阿胶补肾健骨方治疗大鼠骨质疏松症的作用机制研究
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摘要
目的：
通过理论研究认识骨质疏松症（Osteoporosis，OP），运用中西医不同的治疗方法，观察去卵巢大鼠骨质疏松症模型的一般情况，骨病理学特征，骨密度(bone mineral density，BMD)，骨生物力学，骨代谢的血生化指标以及维生素D受体（Vitamin D receptor，VDR）的基因表达等变化，探讨阿胶补肾健骨方对骨质疏松症的影响及作用机制，为运用中医药治疗骨质疏松症提供科学依据。
方法：
采用8月龄雌性Sprague-Dawley（SD）大鼠建造骨质疏松症模型，共70只。实验前置动物于室内适应环境一周，室温18-22℃。将大鼠随机分为以下4组：假手术组（17只）、模型组（17只）、阿胶补肾健骨方组（18只）、阿仑膦酸钠（alendronate sodium）组（18只）。假手术组正常饲养；模型组给予生理盐水灌胃90天，其他各组动物灌胃治疗90天，停止治疗1天，备用。
观察活体大鼠一般表现情况，体重变化。观察治疗三个月后，各组动物经腹腔麻醉，股动脉取血备用。记录双肾重量、右侧股骨重量及子宫重量。将其右侧股骨上的软组织剥离、剔净，用LUNAR-Prodigy双能X线BMD仪测定大鼠全股骨及股骨两端BMD，然后置于生物力学测定仪上测定最大载荷(maximum load，ML)和刚度(stiffness)。取其左侧股骨置于中性福尔马林溶液中固定，常规苏木素-伊红（Hematoxylin-Eosin，HE）染色，按要求制备病理片，显微镜下观察骨组织（bone tissue）结构变化。采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)对大鼠血清骨钙素（osteocalcin，OCN）、Ⅰ型前胶原羧基端前肽(procollagen I carboxyterminal propeptide，PICP)、25羟基维生素D_3（25-hydroxyvitamin D_3，25(OH)D_3）、1,25二羟基维生素D_3（1,25-dihydroxyvitamin D_3，1,25(OH)_2D_3）测定研究。用聚合酶链反应（Polymerase Chain Reaction，PCR）测定肾脏VDR的基因表达。
统计学处理：采用SPSS15.0统计软件包对各项检测数据进行方差分析，所有数据结果均以均数加减标准差（x±s）表示，以P＜0.05为差异，具有统计学意义。
结果：
1大鼠一般表现情况：与假手术组大鼠相比，模型组大鼠精神相对萎靡呆滞或见有狂躁不安，反应较迟钝，毛发无光泽而凌乱，少动嗜睡；阿胶补肾健骨方组、阿仑膦酸钠组大鼠在给药治疗过程中，情绪较稳定，精神状态较佳、较活跃，反应较敏捷，毛发顺整。
2大鼠体重变化：与假手术组大鼠相比，模型组于造模后两个月起，体重明显高于假手术组；阿胶补肾健骨方组体重渐增，与假手术组相近，具有统计学意义；阿仑膦酸钠组体重增加较为明显。
3大鼠子宫指数：与假手术组大鼠相比，各组均明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，均有明显提高，其中阿胶补肾健骨方组明显优于阿仑膦酸钠组，有统计学意义。
4大鼠双肾指数：与假手术组大鼠相比，模型组大鼠双肾指数明显减轻，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，双肾指数均有提高，其中阿胶补肾健骨方组明显优于阿仑膦酸钠组，有统计学意义。
5大鼠股骨重量：与假手术组大鼠相比，各组均明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，均有明显提高，其中阿胶补肾健骨方组明显优于阿仑膦酸钠组，有统计学意义。
6骨组织病理学切片观察：与假手术组大鼠相比，模型组大鼠骨松质部位骨小梁明显减少，排列稀疏、不整齐，部分断裂；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，骨小梁数量减少不明显，排列较规则，连续性、完整性较好；两组与模型组比较，骨形态学结构变化具有统计学意义。
7BMD变化：与假手术组大鼠相比，模型组大鼠的股骨BMD明显偏低，经统计学分析，差异显著(P＜0.05)；给药治疗三个月后，各给药组大鼠的股骨BMD均有明显提高，与模型组比较，P＜0.05；阿胶补肾健骨方组与阿仑膦酸钠组相比较，BMD变化不明显，升高幅度接近，差异无统计学意义。
8骨生物力学测定：与假手术组大鼠相比，模型组大鼠股骨的最大载荷、刚度都明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，大鼠股骨的最大载荷、刚度均明显增高，其中阿胶补肾健骨方组优于阿仑膦酸钠组，有统计学意义。
