己烯雌酚与淫羊藿总黄酮联合用药对去卵巢大鼠腰椎和股骨的影响

背景以往实验结果提示淫羊藿总黄酮有提高雌激素预防去卵巢大鼠胫骨上段松质骨丢失的作用,又可减少雌激素在应用于防治绝经后骨质疏松症中对子宫的刺激等不良反应,将两者结合应用是否有协同作用?目的观察己烯雌酚与淫羊藿总黄酮联合用药对去卵巢大鼠腰椎和股骨的代谢生化指标和骨组织形态计量学的影响.设计随机对照动物实验.单位广东医学院药理教研室.材料4月龄未交配的雌性SD大鼠,体质量(225±15)g,由中国科学院上海实验动物中心提供,动物级别为清洁级(合格证号中科动管第003号).大鼠随机分为5组假手术组、去卵巢组、己烯雌酚组、淫羊藿总黄酮组与联合用药组,每组10只.方法除假手术组大鼠外,其余行双侧卵巢切除术,假手术组和去卵巢组给予溶剂对照,其余分别为己烯雌酚组22.5 μg/(kg·d),淫羊藿总黄酮组300 mg/(kg·d),联合用药组己烯雌酚22.5μg/(kg·d)+淫羊藿总黄酮300 mg(kg·d)],均灌胃给药90天.应用骨组织形态计量学方法测量第5腰椎松质骨的动态参数(周长百分数、骨矿化沉积率、骨表面形成率、骨量形成率、骨组织形成率)和静态参数(骨小梁面积百分数、骨小梁厚度、骨小梁数量、骨小梁分离度、单位骨小梁面积破骨细胞数),同时测量股骨钙、磷及羟脯氨酸含量.主要观察指标①己烯雌酚和淫羊藿总黄酮联合用药对去卵巢大鼠腰椎松质骨的动态参数和静态参数的影响.②己烯雌酚和淫羊藿总黄酮联合用药对去卵巢大鼠股骨生化指标的影响.结果5组大鼠,每组10只均纳入结果分析.①己烯雌酚和淫羊藿总黄酮联合用药对去卵巢大鼠股骨生化指标的影响与假手术组比较,去卵巢组大鼠股骨湿重/体质量比、干重/体质量比均明显降低(P＜0.01),骨钙、磷含量及钙/羟脯氨酸比值均降低(P＜0.05).与去卵巢组比较,己烯雌酚组大鼠股骨生化指标无明显变化(P＞0.05);淫羊藿总黄酮组大鼠骨钙、骨磷含量及钙/羟脯氨酸比值明显增加(P＜0.01).联合用药组骨钙、骨磷含量及钙/羟脯氨酸比值均明显增加(P＜0.01),而且高于己烯雌酚组和淫羊藿总黄酮组(P＜0.05).②己烯雌酚和淫羊藿总黄酮联合用药对去卵巢大鼠腰椎静态和动态参数的影响与假手术组比较,去卵巢组大鼠腰椎静态参数中骨小梁面积百分数、骨小梁厚度分别减少35.0%,31.0%,而骨小梁分离度、破骨细胞数分别增加31.6%,106.9%,差异均有显著性(P＜0.05～0.01);动态参数标记周长百分数、骨量形成率分别增加126.8%,36.6%,差异有显著性(P＜0.01).与去卵巢组比较,己烯雌酚使去卵巢大鼠腰椎的骨小梁面积百分数增加20.3%,但差异无显著性(P＞0.05),骨小梁分离度、破骨细胞数分别减少15.1%,47.1%,差异有显著性(P＜0.05);动态参数标记周长百分数、骨矿化沉积率、骨量形成率分别减少44.3%,28.0%,64.1%,差异有显著性(P＜0.05).淫羊藿总黄酮对去卵巢大鼠腰椎静态参数和动态参数均无明显影响(P＞0.05).联合用药使去卵巢大鼠腰椎松质骨的骨小梁面积百分数、骨小梁厚度分别增加52.7%,36.6%,而骨小梁分离度、破骨细胞数分别减少25.5%,61.0%,差异均有显著性(P均＜0.01);动态参数标记周长百分数、骨矿化沉积率、骨量形成率分别减少56.1%,39.2%和2 80.1%,差异均有显著性(P均＜0.01).联合用药组的作用明显强于己烯雌酚组和淫羊藿总黄酮组,差异有显著性(P＜0.05).结论己烯雌酚和淫羊藿总黄酮联合用药明显增加去卵巢大鼠腰椎的骨量和骨小梁厚度,减少骨小梁分离度,其增加骨量的效应比单用己烯雌酚和单用淫羊藿总黄酮的作用明显,且可使股骨钙、磷含量及钙/羟脯氨酸比值增加,提示己烯雌酚与淫羊藿总黄酮联合用药对去卵巢大鼠腰椎和股骨的作用具有协同作用.

