补肾宁心方及脱氢表雄酮防治绝经后骨质疏松的作用机制
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摘要
骨质疏松与相关的骨折是全球性中老年妇女死亡率和致残率升高的主要原因，随着世界人口的老年化，骨质疏松将成为前所未有的公共卫生问题。
雌激素撤退与绝经后骨质疏松(PMOP)有密切的关系，但激素替代治疗(HRT)对于PMOP的防治作用缺乏有力的证据，而且增加了心脏疾患、中风及乳腺癌的发病率，这使HRT的应用受到了限制。目前应用的经典抗骨质疏松药物，如降钙素、二磷酸盐、雌激素等，多为抑制破骨细胞活性的药物，缺乏刺激成骨细胞活性或既刺激成骨又抑制破骨的药物。因此，在深入研究和发掘化学合成药物的同时，更要注重天然植物化合物的开发研究。在补肾宁心方能有效改善临床症状的基础上，探讨其对绝经后骨质疏松的作用机制具有深远的意义。
第一部分 补肾宁心方及脱氢表雄酮对绝经后骨质疏松小鼠的防治作用
目的 探讨补肾宁心方及脱氢表雄酮防治绝经后骨质疏松的作用。
方法 建立BALB／c小鼠绝经后骨质疏松模型，灌服脱氢表雄酮(DHEA组)或补肾宁心方浓缩液(BSNXD组)。以灌服17β-雌二醇组(E_2组)为阳性对照，灌服生理盐水组(Saline组)为阴性对照。手术相同但不切除卵巢小鼠为假手术对照(Sham组)。12周后处死，心脏取血制备药物血清，测定Th_1／Th_2型细胞因子和DHEA、DHEA-S。取椎骨(L3-4)和左股骨测定骨密度(BMD)；椎骨(L1-2)和右股骨行组织形态计量分析；右胫骨行透射电镜观察，测定护骨素(OPG)的mRNA表达，分析芳香化酶活性；取左胫骨分析凋亡的成骨细胞(OB)；取颅骨植块法培养OB，分析OB增殖能力的变化。取子宫称量湿重，行子宫组织切片观察，分析芳香化酶活性及雌激素受体(ER)亚型的表达。
结果 BSNXD及DHEA对椎骨与股骨的BMD、骨小梁面积均有改善作用(P＜0.05和P＜0.01)；与E_2比较无显著差异(P＞0.05)。BSNXD和DHEA组的DHEA、DHEA-S和IFN-γ水平明显高于Saline组(P＜0.01)，而IL-4水平低于Saline组(P＜0.05)；另外，BSNXD组小鼠的DHEA水平与IFN-γ水平呈
摘要
(P<0.01)，与IL一4水平呈负相关(P<0.05)。BSNXD和DHEA组的子宫重量、
子宫/体重比值明显小于EZ组(P<0.01)，与Sal ine组无差异。BSNXD和DHEA
可提高骨0B的增殖能力，降低0B的凋亡(P<0.05)。BSNXD能提高骨组织的
芳香化酶活性(P<0.05)，而对子宫的芳香化酶活性无影响。与Saline组相
比，BSNXD和DHEA组的骨OPG、子宫ERa和ERp的转录水平均有显著增加
(分别为P<0.05和P<0.01)。
结论补肾宁心方可提高外周血DHEA(一S)的水平，增强骨中芳香酶的活
性。补肾宁心方及DHEA能够防治绝经后骨质疏松而对子宫无/或仅有轻微的
增生作用;其作用机制与增强骨中芳香化酶及0B活性，提高骨OPG表达及调
节Thl/ThZ偏移有关。
Part I Action of BSNXD and DHEA on mice of PMOP
Objective To investigate the effect of Bu-shen-ning-xin decoction (BSNXD) and dehydroepiandrosterone (DHEA) on postmenopausal osteoporosis (PMOP).