9大鼠血清OCN、PICP水平变化：与假手术组大鼠相比，模型组大鼠血清OCN、PICP水平都明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，大鼠血清OCN、PICP水平均明显增高，P＜0.05；阿仑膦酸钠组对大鼠血清OCN的影响略优于阿胶补肾健骨方组，阿胶补肾健骨方组对大鼠血清PICP的影响优于阿仑膦酸钠组，有统计学意义。
10大鼠血清25(OH)D_3、1,25(OH)_2D_3水平：与假手术组大鼠相比，模型组大鼠血清25(OH)D_3、1,25(OH)_2D_3都明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，大鼠血清25(OH)D_3、1,25(OH)_2D_3水平均明显增高，P＜0.05；阿胶补肾健骨方组对大鼠血清25(OH)D_3、1,25(OH)_2D_3水平的影响优于阿仑膦酸钠组，具有统计学意义。
11大鼠肾脏VDR-mRNA的基因表达：与假手术组大鼠相比，模型组大鼠VDR的基因表达水平有显著性差异（P＜0.05）；给药治疗三个月后，各给药组大鼠VDR的基因表达水平均有明显提高，与模型组比较，P＜0.05；阿胶补肾健骨方组对大鼠VDR的基因表达水平优于阿仑膦酸钠组，差异有统计学意义。
结论：
1大鼠去卵巢术建造骨质疏松症模型复制成功。
2实验表明：阿胶补肾健骨方可以改善骨质疏松症大鼠一般表现情况；阿胶补肾健骨方有类似雌激素样作用，调节脂代谢，不致于使其体重迅速增加；阿胶补肾健骨方可以增加子宫指数、双肾指数、股骨重量；阿胶补肾健骨方能延缓骨量丢失，提高BMD，改善骨超微结构和骨强度；阿胶补肾健骨方能增加去卵巢大鼠血清中OCN、PICP、25(OH)D_3、1,25(OH)_2D_3的浓度，同时上调肾中VDR的基因表达水平。说明阿胶补肾健骨方对大鼠骨质疏松症有较好的预防和治疗作用，其作用机制体现多靶点、多途径、多环节促进骨钙磷的代谢，促进骨形成和骨重建，抑制破骨细胞（osteoclast，OC）增殖、骨细胞（osteocyte）和成骨细胞（osteoblast，OB）凋亡。
Objective:
The aim of this study is to investigate the osteoporosis from atheoretical point of view with different traditional Chinese and westernmedical methods，to observe the usual manifestations of rats whichwere completely removal of ligation and ovariectomy，the changes ofbone pathological feature，bone mineral density，bone biomechanics，blood biochemistry and the expression of25(OH)D_3、1,25(OH)_2D_3withthe osteoporotic animal models through experimental study，to explorethe effects and mechanism of treating osteoporosis and provide ascientific basis for treating osteo-porosis with E Jiao Bu Shen Jian GuFormula.
Methods:
Seventy female SD rats aged8months， postmenopausalosteoporotic animal model was established. One week after the feedingadaption，rats were randomly divided into four groups: sham-operatedgroup and model group，17in each group.36rats were randomlydivided into group A of osteoporotic animal model with Alendronatesodium and group E of osteoporotic animal model with E Jiao Bu ShenJian Gu Formula，18in each group. Rats in sham-operated group were fed as usual；rats in model group were intragastrically administratednormal physiological saline for90days，rats in other groups wereintragastrically administrated for90days. All rats were stopped treatingfor a day for containing blood.
