续断抗绝经后骨质疏松作用及其机制研究
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摘要
研究背景
原发性骨质疏松症是以骨量减少、骨的微结构退化为特征的,致使骨的脆性增加以及易于发生骨折的一种全身性骨骼疾病。在原发性骨质疏松症中,绝经后骨质疏松症(PMO)占有很大比例。原发性骨质疏松是一个全球性的健康问题,在我国骨质疏松症患者数达9000万,约占总人口的7%,其中50岁以上妇女发病率高达50%;美国国家骨质疏松症基金会最新数据显示,在美国有1000万人已经罹患骨质疏松症,同时还有3400万人正面临骨丢失而致骨质疏松的威胁。骨质疏松症的并发症相当严重,极大地影响了患者的生活质量。骨质疏松症相关性骨折不但发生率高,而且后果十分严重,甚至引起患者死亡;骨质疏松症的治疗和康复需要耗费大量的经济和卫生资源,给家庭和社会造成极大负担。长期以来,PMO的预防和治疗采用以雌激素和孕激素为主的激素替代疗法(HRT);但是多项大规模临床试验结果显示,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,某些试验甚至因此而叫停,严重限制了HRT在PMO预防和治疗中的应用。针对HRT副作用开发的选择性雌激素受体调节剂(SERMs)引起医疗工作者和患者的关注,此类药物在发挥骨保护作用的同时对生殖系统刺激很小,心血管意外和生殖系统肿瘤发生风险降低,有些还可以用来治疗某些肿瘤;也存在消化道不良反应和增加深静脉血栓发生率等危险。其它新的抗骨质疏松药物,如二膦酸盐类、鲑鱼降钙素(CT)和重组人甲状旁腺素(rhPTH)等,临床试验结果显示,具有确切的PMO预防或者治疗作用;但同样存在诸多缺点。因此,发掘更加安全、有效、经济的PMO防治药物,引起了全世界的广泛关注。中药在治疗骨质疏松症方面具有整体治疗及副作用小等优点,受到国内外广泛关注。此外,中药资源丰富、价格低廉,更适合我国的国情。
研究目的
本课题的目的旨在本实验室中药黄酮抗骨质疏松研究的基础上,对中医骨科临床常用药物续断的抗绝经后骨质疏松症作用进行系统深入的研究,发掘、分离得到重要活性成分—续断总皂苷;在体外原代培养成骨细胞模型上阐明续断防治绝经后骨质疏松症的物质基础及其机制。为续断的深入研究提供理论依据,为天然活性产物防治骨丢失、骨质疏松症提供新线索。
研究方法
1.本课题采用3月龄Sprague-Dawley大鼠,以模拟手术作为对照,行双侧卵巢切除术(OVX)建立大鼠绝经后骨质疏松症模型,以雌二醇为阳性对照药物,考察续断提取物对去卵巢引起的大鼠绝经后骨质疏松症的防治效果。OVX术后4周将动物随机分组,分别给予续断提取物100,300,500mg/kg/d和17β-雌二醇25μg/kg/d进行灌胃治疗。给药16周收集大鼠24h尿液,麻醉动物采集血清,处死动物取子宫、心脏、肝脏、脾、肺、肾、胸腺、脑和左右股骨等样本,脏器称重计算脏器系数,对子宫进行病理组织学检查。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MTS材料测试系统对大鼠股骨生物力学特性进行分析,采用骨密度仪及MicroCT对股骨的BMD、BMC及干骺端骨小梁微结构进行分析。
2. 60%乙醇回流提取续断总皂苷,D101型大孔树脂对续断总皂苷进行分离纯化,以续断皂苷Ⅵ作为对照品,采用比色法进行分析。
3.采用酶消化法分离得到大鼠原代成骨细胞,并从形态、组织化学及功能学等方面对细胞进行鉴定。
4.采用MTT、茜素红染色等方法,观察续断总皂苷对大鼠原代成骨细胞的增殖、分化及矿化过程的影响;采用RT-PCR的方法对续断总皂苷对大鼠原代成骨细胞OPG和RANKLmRNA表达的影响进行分析。
实验结果
1.续断提取物可剂量依赖性地抑制OVX引起的大鼠体重增加;降低OVX所致的大鼠尿Ca、P的升高;降低骨转换指标ALP、OCN和DPD的含量,减慢骨转换率;续断提取物还可以增加大鼠股骨BMD,改善其生物力学特性中的骨应力和弹性模量等内在特性;同时可以改善大鼠股骨干骺端骨小梁的微结构,包括增加骨组织容量,增加骨小梁的数量、厚度以及连接密度,降低骨小梁间隙和SMI指数。长期应用续断提取物进行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.成功分离纯化得到续断总皂苷,纯度为76.5%。
3.成功分离得到大鼠原代成骨细胞,鉴定结果显示,该细胞具有成骨细胞的形态学、生物化学和功能学特性。
4.续断总皂苷可剂量依赖性地促进大鼠原代成骨细胞的增殖,促进率可达180%;可剂量依赖性地提高大鼠原代成骨细胞ALP活性,最多可达2.5倍;还可增加大鼠原代成骨细胞矿化结节的形成。
5.续断皂苷可剂量依赖性地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8倍以上。
结论
1.续断对大鼠OVX所致绝经后骨质疏松症具有明显的防治作用,能对抗去势引起的大鼠体重增加,减少骨丢失、保持骨矿密度、增加骨强度、防止骨折发生,与雌激素相比较具有无子宫刺激的优点。
2.续断总皂苷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。这是续断防治绝经后骨质疏松症的物质基础,也是其抗骨质疏松症的细胞学机制。
3.续断总皂苷剂量依赖性地提高大鼠原代成骨细胞OPG/RANKLmRNA表达水平的比值,表明续断总皂苷可能通过调节OPG/RANKL表达对破骨过程产生影响。
4.本研究揭示了中药续断防治绝经后骨质疏松症的作用及其机制,为续断的临床应用提供了实验依据;发现续断皂苷防治绝经后骨质疏松作用,证明续断皂苷抗骨质疏松作用可能与其促进成骨细胞增殖、分化与成熟,并与通过调节OPG/RANKL表达对破骨过程产生影响有关。本研究不仅揭示了续断防治绝经后骨质疏松的物质基础及其机制,而且发现了续断皂苷新的作用及其潜在的作用靶点;不仅为续断及其皂苷的应用提供了实验依据,而且为发掘新的抗骨质疏松药物提供了线索。
Background
