杜仲防治绝经后骨质疏松及其机理研究
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摘要
研究背景
中年以后,生理性退行性改变所致的骨质疏松(osteoporosis, OP)的发病率随年龄的增长而增加,以骨量减少、骨组织显微结构退化,骨脆性增高,诱发骨折为特征。OP是一个全球性的健康问题,严重影响中老年人的生活质量。雌激素在OP的发生过程中起着极其重要的作用,尤其是绝经后骨质疏松症(postmenopausal osteoporosis, PMO),因此,激素替代疗法(hormonereplacement therapy, HRT)一直被视为是防治PMO的有效手段。妇女健康倡议研究结果显示,HRT的长期应用具有增加患者的心血管意外事件和生殖系统肿瘤发生的风险,严重限制了HRT在PMO预防和治疗中的应用。其它的抗骨质疏松药物,包括促进骨形成和抑制骨吸收的药物,虽然具有确切的PMO预防或者治疗作用,但是长期服用不仅其价格昂贵,而且可能产生副作用。因此,迫切需要寻找安全、有效的防治OP的药物,更好地克服现有药物存在的问题。
临床实践证明,许多天然药物可以有效、安全地防治与OP相类似的临床症状。某些植物富含许多天然植物化学物质,这些物质具有重要的生理活性。大豆异黄酮是天然的选择性雌激素受体调节剂(selectiveestrogenreceptormodulators, SERMs),其对骨组织药理作用类似于雷洛昔芬,能够防止绝经妇女或卵巢切除(ovariectomy, OVX)大鼠的骨丢失。近年来,植物中所富含的多酚类物质也引起了研究者的关注。木脂素类,如从亚麻子中分离得到的闭联异松树脂醇二酯能够有效的防治妇女绝经导致的骨质丢失的发生;从红豆杉中分离得到的异紫杉脂素在动物OVX后能有效延缓骨质丢失;从红楠中分离的木脂素类能够显著的促进小鼠成骨细胞(osteoblast, OB)的增殖和分化。此外,也有研究显示黄酮类如芦丁能够有效的抑制绝经大鼠骨量的减少和骨强度的下降,可能与其抑制骨吸收和促进骨形成有关。
研究目的
杜仲是一富含木脂素、酚酸和黄酮类等化学物质的天然“补肾”良药,其防治与OP相类似的临床症状的骨症已有千余年的历史。因此,本课题在复制OVX大鼠骨质疏松模型的基础上,系统地研究了杜仲防治PMO作用;发掘、分离得到重要活性成分——杜仲总木脂素;体外培养大鼠原代成骨细胞,在细胞水平上对药物活性成分进行研究,分析杜仲防治PMO的物质基础及其机制。
研究方法
1.选取3月龄未经产雌性二级Sprague--Dawley(SD)大鼠,随机分为假手术(SHAM)组,去卵巢(OVX)组,杜仲皮提取物组(DZCE)及17β-雌二醇组(E2)。术后第4周开始灌胃给药,连续12周。期间每周观察一次大鼠体质量变化。动物处死前,收集大鼠24h尿液,麻醉动物采集血清。全部动物处死并解剖,采集以下标本并进行测定分析:观察大鼠子宫及其它脏器的体质量及子宫病理变化,分析药物对子宫及长期用药对脏器的影响;采用全自动生化分析仪对大鼠血清和尿液中的Ca、P和ALP等骨代谢指标及肝肾功能指标进行分析;采用ELISA等免疫学方法对骨转换指标尿DPD和血清OCN进行测定;采用三点弯曲实验对大鼠股骨生物力学性能进行分析;采用DEXA及μCT对股骨的BMD、BMC及干骺端骨小梁微结构特性进行分析。
2. 50%乙醇加热回流提取杜仲总木脂素,采用AB-8型大孔树脂对杜仲总木脂素进行分离纯化,依次用去离子水、50%乙醇洗脱。收集50%乙醇洗脱部分,浓缩,以松脂醇二葡糖苷作为对照品,采用比色法进行分析。
3.采用多次酶消化法进行大鼠原代成骨细胞体外培养,通过相差显微镜观察细胞形态,并用碱性磷酸酶染色和钙结节茜素红染色等方法对所获得的细胞进行鉴定。
4.采用MTT、矿化结节茜素红染色等方法,观察杜仲总木脂素对大鼠原代成骨细胞的增殖、分化及矿化过程的影响。
5.采用RT-PCR的方法观察杜仲总木脂素对大鼠原代成骨细胞OPG和RANKLmRNA表达的影响。
实验结果
1. 300和500mg/kg/day治疗剂量的DZCE连续治疗16周,能够显著的改善大鼠股骨的结构力学参数,包括骨应力和弹性模量。这种生物力学性能的改变与DZCE增加BMD和改善骨小梁微结构特性密切相关。DZCE连续治疗16周后能够剂量依赖性的抑制OVX造成的BMD的下降,同时也伴随着反映骨重建的生化指标的下降,DZCE显著降低ALP、OCN和DPD的水平,降低OVX所致的大鼠尿Ca、P的升高。μCT分析结果显示500mg/kg/day治疗剂量的DZCE显著的增加BV/TV、Tb.N、Tb.Th以及Conn.D,降低Tb.Sp和SMI指数。16周的DZCE连续治疗,未观察到子宫刺激作用,对其它脏器和肝肾功能亦未产生明显影响。