To observe the usual manifestations and the changes of weightfrom living rats.After treated for3months，2％pentobarbital sodiumintraperitoneal anesthesia，getting blood from the arteria femoralis forreserve and writing down the weight of both kidneys，offside femur anduterus after three months. Soft tissue was removed completely from therats’ offside femur. To use of LUNAR-Prodigy X-ray absorptiometry ofoffside femur and the bone mineral density of bothends of femurmeasured in rats.Then biomechanical detector to determine themaximum load (ML) and stiffness. Femur of left side was putted andfixed in neutral Formaldehyde solution. And then conventional HEstaining，making pathological section as required，observing thechanges of bone tissue under the Microscope. The serumosteocalcin(OCN)，the procollagen I carboxyterminal propeptide(PICP)，25-hydroxyvitamin D_3，[25(OH)D_3]，1,25-dihydroxyvitaminD_3[1,25(OH)_2D_3] were detected by enzyme-linked immunosorbentassay (ELISA). The expression of Vitamin D receptor（VDR） in kidneytissue was detected by Polymerase Chain Reaction（PCR） method.
Statistical analysis: SPSS15.0statistical software were used foranalysis of variance，all data were represented by mean（x±s）standarddeviation，P values of <0.05were considered statistically significant.
Results:
（1）The usual manifestations of rats
Compared with sham-operated rats，spirits of in model group weresagged、furious anxiety and were slow in reacting；Their hair was not brightness，untidy；Some rats were less physically active and somnolent.Compared with group A and group E，rats in the two groups wereemotional stability，in good spirits，lively，responsive and their hair wassupple，brightness during treatment.
（2）Changes of weight of rats
Compared with sham-operated，weight of rats in model group wasapparently heavier than sham-operated rats after two months. Weightof rats in model group E was gradual increase，which was similar to thesham-operated rats.There was statistically significant difference.However，rats in group A gain weight obviously.
（3）Index of uterus of rats
Compared with sham-operated group，index of uterus wassignificantly decreased in other groups（P＜0.05）；Index of uterus ofgroup E was superior to that of the group A.There was obviousdifferences in statistical significance.
（4）Index of both kidneys of rats
Index of both kidneys of model group was significantly lower thansham-operated group （P＜0.05）； Index of both kidneys of group Eand group A was significantly higher than model group. Index of bothkidneys of group E was superior to that of the group A. There wasobvious differences in statistical significance.
（5）Weight of femur of rats
Compared with sham-operated group，weights of femur weresignificantly decreased in other groups（P＜0.05）； Weights of femur ofgroup E and group A were higher than model group. And the weight offemur of group E was superior to group A. There was obviousdifferences in statistical significance.
（6）Observation of pathological section of bone tissue
Sham-operated rats femur bone were rich，continuous，dense，however，rats in model group were sparse trabeculae，irregular；Compared with model group，there was no obvious decrease intrabeculae bone，arranged regularly，continuous and intact. Comparedwith model group，bstructural changes of bone morphology wassignificantly difference in group E and group A.
（7）Differences of BMD of rats
BMD of femur of model group was significantly lower thansham-operated group. From statistical analysis，there was obviousdifferences in statistical significance（P＜0.05）. After treated for3months，compared with model group，bone mineral density of femur ofother groups was significantly increased（P＜0.05）.Compared group E and group A，the changes of bone mineral densitywas unconspicuous，the scope was similar. There was no significantlydifference between group E and group A in BMD of femur.
（8）Results of Biomechanics of rats
Compared with sham-operated group，the largest compressivestress and stiffness were obvious decreased in femur of rats in modelgroup，P＜0.05；compared with model group，the largest compressivestress and stiffness were significantly increased in femur of rats ingroup E and group A. And the largest compressive stress and stiffnessof group E was superior to group A. There was statistically significantdifference.
（9）Changes of OCN and PICP of rats
OCN and PICP were lower than sham-operated group，P＜0.05；and OCN and PICP of group E and group A were higher than modelgroup P＜0.05. Effect of OCN of group A was a little higher than groupE；however，effect of PICP of group E was superior to group A. There was statistically significant difference.