Osteoporosisisakindofsystematicskeletondiseaseinwhichbonemass isreduced, bone microstructure is degenerated, bones become fragile and morelikely to break. Postmenopausal osteoporosis is the main type of primarycultured osteoporosis. Osteoporosis has become a major public health threat allthe country of the world. Up to now, in our country, 90 million chineses or 7%of the people have osteoporosis. Almost 50% women over 50 years old wereaffected by the disease. The up-to-date data of the National OsteoporosisFoundationshows that in theUSAtoday,10 millionindividuals are estimatedtoalready have the disease and almost 34 million more are estimated to have lowbone mass, placing them at increased risk for osteoporosis. Osteoporosis oftencauses serious complications and lowers a patient’s quality of life. The rate ofosteoporosis-related fracture is very high, fractures due to osteoporosis causevery serious results even cause the death of the patient. Osteoporosis-relatedfractures cost alargeamount ofeconomicand healthresources. For a longtime,hormone replacement therapy (HRT) by estrogen and progestogen has been themain route for the prevention and treatment of postmenopausal osteoporosis. But several large scale clinical trials showed that long-term application of HRTincreased the risks of cardiovascular events and cancers of reprodutive system.Some trials were ceased for the serious side effects. This limited the applicationof HRT for the prevention and treatment of postmenopausal osteoporosis.Researchers and patients put their eyes on the selective estrogen receptormodulators (SERMs). This kind of drugs can prevent bone loss withoutstimulating effects on the reprodutive system. Also the risks of cardiovascularevents and cancers of reprodutive system are not increased. These drugs evencanbeusedtotreatsomecancers.Buttheystillhavegastrointestinalsideeffectsand can cause thrombus in deep vein. A series of new drugs such asbisphosphonates, salmon calcitonin (CT) and recombinated human parathyroidhormone (rhPTH) show good effects on the prevention or treatment of thepostmenopausal osteoporosis in clinical trials. The shortages such as only targetone site, can not be applicated in combination due to conflict mechanism, andhave very high price limit the application of these drugs. Thus alternative drugsof proven efficacy and more safety and economic should be developed for theprevention and treatment of postmenopausal osteoporosis. Traditional ChineseMedicine (TCM) treat postmenopausal osteoporosis from multiple aspects andhas less side effects. The principle of TCM is consistent with the desire ofpeople to return to the nature. What’s more TCM has more abundant resourcesand lower price compared with chemical drugs, and fit the situation of ourcountry.Objective
The objective of this subject is that observe the effects of Radix Dipsaci,which is often used in the clinical practice in orthopaedics, on OVX-induced postmenopausal osteoporosis comprehensively, systemically and scientifically;isolate and purify the Radix Dipsaci total saponins and observe the effects ofRadix Dipsaci total saponins on osteoblast; discuss the mechanism of theantiosteoporotic effects of Radix Dipsaci on postmenopausal osteoporosis;provide the theoretic witness for the advanced research of Radix Dipsaci andexperimentalevidenceforthenatureproductbasednewdrugdevelopment.