Methods Female BALB/c mice (8-9 months age) were divided randomly into six groups containing ten mice per group. Mice of group 1 were left intact and served as a normal control group (NC group), mice of group 2 were operated without lesion of ovary (Sham operation group). Mice of groups 3~6 were ovariectbmized and, after a 7-day delay to allow for foundation of osteoporosis, were afforded with the same volume of Chinese traditional medicine (BSNXD group), dehydroepiandrosterone (DHEA group), 17p-estradiol (E2 group) and saline (Saline group), respectively. The skeleton, blood and uterus of each group were collected after 12 weeks of treatment. The uteri were weighed and analyzed histomorphometrically for differences. The level of IL-4/IFN-r, DHEA and DHEA-S in serum was detected by ELISA or EIA. The excised vertebra (L3-4) and left femurs were measured for the bone mineral density (BMD) by double energy x ray absorption (DEXA), the right femurs were measured for norphometry, the left tibia were analyzed for
osteoblastic apoptosis. The semi-quantitative RT-PCR was performed to compare the mRNA level of osteoprotegerin (OPG) from tibia and ERa/p from uterus. The osteoblasts were isolated from calvaria and cultured for analyzing the cell proliferation. Both bone and uterus from every group were processed for the activity of aromatase.
Results The level of BMD and area of bone trabecula of BSNXD group and DHEA group were higher than that of Saline group (P < 0.05 and P < 0.01, respectively). The level of IFN-r, DHEA and DHEA-S in BSNXD group and DHEA group were improved significantly, the IL-4 level was reduced comparing to that of Saline group (P < 0.05 and P < 0.01, respectively). In BSNXD group,
moreover, the level of DHEA was positively related to that of IFN-r (P < 0.01), negatively related to that of IL-4 (P < 0.05). The weight of uterus and the ratio of uterus to body of BSNXD group and DHEA group were dominantly lower than that of E2 group (P < 0.01), the same to that of Saline group. More potent viability and less apoptosis of osteoblasts were seen in bone of BSNXD and DHEA group. BSNXD could improve the aromatase activity not of uterus but of bone. The level of OPG mRNA in bone, ERa and ERB mRNA in uterus from BSNXD and DHEA group were all higher than that from Saline group.
Conclusions BSNXD could improve the level of DHEA (-s) in blood and enhance the aromatase activity in bone. Both BSNXD and DHEA could prevent and cure the PMOP with little side-effect on uterus. The mechanism of BSNXD for PMOP was related to that enhancing the osteoblastic viability and aromatase activity of bone, regulating the bias of Th1/Th2, improving the expression of OPG which could inhibit the activity of osteoclasts.
雌激素撤退与绝经后骨质疏松(PMOP)有密切的关系，但激素替代治疗(HRT)对于PMOP的防治作用缺乏有力的证据，而且增加了心脏疾患、中风及乳腺癌的发病率，这使HRT的应用受到了限制。目前应用的经典抗骨质疏松药物，如降钙素、二磷酸盐、雌激素等，多为抑制破骨细胞活性的药物，缺乏刺激成骨细胞活性或既刺激成骨又抑制破骨的药物。因此，在深入研究和发掘化学合成药物的同时，更要注重天然植物化合物的开发研究。在补肾宁心方能有效改善临床症状的基础上，探讨其对绝经后骨质疏松的作用机制具有深远的意义。
第一部分 补肾宁心方及脱氢表雄酮对绝经后骨质疏松小鼠的防治作用
目的 探讨补肾宁心方及脱氢表雄酮防治绝经后骨质疏松的作用。
方法 建立BALB／c小鼠绝经后骨质疏松模型，灌服脱氢表雄酮(DHEA组)或补肾宁心方浓缩液(BSNXD组)。以灌服17β-雌二醇组(E_2组)为阳性对照，灌服生理盐水组(Saline组)为阴性对照。手术相同但不切除卵巢小鼠为假手术对照(Sham组)。12周后处死，心脏取血制备药物血清，测定Th_1／Th_2型细胞因子和DHEA、DHEA-S。取椎骨(L3-4)和左股骨测定骨密度(BMD)；椎骨(L1-2)和右股骨行组织形态计量分析；右胫骨行透射电镜观察，测定护骨素(OPG)的mRNA表达，分析芳香化酶活性；取左胫骨分析凋亡的成骨细胞(OB)；取颅骨植块法培养OB，分析OB增殖能力的变化。取子宫称量湿重，行子宫组织切片观察，分析芳香化酶活性及雌激素受体(ER)亚型的表达。