（10）Gene level of25(OH)D_3、1,25(OH)_2D_3of rats
Concentration of25(OH)D_3，1,25(OH)_2D_3o in model group wasobvious lower than sham-operated group (P＜0.05)；however theconcentration of25(OH)D_3，1,25(OH)_2D_3in group E and group A washigher than model group (P＜0.05). And group E was superior to groupA. There was statistically significant difference.
（11）Expression of VDR-mRNA in kidneys tissue
Compared with sham-operated group，expression of VDR-mRNA inkidneys tissue of model group were statistically significant difference.After treated for3months，expression of VDR-mRNA on gene level inkidneys tissue of groups were higher than model group (P＜0.05)；Andexpression of VDR-mRNA on gene level in kidneys tissue of group Ewas superior to group A. There was statistically significant difference.
Conclusion:
（1）There is significantly difference between sham-operated groupand ovariectomized rats，as a result，to build a animal model ofosteoporosis to meet the requirements of this experiment successfully.
（2）The result shows that E Jiao Bu Shen Jian Gu Formula canimprove general performance of ovariectomized rats. Its effects whichadjust lipid metabolism and prevent gain weight rapidly are similar toEstrogen. E Jiao Bu Shen Jian Gu Formula can not only increase indexof uterus，both kidneys and weight of bone but also put off a loss ofbone mass，increase BMD and improve bone ultrastructure，strength ofbone. E Jiao Bu Shen Jian Gu Formula can not only improve theconcentration of OCN，PICP，25(OH)D_3and1,25(OH)_2D_3but alsoincreasing regulate the VDR-mRNA. There was a better prevention andtreatment that E Jiao Bu Shen Jian Gu Formula can be treated for osteoporosis. The mechanism may be multi-channel，multi-target andmulti-step not only to promote metabolism of calcium and phosphorus inbone， bone formation and bony remodeling but also to controlosteoclast proliferation，osteocyte and osteoblast apoptosis.
通过理论研究认识骨质疏松症（Osteoporosis，OP），运用中西医不同的治疗方法，观察去卵巢大鼠骨质疏松症模型的一般情况，骨病理学特征，骨密度(bone mineral density，BMD)，骨生物力学，骨代谢的血生化指标以及维生素D受体（Vitamin D receptor，VDR）的基因表达等变化，探讨阿胶补肾健骨方对骨质疏松症的影响及作用机制，为运用中医药治疗骨质疏松症提供科学依据。
方法：
采用8月龄雌性Sprague-Dawley（SD）大鼠建造骨质疏松症模型，共70只。实验前置动物于室内适应环境一周，室温18-22℃。将大鼠随机分为以下4组：假手术组（17只）、模型组（17只）、阿胶补肾健骨方组（18只）、阿仑膦酸钠（alendronate sodium）组（18只）。假手术组正常饲养；模型组给予生理盐水灌胃90天，其他各组动物灌胃治疗90天，停止治疗1天，备用。
观察活体大鼠一般表现情况，体重变化。观察治疗三个月后，各组动物经腹腔麻醉，股动脉取血备用。记录双肾重量、右侧股骨重量及子宫重量。将其右侧股骨上的软组织剥离、剔净，用LUNAR-Prodigy双能X线BMD仪测定大鼠全股骨及股骨两端BMD，然后置于生物力学测定仪上测定最大载荷(maximum load，ML)和刚度(stiffness)。取其左侧股骨置于中性福尔马林溶液中固定，常规苏木素-伊红（Hematoxylin-Eosin，HE）染色，按要求制备病理片，显微镜下观察骨组织（bone tissue）结构变化。采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)对大鼠血清骨钙素（osteocalcin，OCN）、Ⅰ型前胶原羧基端前肽(procollagen I carboxyterminal propeptide，PICP)、25羟基维生素D_3（25-hydroxyvitamin D_3，25(OH)D_3）、1,25二羟基维生素D_3（1,25-dihydroxyvitamin D_3，1,25(OH)_2D_3）测定研究。用聚合酶链反应（Polymerase Chain Reaction，PCR）测定肾脏VDR的基因表达。