2.分离纯化杜仲总木脂素,50%乙醇洗脱液干燥后总固物中杜仲总木脂素纯度可达58.5%。
3.多次酶消化法获得数量丰富的细胞,而且具有典型的OB形态特征,碱性磷酸酶染色和矿化结节染色均呈阳性。
4.在30~500μg/mL浓度范围内,杜仲总木质素剂量依赖性地促进大鼠原代成骨细胞的增殖;100和300μg/mL杜仲总木脂素能够显著的增加大鼠原代成骨细胞ALP的活性和矿化结节形成的数量。
5.100和300μg/mL杜仲总木质素能够显著的调节大鼠原代成骨细胞OPG和RANKL基因表达,增加两者mRNA表达水平的比值达4倍以上。
结论
1.杜仲治疗16周能够通过增加大鼠股骨的BMD和改善骨小梁微结构特性进而阻止OVX导致的股骨生物力学性能的下降,与雌激素相比较具有无子宫刺激的优点。
2.杜仲总木脂素能剂量依赖性地促进大鼠原代培养成骨细胞的增殖、分化及成熟。木脂素类化合物可能是杜仲发挥抗大鼠PMO的活性成分。
3.杜仲总木脂素剂量依赖性地提高大鼠原代成骨细胞OPG/RANKL mRNA表达水平的比值,表明杜仲总木脂素可能通过调节OPG/RANKL表达对破骨细胞(osteoclast,OC)分化和成熟产生影响。
4.杜仲富含多酚类化合物——木脂素,能够有效地阻止大鼠PMO的骨量丢失、骨小梁微结构的破坏及生物力学性能的下降,这种药理学作用可能与杜仲活性成分促进OB骨形成或通过调节OB分泌OPG和RANKL来间接抑制OC的分化和成熟有关。杜仲及其活性成分木脂素类化合物可能成为防治OP的新的天然药物。
Background
The incidence of osteoporosis increases dramatically with life expectancy. Thisdisease is characterized by a reduction in bone mass and microarchitecturaldeterioration of bone tissue, resulting in skeletal fragility and susceptibility tofractures. Osteoporosis has become increasingly recognised as a major healthcareproblem which will affect the lives of a considerable number of individuals.Because hypoestrogenemia after menopause is an important cause of osteoporosis,hormone replacement therapy (HRT) was used to be a popular regime forprevention and treatment of postmenopausal osteoporosis. Ironically, data fromthe Women’s Health Initiative (WHI) Trial indicated that long-term acceptanceand/or compliance of HRT is low due to potentially malignant effects onreproductive tissue. Although traditional therapic agents that stimulated boneformation (e.g., sodium fluoride, growth hormone, and anabolic steroides) andantiresorptive agents (e.g., calcitonin and bisphosphonates) may prevent furtherbone loss in established osteoporosis, their costs are too high to benefit a largepopulation in the developing or even the developed countries for prevention andtreatment of osteoporosis. Consequently, it is necessary to develop“natural”products or synthetic substance with less undeirable side effects that cansubstitute or reduce the need for drugs used currently.
