）. ②ERbeta in blank control group was expressed weakly only in proferative zone while almost no positive cells were observed in the resting and proliferative zones. The expression significantly increased in hypertrophic region, meantime, the positive cells were also observed in the resting and proliferative zones after being affected by puerarin and 1×10^-6 mol/L estradiol. Number and area of receptor-positive cells was less in the 1×10^-6,1×10^-5,1×10^-4 mol/L puerarin groups than in the positive control group [29.8 ±2.9,43.5 ±5.1,63.3 ±5.8,83.3 ±6.9, （819.78±164.53）,（1 175.76±188.73）,（1 585.15±198.93）, （2 036.12 ±384.23） μm^2,P 〈 0.01-0.05]. The effect of puerarin on bone was weaker markedly than that of estradiol, and showed dosage-dependent tendency. CONCLUSION： Puerarin can enhance bone formation in vitro. As estradiol, puerarin can also up-regulate the expression of ERbeta in epiphyseal plate. It suggests that the effect of puerarin on endochondral bone formation may be associated with its upregulating the expression of ERbeta in epiphyseal plate.