统计学处理：采用SPSS15.0统计软件包对各项检测数据进行方差分析，所有数据结果均以均数加减标准差（x±s）表示，以P＜0.05为差异，具有统计学意义。
结果：
1大鼠一般表现情况：与假手术组大鼠相比，模型组大鼠精神相对萎靡呆滞或见有狂躁不安，反应较迟钝，毛发无光泽而凌乱，少动嗜睡；阿胶补肾健骨方组、阿仑膦酸钠组大鼠在给药治疗过程中，情绪较稳定，精神状态较佳、较活跃，反应较敏捷，毛发顺整。
2大鼠体重变化：与假手术组大鼠相比，模型组于造模后两个月起，体重明显高于假手术组；阿胶补肾健骨方组体重渐增，与假手术组相近，具有统计学意义；阿仑膦酸钠组体重增加较为明显。
3大鼠子宫指数：与假手术组大鼠相比，各组均明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，均有明显提高，其中阿胶补肾健骨方组明显优于阿仑膦酸钠组，有统计学意义。
4大鼠双肾指数：与假手术组大鼠相比，模型组大鼠双肾指数明显减轻，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，双肾指数均有提高，其中阿胶补肾健骨方组明显优于阿仑膦酸钠组，有统计学意义。
5大鼠股骨重量：与假手术组大鼠相比，各组均明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，均有明显提高，其中阿胶补肾健骨方组明显优于阿仑膦酸钠组，有统计学意义。
6骨组织病理学切片观察：与假手术组大鼠相比，模型组大鼠骨松质部位骨小梁明显减少，排列稀疏、不整齐，部分断裂；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，骨小梁数量减少不明显，排列较规则，连续性、完整性较好；两组与模型组比较，骨形态学结构变化具有统计学意义。
7BMD变化：与假手术组大鼠相比，模型组大鼠的股骨BMD明显偏低，经统计学分析，差异显著(P＜0.05)；给药治疗三个月后，各给药组大鼠的股骨BMD均有明显提高，与模型组比较，P＜0.05；阿胶补肾健骨方组与阿仑膦酸钠组相比较，BMD变化不明显，升高幅度接近，差异无统计学意义。
8骨生物力学测定：与假手术组大鼠相比，模型组大鼠股骨的最大载荷、刚度都明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，大鼠股骨的最大载荷、刚度均明显增高，其中阿胶补肾健骨方组优于阿仑膦酸钠组，有统计学意义。
9大鼠血清OCN、PICP水平变化：与假手术组大鼠相比，模型组大鼠血清OCN、PICP水平都明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，大鼠血清OCN、PICP水平均明显增高，P＜0.05；阿仑膦酸钠组对大鼠血清OCN的影响略优于阿胶补肾健骨方组，阿胶补肾健骨方组对大鼠血清PICP的影响优于阿仑膦酸钠组，有统计学意义。
10大鼠血清25(OH)D_3、1,25(OH)_2D_3水平：与假手术组大鼠相比，模型组大鼠血清25(OH)D_3、1,25(OH)_2D_3都明显降低，P＜0.05；阿胶补肾健骨方组、阿仑膦酸钠组与模型组大鼠相比较，大鼠血清25(OH)D_3、1,25(OH)_2D_3水平均明显增高，P＜0.05；阿胶补肾健骨方组对大鼠血清25(OH)D_3、1,25(OH)_2D_3水平的影响优于阿仑膦酸钠组，具有统计学意义。
11大鼠肾脏VDR-mRNA的基因表达：与假手术组大鼠相比，模型组大鼠VDR的基因表达水平有显著性差异（P＜0.05）；给药治疗三个月后，各给药组大鼠VDR的基因表达水平均有明显提高，与模型组比较，P＜0.05；阿胶补肾健骨方组对大鼠VDR的基因表达水平优于阿仑膦酸钠组，差异有统计学意义。
结论：
1大鼠去卵巢术建造骨质疏松症模型复制成功。
2实验表明：阿胶补肾健骨方可以改善骨质疏松症大鼠一般表现情况；阿胶补肾健骨方有类似雌激素样作用，调节脂代谢，不致于使其体重迅速增加；阿胶补肾健骨方可以增加子宫指数、双肾指数、股骨重量；阿胶补肾健骨方能延缓骨量丢失，提高BMD，改善骨超微结构和骨强度；阿胶补肾健骨方能增加去卵巢大鼠血清中OCN、PICP、25(OH)D_3、1,25(OH)_2D_3的浓度，同时上调肾中VDR的基因表达水平。说明阿胶补肾健骨方对大鼠骨质疏松症有较好的预防和治疗作用，其作用机制体现多靶点、多途径、多环节促进骨钙磷的代谢，促进骨形成和骨重建，抑制破骨细胞（osteoclast，OC）增殖、骨细胞（osteocyte）和成骨细胞（osteoblast，OB）凋亡。
Objective:
The aim of this study is to investigate the osteoporosis from atheoretical point of view with different traditional Chinese and westernmedical methods，to observe the usual manifestations of rats whichwere completely removal of ligation and ovariectomy，the changes ofbone pathological feature，bone mineral density，bone biomechanics，blood biochemistry and the expression of25(OH)D_3、1,25(OH)_2D_3withthe osteoporotic animal models through experimental study，to explorethe effects and mechanism of treating osteoporosis and provide ascientific basis for treating osteo-porosis with E Jiao Bu Shen Jian GuFormula.