Through thousands of years of human experimentation, belief in the safetyof“natural”products has contributed to the fairly widespread use ofcomplementary therapies among women to relieve postmenopausal symptoms. Indeed, many of commonly consumed foods, herbs and spices contain a complexarray of naturally occurring bioactive molecules called phytochemicals, whichmay confer health benefits. Soy food and isoflavones have received considerableattention for their potential role in preventing osteopenia induced by ovariectomyin rats or by menopause in women. They have been characterized as naturallyoccurring selective estrogen receptor modulators (SERMs) with similar beneficialeffects to raloxifene on bone. Very recently, attention also has focused on thepossible role of other polyphenols. Lignans, secoisolariciresinol diglycoside fromflaxseed and isotaxiresinol from Taxux yunnanensis prevented bone loss inpostmenopausal women or ovariectomized model, respectively. Arylnaphthalenelignans isolated from Machilus thunbergii increased mouse osteoblastdifferentiation by increasing ALP activity, collagen content and mineralization.Flavonoids, rutin have been shown to inhibits estrogen deficiency-induced boneloss in OVX rats, both by slowing resorption and by increasing osteoblasticactivity, resulting in increased femoral strength.
Objective
Du-Zhong, be rich in polyphenolic compounds such as lignans, phenolicacid, and flavonoids, is a kidney-tonifying herbal medicine with a long history ofsafe use for treatment of bone fractures and joint diseases in China. Consequently,the aim of the present study was to systematically evaluate the ability ofDu-Zhong cortex extract (DZCE) consumption to prevent osteoporosis inducedby ovariectomy (OVX) in rats, and isolate and purify Du-Zhong total lignans andobserve the effects of total lignans on osteoblast; discuss the mechanism of theantiosteoporotic effects of Du-Zhong on postmenopausal osteoporosis.
Methods
1. Eighty 3-month-old female Sprague-Dawley rats were used and randomly assigned into sham-operated group (SHAM) and five ovariectomy (OVX)subgroups, i.e. OVX with vehicle (OVX); OVX with 17α-ethinylestradiol (E2,25ug/kg/day); OVX with DZCE of graded doses (100, 300, or 500mg/kg/day).Daily oral administration of DZCE or E2 started on week 4 after OVX for 16weeks. The body weight of the animals was recorded weekly during theexperimental period. efore euthanizing the animals, urine and serum sampleswere collected for biochemical analysis. The femur were dissected for evaluationof bone mineral content (BMC) and bone mineral density (BMD), bonemechanical competence and trabecular microarchitectural properties.
2. Dried and powder Du-zhong cortex were refluxed with 50% alcohol. Then theextract was concentrated and subjected to AB-8 macroporous adsorptive resinseluted using distilled water, 50% alcohol. The total lignans (TL) wereconcentrated in the 50% fraction quantitativly determined using colorimetricmethod by using pinoresinol diglucoside as stand.
3. Primary cultures of rats osteoblasts were obtained by enzyme digestion andidentified by morphological features, the alkaline phosphatase staining andAlizarin Red S staining of calcified nodules.
4. The effects of TL on the proliferation and differentiation of rat osteoblast wereevaluated by the MTT method, measuring the activity of alkaline phosphatase(ALP activity) and Alizarin red staining.
5. The effects of TL on the expression of OPG and RANKL mRNA in rat primarycultured osteoblasts by RT-PCR.
Results
1. Treatment with DZCE at higher doses (300 or 500 mg/kg/day) was found to beable to significantly prevent OVX-induced decrease in biomechanical quality offemur such as maximum stress and Young's modulus. The mechanical changes were associated with the prevention of a further BMD decrease or even withsome improvements in microarchitecture. DZCE dose-dependently inhibited totalBMD decrease in the femur caused by OVX, which was accompanied by asignificant decrease in skeletal remodeling, as was evidenced by the decreasedlevels of the bone turnover markers osteocalcin (OC), alkaline phosphatese(ALP), deoxypyridinoline (DPD), and urinary Ca and P excretions.μCTanalysis of the femoral metaphysis showed that DZCE at the highest doses(500mg/ kg /day) significantly prevent decrease in bone volume/tissue volum(BV/TV), connect density (Conn.D), trabecula number (Tb.N) and trabeculathickness (Tb.Th), and increase in trabecular separation (Tb.Sp) and structuremodel index (SMI) in OVX rats. DZCE at all dose levels did not prevent theincrease in body weight induced by ovariectomy in rats. Long-term using ofDZCE had not influcence on uters, other organs and the liver and kidneyfunctions.