结果 BSNXD及DHEA对椎骨与股骨的BMD、骨小梁面积均有改善作用(P＜0.05和P＜0.01)；与E_2比较无显著差异(P＞0.05)。BSNXD和DHEA组的DHEA、DHEA-S和IFN-γ水平明显高于Saline组(P＜0.01)，而IL-4水平低于Saline组(P＜0.05)；另外，BSNXD组小鼠的DHEA水平与IFN-γ水平呈
摘要
(P<0.01)，与IL一4水平呈负相关(P<0.05)。BSNXD和DHEA组的子宫重量、
子宫/体重比值明显小于EZ组(P<0.01)，与Sal ine组无差异。BSNXD和DHEA
可提高骨0B的增殖能力，降低0B的凋亡(P<0.05)。BSNXD能提高骨组织的
芳香化酶活性(P<0.05)，而对子宫的芳香化酶活性无影响。与Saline组相
比，BSNXD和DHEA组的骨OPG、子宫ERa和ERp的转录水平均有显著增加
(分别为P<0.05和P<0.01)。
结论补肾宁心方可提高外周血DHEA(一S)的水平，增强骨中芳香酶的活
性。补肾宁心方及DHEA能够防治绝经后骨质疏松而对子宫无/或仅有轻微的
增生作用;其作用机制与增强骨中芳香化酶及0B活性，提高骨OPG表达及调
节Thl/ThZ偏移有关。
Part I Action of BSNXD and DHEA on mice of PMOP
Objective To investigate the effect of Bu-shen-ning-xin decoction (BSNXD) and dehydroepiandrosterone (DHEA) on postmenopausal osteoporosis (PMOP).
Methods Female BALB/c mice (8-9 months age) were divided randomly into six groups containing ten mice per group. Mice of group 1 were left intact and served as a normal control group (NC group), mice of group 2 were operated without lesion of ovary (Sham operation group). Mice of groups 3~6 were ovariectbmized and, after a 7-day delay to allow for foundation of osteoporosis, were afforded with the same volume of Chinese traditional medicine (BSNXD group), dehydroepiandrosterone (DHEA group), 17p-estradiol (E2 group) and saline (Saline group), respectively. The skeleton, blood and uterus of each group were collected after 12 weeks of treatment. The uteri were weighed and analyzed histomorphometrically for differences. The level of IL-4/IFN-r, DHEA and DHEA-S in serum was detected by ELISA or EIA. The excised vertebra (L3-4) and left femurs were measured for the bone mineral density (BMD) by double energy x ray absorption (DEXA), the right femurs were measured for norphometry, the left tibia were analyzed for
osteoblastic apoptosis. The semi-quantitative RT-PCR was performed to compare the mRNA level of osteoprotegerin (OPG) from tibia and ERa/p from uterus. The osteoblasts were isolated from calvaria and cultured for analyzing the cell proliferation. Both bone and uterus from every group were processed for the activity of aromatase.
Results The level of BMD and area of bone trabecula of BSNXD group and DHEA group were higher than that of Saline group (P < 0.05 and P < 0.01, respectively). The level of IFN-r, DHEA and DHEA-S in BSNXD group and DHEA group were improved significantly, the IL-4 level was reduced comparing to that of Saline group (P < 0.05 and P < 0.01, respectively). In BSNXD group,
moreover, the level of DHEA was positively related to that of IFN-r (P < 0.01), negatively related to that of IL-4 (P < 0.05). The weight of uterus and the ratio of uterus to body of BSNXD group and DHEA group were dominantly lower than that of E2 group (P < 0.01), the same to that of Saline group. More potent viability and less apoptosis of osteoblasts were seen in bone of BSNXD and DHEA group. BSNXD could improve the aromatase activity not of uterus but of bone. The level of OPG mRNA in bone, ERa and ERB mRNA in uterus from BSNXD and DHEA group were all higher than that from Saline group.
Conclusions BSNXD could improve the level of DHEA (-s) in blood and enhance the aromatase activity in bone. Both BSNXD and DHEA could prevent and cure the PMOP with little side-effect on uterus. The mechanism of BSNXD for PMOP was related to that enhancing the osteoblastic viability and aromatase activity of bone, regulating the bias of Th1/Th2, improving the expression of OPG which could inhibit the activity of osteoclasts.
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