Methods
1. OVX or sham operations were performed on sixty 3-month-old virginSprague-Dawley rats. Observe the effects of Radix Dipsaci extract (RDE) onpostmenopausal osteoporosis by using 17β-estrodiol as positive control. Ratsthat were divided into six groups: sham control group (sham, n=10); OVXcontrol group (OVX, n=10); 17β-estradiol treatment group (E2, n=10); threeRadix Dipsaci extract treatment groups RDE100 (n=10), RDE300 (n=10) andRDE500 (n=10). The treatment began 4 weeks after the surgery and lasted for16 weeks. The rats received RDE 100, 300, 500mg/kg/d or 17β-estrodiol25μg/kg/d respectively. Collect 24h urine, serum, uterus, heart, liver, spleen,lung, kidney, thymus, brain and femur samples. Organs were weighed andcomputed the organ index. Biochemical parameters and bone turnover markerswere analyzed with automatic biochemical analyzer or ELISAassay. Bone masswas analyzed by DEXA, bone biomechanical properties was measured by threepoint bending test and the trabecular bone microarchitecture was evaluated byMicroCT.
2. Radix Dipsaci total saponins was isolated and purified by 60% ethanolrefluxing and D101 macroporous resin. Total saponins was analyzed bycolorimetricmethodbyusingasperosaponinⅥasstand.
3. Rat primarycultured osteoblasts were obtained byenzyme digestion andidentifiedbymorphological,histochemicalandfunctionalproperties.
4. Observed the effects of Radix Dipsaci total saponins on proliferation,differentiation and calcification of rat primary cultured osteoblasts by usingMTT assay and Alizarin Red S staining. Observed the effects of Radix Dipsacitotal saponins on the expression of OPG and RANKL mRNA in rat primaryculturedosteoblasts.
Results
1. 16 weeks treatment of RDE slowed down the body weight gain inducedby the OVX; reversed the increasing of urinary Ca, P excretion and boneturnover markers such as serum ALP, OCN and urinary DPD induced by theOVX; The treatment could also enhance the bone strength and prevent thedeterioration of trabecular microarchitecture; increased the bone volume,trabecular number, thickness and connected density, decreased the trabecularspace and SMI. The effects mentioned above were not accompanied withstimulating effects on uterus. Long-term using of RDE had not influcence on
otherorgansandtheliverandkidneyfunctions.2. Isolated and purified Radix Dipsaci total saponins successfully, and thepurityoftheRadixDipsacitotalsaponinsis76.5%.
3. Obtained the rat primary cultured osteoblasts successfully, and the cellshadthemorphological,histochemicalandfunctionalpropertiesofosteoblast.