2. Extracted and purified Du-zhong total lignans successfully, and the purity oftotal lignans was up to 58.5 % in the dried part of 50 % ethanolic elution.
3. The cultured cells possessed exhibited the typical morphological featurehistochemical and functional properties of osteoblast.
4. The osteoblast proliferation was significantly increased dose-dependentlywhen the cells were treated with TL (30μg/mL to 500μg/mL). The activity ofALP of the osteoblasts in the presence of TL at 100μg/mL or 300μg/ml for 3days increased significantly. TL at 100μg/mL or 300μg/mL could significantlystimulate the formation of calcified nodules.
5. TL modulate the expression of OPG and RANKL mRNA in the rat primarycultured osteoblasts, the ratio of OPG/RANKL mRNA were significantlyincreased by 100μg/mL or 300μg/ml TL.
Conclusions
1. 16 weeks of DZCE treatment improve bone biomechanical quality throughmodifications of BMD, and trabecular microarchitecture without hyperplasticeffect on uterus.
2. Total lignans from Du-zhong could promote the proliferation, differentiationand of rat primary cultured osteoblasts in a dose-dependent manner, and totallignans might be one of the active constituents facilitating antiosteoporotic effectson OVX induced postmenopausal osteoporosis.
3. Total lignans from Du-zhong increased the ratio of OPG/RANKL mRNA in adose-dependent manner. It may affect the osteoclast formation and activation viathis pathway.
4. Du-Zhong, be rich in polyphenolic compounds such as lignans, can preventsthe estrogen deficiency-induced bone loss and deterioration of trabecularmicroarchitecture, thereby maintaining biomechanical competence of bone, bothby slowing down resorption and increasing osteoblastic activity. Du-Zhong andlignans might be a potential alternative medicine for treatment of postmenopausalosteoporosis.
中年以后,生理性退行性改变所致的骨质疏松(osteoporosis, OP)的发病率随年龄的增长而增加,以骨量减少、骨组织显微结构退化,骨脆性增高,诱发骨折为特征。OP是一个全球性的健康问题,严重影响中老年人的生活质量。雌激素在OP的发生过程中起着极其重要的作用,尤其是绝经后骨质疏松症(postmenopausal osteoporosis, PMO),因此,激素替代疗法(hormonereplacement therapy, HRT)一直被视为是防治PMO的有效手段。妇女健康倡议研究结果显示,HRT的长期应用具有增加患者的心血管意外事件和生殖系统肿瘤发生的风险,严重限制了HRT在PMO预防和治疗中的应用。其它的抗骨质疏松药物,包括促进骨形成和抑制骨吸收的药物,虽然具有确切的PMO预防或者治疗作用,但是长期服用不仅其价格昂贵,而且可能产生副作用。因此,迫切需要寻找安全、有效的防治OP的药物,更好地克服现有药物存在的问题。
临床实践证明,许多天然药物可以有效、安全地防治与OP相类似的临床症状。某些植物富含许多天然植物化学物质,这些物质具有重要的生理活性。大豆异黄酮是天然的选择性雌激素受体调节剂(selectiveestrogenreceptormodulators, SERMs),其对骨组织药理作用类似于雷洛昔芬,能够防止绝经妇女或卵巢切除(ovariectomy, OVX)大鼠的骨丢失。近年来,植物中所富含的多酚类物质也引起了研究者的关注。木脂素类,如从亚麻子中分离得到的闭联异松树脂醇二酯能够有效的防治妇女绝经导致的骨质丢失的发生;从红豆杉中分离得到的异紫杉脂素在动物OVX后能有效延缓骨质丢失;从红楠中分离的木脂素类能够显著的促进小鼠成骨细胞(osteoblast, OB)的增殖和分化。此外,也有研究显示黄酮类如芦丁能够有效的抑制绝经大鼠骨量的减少和骨强度的下降,可能与其抑制骨吸收和促进骨形成有关。