Methods:
Seventy female SD rats aged8months， postmenopausalosteoporotic animal model was established. One week after the feedingadaption，rats were randomly divided into four groups: sham-operatedgroup and model group，17in each group.36rats were randomlydivided into group A of osteoporotic animal model with Alendronatesodium and group E of osteoporotic animal model with E Jiao Bu ShenJian Gu Formula，18in each group. Rats in sham-operated group were fed as usual；rats in model group were intragastrically administratednormal physiological saline for90days，rats in other groups wereintragastrically administrated for90days. All rats were stopped treatingfor a day for containing blood.
To observe the usual manifestations and the changes of weightfrom living rats.After treated for3months，2％pentobarbital sodiumintraperitoneal anesthesia，getting blood from the arteria femoralis forreserve and writing down the weight of both kidneys，offside femur anduterus after three months. Soft tissue was removed completely from therats’ offside femur. To use of LUNAR-Prodigy X-ray absorptiometry ofoffside femur and the bone mineral density of bothends of femurmeasured in rats.Then biomechanical detector to determine themaximum load (ML) and stiffness. Femur of left side was putted andfixed in neutral Formaldehyde solution. And then conventional HEstaining，making pathological section as required，observing thechanges of bone tissue under the Microscope. The serumosteocalcin(OCN)，the procollagen I carboxyterminal propeptide(PICP)，25-hydroxyvitamin D_3，[25(OH)D_3]，1,25-dihydroxyvitaminD_3[1,25(OH)_2D_3] were detected by enzyme-linked immunosorbentassay (ELISA). The expression of Vitamin D receptor（VDR） in kidneytissue was detected by Polymerase Chain Reaction（PCR） method.
Statistical analysis: SPSS15.0statistical software were used foranalysis of variance，all data were represented by mean（x±s）standarddeviation，P values of <0.05were considered statistically significant.
Results:
（1）The usual manifestations of rats
Compared with sham-operated rats，spirits of in model group weresagged、furious anxiety and were slow in reacting；Their hair was not brightness，untidy；Some rats were less physically active and somnolent.Compared with group A and group E，rats in the two groups wereemotional stability，in good spirits，lively，responsive and their hair wassupple，brightness during treatment.
（2）Changes of weight of rats
Compared with sham-operated，weight of rats in model group wasapparently heavier than sham-operated rats after two months. Weightof rats in model group E was gradual increase，which was similar to thesham-operated rats.There was statistically significant difference.However，rats in group A gain weight obviously.