Influence of diethylstilbestrol combined with epimedium pubescens flavonoids on lumbar vertebral body and femur in ovariectomized rats

BACKGROUND: Previous experiments indicate that epimedium pubesceus flavonoids (EPF) could enhance protective effect of estrogen on proximal tibial metaphysis (PTM) and reduce its adverse effect on uterine tissue in ovariectomized rats. Do they have synergetic effects on lumbar vertebral body and femur in ovariectomized rats when used in combination?OBJECTIVE: To observe the influence of diethylstilbestrol combined with EPF on metabolic biochemical indexes and bone histomorphometry of lumbar and femur in ovariectomized rats.DESIGN: Randomized controlled study on experimental animals.SETTING: Department of Pharmacology, Guangdong Medical College.MATERIALS: Female unmated SD rats of 4 months old, with the body mass of (225±15)g, were provided by Shanghai Experimental Animal Centre of the Chinese Academy of Sciences. They were of clean grade (Certification No. CSAM003). All rats were randomized into 5 groups: namely,sham- operation group, ovariectomy group, diethylstilbestrol group, EPF group, and combination group with 10 rats in each group.METHODS: Rats in all the groups except sham operation group were subjected to bilateral ovariectomy. Those in sham operation group and ovariectomy group were given solvent as control, while rats in the other groups were given gastric perfusion of: diethylstilbestrol at the dosage of 22.5 μg/metric analysis was used to assess the static parameters percent trabecular area(%Tb. Ar), trabecular number(Tb.N), trabecular thickness (Tb.Th), trabecular seperation(Tb. Sp), osteoclast number per mm2 (Oc.N)] and dynamic parameterspercent labeled perimeter(%L.Pm), mineral apposition rate (MAR), bone formation rate(BFR)per unit of bone volume (BFR/BV)] of the fifth lumbar vertebral body(LV5). Meanwhile the contents of femur calcium,phosphorus and hydroxyproline were also measured.static parameters of LV5 of ovariectomized rats due to the combined adethylstilbestrol and EPF on femur biochemical indexes in ovariectomized rats.RFSULTS: Five groups with 10 rats in each were included in the result chemical indexes of ovariectomized rats: In contrast with those of sham operation group, ratios of femur wet weight/body mass and dry weight/body mass were obviously decreased in groups of ovariectomized rats (P ＜ 0.01).Meanwhile the content of bone calcium and phosphorus, and calcium /hydroxyproline were also markedly reduced (P ＜ 0.05). The biochemical indexes did not change obviously in diethylstilbestrol group compared to ovariectomy group (P ＞ 0. 05); however, in EPF group, there was an obvious increment in the content of bone calcium and phosphorus, as well as the ratio of calcium/hydroxyproline (P ＜ 0.01). The same results were also observed in combination group, with the increment significantly highin the dynamic and static parameters of lumbar spongy bone of ovariectomized rats due to the combined administration of diethylstilbestrol and EPF: Compared with those of sham operation group, the %Tb. Ar and Tb.Th of ovariectomized rat lumbar were reduced by 35.0% and 31.0%, respectively; the Tb. Sp and the Oc.N increased significantly by 31.6% and 106.9% (P ＜ 0.05-0.01). The %L.Pm and BFR increased by 126.8% and 36.6%, respectively, with significant differences (P ＜ 0.01). Compared with that of ovariectomy group, %Tb.Ar of ovariectomized lumbar was increased by 20% due to diethylstilbestrol, but the difference was of no statistical meaning (P ＞ 0.05). Meanwhile the Tb. Sp and the Oc.N were significantly reduced by 15.1% and 47.1% (P ＜ 0.05). However, EPF did not obviously affect the dynamic and static parameters of ovariectomized rat lumbar (P ＞ 0.05). In combination group, the %Tb.Ar and Tb. Th of ovariectomized rat lumbar were increased by 52.7% and 36.6%, while Tb.Sp and the Oc.N were reduced by 25.5% and 61.0%, with significant differences (P ＜ 0.01). As to the dynamic parameters, %L.Pm, MAR and BFR were reduced by 56.1%, 39.2% and 2 80.1%, with significant differences (P ＜ 0.01). The combined influence was proved obviously stronger than that of diethylstilbestrol and EPF groups (P ＜ 0.05).CONCLUSION: The combination of d iethylstilbestrol and EPF can obviously increase bone mass and Tb.Th of ovariectomized rat lumbar, and reduce the Tb. Sp, with the enhancement on bone mass obviously stronger than using diethylstilbestrol or EPF only. Moreover, the content of femur calcium and phosphorus, and the ratio of calcium/hydroxyproline were also increased, suggesting that diethylstilbestrol and EPF have a synergetic effect on ovariectomized lumbar and femur.