研究目的
杜仲是一富含木脂素、酚酸和黄酮类等化学物质的天然“补肾”良药,其防治与OP相类似的临床症状的骨症已有千余年的历史。因此,本课题在复制OVX大鼠骨质疏松模型的基础上,系统地研究了杜仲防治PMO作用;发掘、分离得到重要活性成分——杜仲总木脂素;体外培养大鼠原代成骨细胞,在细胞水平上对药物活性成分进行研究,分析杜仲防治PMO的物质基础及其机制。
研究方法
1.选取3月龄未经产雌性二级Sprague--Dawley(SD)大鼠,随机分为假手术(SHAM)组,去卵巢(OVX)组,杜仲皮提取物组(DZCE)及17β-雌二醇组(E2)。术后第4周开始灌胃给药,连续12周。期间每周观察一次大鼠体质量变化。动物处死前,收集大鼠24h尿液,麻醉动物采集血清。全部动物处死并解剖,采集以下标本并进行测定分析:观察大鼠子宫及其它脏器的体质量及子宫病理变化,分析药物对子宫及长期用药对脏器的影响;采用全自动生化分析仪对大鼠血清和尿液中的Ca、P和ALP等骨代谢指标及肝肾功能指标进行分析;采用ELISA等免疫学方法对骨转换指标尿DPD和血清OCN进行测定;采用三点弯曲实验对大鼠股骨生物力学性能进行分析;采用DEXA及μCT对股骨的BMD、BMC及干骺端骨小梁微结构特性进行分析。
2. 50%乙醇加热回流提取杜仲总木脂素,采用AB-8型大孔树脂对杜仲总木脂素进行分离纯化,依次用去离子水、50%乙醇洗脱。收集50%乙醇洗脱部分,浓缩,以松脂醇二葡糖苷作为对照品,采用比色法进行分析。
3.采用多次酶消化法进行大鼠原代成骨细胞体外培养,通过相差显微镜观察细胞形态,并用碱性磷酸酶染色和钙结节茜素红染色等方法对所获得的细胞进行鉴定。
4.采用MTT、矿化结节茜素红染色等方法,观察杜仲总木脂素对大鼠原代成骨细胞的增殖、分化及矿化过程的影响。
5.采用RT-PCR的方法观察杜仲总木脂素对大鼠原代成骨细胞OPG和RANKLmRNA表达的影响。
实验结果
1. 300和500mg/kg/day治疗剂量的DZCE连续治疗16周,能够显著的改善大鼠股骨的结构力学参数,包括骨应力和弹性模量。这种生物力学性能的改变与DZCE增加BMD和改善骨小梁微结构特性密切相关。DZCE连续治疗16周后能够剂量依赖性的抑制OVX造成的BMD的下降,同时也伴随着反映骨重建的生化指标的下降,DZCE显著降低ALP、OCN和DPD的水平,降低OVX所致的大鼠尿Ca、P的升高。μCT分析结果显示500mg/kg/day治疗剂量的DZCE显著的增加BV/TV、Tb.N、Tb.Th以及Conn.D,降低Tb.Sp和SMI指数。16周的DZCE连续治疗,未观察到子宫刺激作用,对其它脏器和肝肾功能亦未产生明显影响。
2.分离纯化杜仲总木脂素,50%乙醇洗脱液干燥后总固物中杜仲总木脂素纯度可达58.5%。
3.多次酶消化法获得数量丰富的细胞,而且具有典型的OB形态特征,碱性磷酸酶染色和矿化结节染色均呈阳性。
4.在30~500μg/mL浓度范围内,杜仲总木质素剂量依赖性地促进大鼠原代成骨细胞的增殖;100和300μg/mL杜仲总木脂素能够显著的增加大鼠原代成骨细胞ALP的活性和矿化结节形成的数量。
5.100和300μg/mL杜仲总木质素能够显著的调节大鼠原代成骨细胞OPG和RANKL基因表达,增加两者mRNA表达水平的比值达4倍以上。
结论
1.杜仲治疗16周能够通过增加大鼠股骨的BMD和改善骨小梁微结构特性进而阻止OVX导致的股骨生物力学性能的下降,与雌激素相比较具有无子宫刺激的优点。
2.杜仲总木脂素能剂量依赖性地促进大鼠原代培养成骨细胞的增殖、分化及成熟。木脂素类化合物可能是杜仲发挥抗大鼠PMO的活性成分。
3.杜仲总木脂素剂量依赖性地提高大鼠原代成骨细胞OPG/RANKL mRNA表达水平的比值,表明杜仲总木脂素可能通过调节OPG/RANKL表达对破骨细胞(osteoclast,OC)分化和成熟产生影响。
4.杜仲富含多酚类化合物——木脂素,能够有效地阻止大鼠PMO的骨量丢失、骨小梁微结构的破坏及生物力学性能的下降,这种药理学作用可能与杜仲活性成分促进OB骨形成或通过调节OB分泌OPG和RANKL来间接抑制OC的分化和成熟有关。杜仲及其活性成分木脂素类化合物可能成为防治OP的新的天然药物。
Background
The incidence of osteoporosis increases dramatically with life expectancy. Thisdisease is characterized by a reduction in bone mass and microarchitecturaldeterioration of bone tissue, resulting in skeletal fragility and susceptibility tofractures. Osteoporosis has become increasingly recognised as a major healthcareproblem which will affect the lives of a considerable number of individuals.Because hypoestrogenemia after menopause is an important cause of osteoporosis,hormone replacement therapy (HRT) was used to be a popular regime forprevention and treatment of postmenopausal osteoporosis. Ironically, data fromthe Women’s Health Initiative (WHI) Trial indicated that long-term acceptanceand/or compliance of HRT is low due to