（3）Index of uterus of rats
Compared with sham-operated group，index of uterus wassignificantly decreased in other groups（P＜0.05）；Index of uterus ofgroup E was superior to that of the group A.There was obviousdifferences in statistical significance.
（4）Index of both kidneys of rats
Index of both kidneys of model group was significantly lower thansham-operated group （P＜0.05）； Index of both kidneys of group Eand group A was significantly higher than model group. Index of bothkidneys of group E was superior to that of the group A. There wasobvious differences in statistical significance.
（5）Weight of femur of rats
Compared with sham-operated group，weights of femur weresignificantly decreased in other groups（P＜0.05）； Weights of femur ofgroup E and group A were higher than model group. And the weight offemur of group E was superior to group A. There was obviousdifferences in statistical significance.
（6）Observation of pathological section of bone tissue
Sham-operated rats femur bone were rich，continuous，dense，however，rats in model group were sparse trabeculae，irregular；Compared with model group，there was no obvious decrease intrabeculae bone，arranged regularly，continuous and intact. Comparedwith model group，bstructural changes of bone morphology wassignificantly difference in group E and group A.
（7）Differences of BMD of rats
BMD of femur of model group was significantly lower thansham-operated group. From statistical analysis，there was obviousdifferences in statistical significance（P＜0.05）. After treated for3months，compared with model group，bone mineral density of femur ofother groups was significantly increased（P＜0.05）.Compared group E and group A，the changes of bone mineral densitywas unconspicuous，the scope was similar. There was no significantlydifference between group E and group A in BMD of femur.
（8）Results of Biomechanics of rats
Compared with sham-operated group，the largest compressivestress and stiffness were obvious decreased in femur of rats in modelgroup，P＜0.05；compared with model group，the largest compressivestress and stiffness were significantly increased in femur of rats ingroup E and group A. And the largest compressive stress and stiffnessof group E was superior to group A. There was statistically significantdifference.
（9）Changes of OCN and PICP of rats
OCN and PICP were lower than sham-operated group，P＜0.05；and OCN and PICP of group E and group A were higher than modelgroup P＜0.05. Effect of OCN of group A was a little higher than groupE；however，effect of PICP of group E was superior to group A. There was statistically significant difference.
（10）Gene level of25(OH)D_3、1,25(OH)_2D_3of rats
Concentration of25(OH)D_3，1,25(OH)_2D_3o in model group wasobvious lower than sham-operated group (P＜0.05)；however theconcentration of25(OH)D_3，1,25(OH)_2D_3in group E and group A washigher than model group (P＜0.05). And group E was superior to groupA. There was statistically significant difference.
（11）Expression of VDR-mRNA in kidneys tissue
Compared with sham-operated group，expression of VDR-mRNA inkidneys tissue of model group were statistically significant difference.After treated for3months，expression of VDR-mRNA on gene level inkidneys tissue of groups were higher than model group (P＜0.05)；Andexpression of VDR-mRNA on gene level in kidneys tissue of group Ewas superior to group A. There was statistically significant difference.
Conclusion:
（1）There is significantly difference between sham-operated groupand ovariectomized rats，as a result，to build a animal model ofosteoporosis to meet the requirements of this experiment successfully.
（2）The result shows that E Jiao Bu Shen Jian Gu Formula canimprove general performance of ovariectomized rats. Its effects whichadjust lipid metabolism and prevent gain weight rapidly are similar toEstrogen. E Jiao Bu Shen Jian Gu Formula can not only increase indexof uterus，both kidneys and weight of bone but also put off a loss ofbone mass，increase BMD and improve bone ultrastructure，strength ofbone. E Jiao Bu Shen Jian Gu Formula can not only improve theconcentration of OCN，PICP，25(OH)D_3and1,25(OH)_2D_3but alsoincreasing regulate the VDR-mRNA. There was a better prevention andtreatment that E Jiao Bu Shen Jian Gu Formula can be treated for osteoporosis. The mechanism may be multi-channel，multi-target andmulti-step not only to promote metabolism of calcium and phosphorus inbone， bone formation and bony remodeling but also to controlosteoclast proliferation，osteocyte and osteoblast apoptosis.
引文
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