potentially malignant effects onreproductive tissue. Although traditional therapic agents that stimulated boneformation (e.g., sodium fluoride, growth hormone, and anabolic steroides) andantiresorptive agents (e.g., calcitonin and bisphosphonates) may prevent furtherbone loss in established osteoporosis, their costs are too high to benefit a largepopulation in the developing or even the developed countries for prevention andtreatment of osteoporosis. Consequently, it is necessary to develop“natural”products or synthetic substance with less undeirable side effects that cansubstitute or reduce the need for drugs used currently.
Through thousands of years of human experimentation, belief in the safetyof“natural”products has contributed to the fairly widespread use ofcomplementary therapies among women to relieve postmenopausal symptoms. Indeed, many of commonly consumed foods, herbs and spices contain a complexarray of naturally occurring bioactive molecules called phytochemicals, whichmay confer health benefits. Soy food and isoflavones have received considerableattention for their potential role in preventing osteopenia induced by ovariectomyin rats or by menopause in women. They have been characterized as naturallyoccurring selective estrogen receptor modulators (SERMs) with similar beneficialeffects to raloxifene on bone. Very recently, attention also has focused on thepossible role of other polyphenols. Lignans, secoisolariciresinol diglycoside fromflaxseed and isotaxiresinol from Taxux yunnanensis prevented bone loss inpostmenopausal women or ovariectomized model, respectively. Arylnaphthalenelignans isolated from Machilus thunbergii increased mouse osteoblastdifferentiation by increasing ALP activity, collagen content and mineralization.Flavonoids, rutin have been shown to inhibits estrogen deficiency-induced boneloss in OVX rats, both by slowing resorption and by increasing osteoblasticactivity, resulting in increased femoral strength.
Objective
Du-Zhong, be rich in polyphenolic compounds such as lignans, phenolicacid, and flavonoids, is a kidney-tonifying herbal medicine with a long history ofsafe use for treatment of bone fractures and joint diseases in China. Consequently,the aim of the present study was to systematically evaluate the ability ofDu-Zhong cortex extract (DZCE) consumption to prevent osteoporosis inducedby ovariectomy (OVX) in rats, and isolate and purify Du-Zhong total lignans andobserve the effects of total lignans on osteoblast; discuss the mechanism of theantiosteoporotic effects of Du-Zhong on postmenopausal osteoporosis.
Methods
1. Eighty 3-month-old female Sprague-Dawley rats were used and randomly assigned into sham-operated group (SHAM) and five ovariectomy (OVX)subgroups, i.e. OVX with vehicle (OVX); OVX with 17α-ethinylestradiol (E2,25ug/kg/day); OVX with DZCE of graded doses (100, 300, or 500mg/kg/day).Daily oral administration of DZCE or E2 started on week 4 after OVX for 16weeks. The body weight of the animals was recorded weekly during theexperimental period. efore euthanizing the animals, urine and serum sampleswere collected for biochemical analysis. The femur were dissected for evaluationof bone mineral content (BMC) and bone mineral density (BMD), bonemechanical competence and trabecular microarchitectural properties.
2. Dried and powder Du-zhong cortex were refluxed with 50% alcohol. Then theextract was concentrated and subjected to AB-8 macroporous adsorptive resinseluted using distilled water, 50% alcohol. The total lignans (TL) wereconcentrated in the 50% fraction quantitativly determined using colorimetricmethod by using pinoresinol diglucoside as stand.
3. Primary cultures of rats osteoblasts were obtained by enzyme digestion andidentified by morphological features, the alkaline phosphatase staining andAlizarin Red S staining of calcified nodules.
4. The effects of TL on the proliferation and differentiation of rat osteoblast wereevaluated by the MTT method, measuring the activity of alkaline phosphatase(ALP activity) and Alizarin red staining.
5. The effects of TL on the expression of OPG and RANKL mRNA in rat primarycultured osteoblasts by RT-PCR.
Results
1. Treatment with DZCE at higher doses (300 or 500 mg/kg/day) was found to beable to significantly prevent OVX-induced decrease in biomechanical quality offemur such as maximum stress and Young's modulus. The mechanical changes were associated with the prevention of a further BMD decrease or even withsome improvements in microarchitecture. DZCE dose-dependently inhibited totalBMD decrease in the femur caused by OVX, which was accompanied by asignificant decrease in skeletal remodeling, as was evidenced by the decreasedlevels of the bone turnover markers osteocalcin (OC), alkaline phosphatese(ALP), deoxypyridinoline (DPD), and urinary Ca and P excretions.μCTanalysis of the femoral metaphysis showed that DZCE at the highest doses(500mg/ kg /day) significantly prevent decrease in bone volume/tissue volum(BV/TV), connect density (Conn.D), trabecula number (Tb.N) and trabeculathickness (Tb.Th), and increase in trabecular separation (Tb.Sp) and structuremodel index (SMI) in OVX rats. DZCE at all dose levels did not prevent theincrease in body weight induced by ovariectomy in rats. Long-term using ofDZCE had not influcence on uters, other organs and the liver and kidneyfunctions.
2. Extracted and purified Du-zhong total lignans successfully, and the purity oftotal lignans was up to 58.5 % in the dried part of 50 % ethanolic elution.
3. The cultured cells possessed exhibited the typical morphological featurehistochemical and functional properties of osteoblast.
4. The osteoblast proliferation was significantly increased dose-dependentlywhen the cells were treated with TL (30μg/mL to 500μg/mL). The activity ofALP of the osteoblasts in the presence of TL at 100μg/mL or 300μg/ml for 3days increased significantly. TL at 100μg/mL or 300μg/mL could significantlystimulate the formation of calcified nodules.
5. TL modulate the expression of OPG and RANKL mRNA in the rat primarycultured osteoblasts, the ratio of OPG/RANKL mRNA were significantlyincreased by 100μg/mL or 300μg/ml TL.
Conclusions
1. 16 weeks of DZCE treatment improve bone biomechanical quality throughmodifications of BMD, and trabecular microarchitecture without hyperplasticeffect on uterus.
2. Total lignans from Du-zhong could promote the proliferation, differentiationand of rat primary cultured osteoblasts in a dose-dependent manner, and totallignans might be one of the active constituents facilitating antiosteoporotic effectson OVX induced postmenopausal osteoporosis.
3. Total lignans from Du-zhong increased the ratio of OPG/RANKL mRNA in adose-dependent manner. It may affect the osteoclast formation and activation viathis pathway.
4. Du-Zhong, be rich in polyphenolic compounds such as lignans, can preventsthe estrogen deficiency-induced bone loss and deterioration of trabecularmicroarchitecture, thereby maintaining biomechanical competence of bone, bothby slowing down resorption and increasing osteoblastic activity. Du-Zhong andlignans might be a potential alternative medicine for treatment of postmenopausalosteoporosis.
引文
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