4. Radix Dipsaci total saponins increased the proliferation of rat primaryculturedosteoblasts upto180%at thelargedosageinadose-dependent manner.Radix Dipsaci total saponins also increased ALPactivityof rat primaryculturedosteoblasts up to 2.5 folds, and also increased the amount of the calcification bonenodulesintheratprimaryculturedosteoblasts.
5. Radix Dipsaci total saponins modulate the expression of OPG andRANKL mRNA in the rat primary cultured osteoblasts, the ratio ofOPG/RANKLmRNAwasincreasedupto8folds.
Conclusions
1. Radix Dipsaci had potential antiosteoporotic effects on OVX inducedpostmenopausal osteoporosis, could slow down the body weight gain due toOVX, prevent bone loss, maintain BMD, enhance the bone strength. Comparedwithestrogenithadnostimulatingeffectsonuterus.
2. Radix Dipsaci total saponins increased the proliferation, differentiationand activation of rat primary cultured osteoblasts in a dose-dependent manner.This may be the cytological mechanism of the antiosteoporotic effects on OVXinducedpostmenopausalosteoporosis.
3.Radix Dipsaci total saponins increased theratioofOPG/RANKLmRNAin a dose-dependent manner. It may affect the osteoclast formation andactivationviathispathway.
4.Ourstudyrevealedtheantiosteoporoticeffects andmechanismsofRadixDipsacionpostmenopausalosteoporosis.Discoveredtheantiosteoporoticeffectsof Radix Dipsaci total saponins and proved that the effects were due to thepromoting effects on proliferation, differentiation and calcification ofosteoblasts. Radix Dipsaci total saponins could act on the bone resorption byregulating the OPG/RANKL system. Our study not only revealed the elementsandmechanism ofthe effects ofRadix Dipsaci onpostmenopausal osteoporosis,also found the new effects and potential target site of the Radix Dipsaci totalsaponins. Our study not only provided the experimental evidences for the application of the Radix Dipsaci and its total saponins, also gave a new clue onthedevelopmentoftheantiosteoporoticdrugs.
原发性骨质疏松症是以骨量减少、骨的微结构退化为特征的,致使骨的脆性增加以及易于发生骨折的一种全身性骨骼疾病。在原发性骨质疏松症中,绝经后骨质疏松症(PMO)占有很大比例。原发性骨质疏松是一个全球性的健康问题,在我国骨质疏松症患者数达9000万,约占总人口的7%,其中50岁以上妇女发病率高达50%;美国国家骨质疏松症基金会最新数据显示,在美国有1000万人已经罹患骨质疏松症,同时还有3400万人正面临骨丢失而致骨质疏松的威胁。骨质疏松症的并发症相当严重,极大地影响了患者的生活质量。骨质疏松症相关性骨折不但发生率高,而且后果十分严重,甚至引起患者死亡;骨质疏松症的治疗和康复需要耗费大量的经济和卫生资源,给家庭和社会造成极大负担。长期以来,PMO的预防和治疗采用以雌激素和孕激素为主的激素替代疗法(HRT);但是多项大规模临床试验结果显示,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,某些试验甚至因此而叫停,严重限制了HRT在PMO预防和治疗中的应用。针对HRT副作用开发的选择性雌激素受体调节剂(SERMs)引起医疗工作者和患者的关注,此类药物在发挥骨保护作用的同时对生殖系统刺激很小,心血管意外和生殖系统肿瘤发生风险降低,有些还可以用来治疗某些肿瘤;也存在消化道不良反应和增加深静脉血栓发生率等危险。其它新的抗骨质疏松药物,如二膦酸盐类、鲑鱼降钙素(CT)和重组人甲状旁腺素(rhPTH)等,临床试验结果显示,具有确切的PMO预防或者治疗作用;但同样存在诸多缺点。因此,发掘更加安全、有效、经济的PMO防治药物,引起了全世界的广泛关注。中药在治疗骨质疏松症方面具有整体治疗及副作用小等优点,受到国内外广泛关注。此外,中药资源丰富、价格低廉,更适合我国的国情。
研究目的
本课题的目的旨在本实验室中药黄酮抗骨质疏松研究的基础上,对中医骨科临床常用药物续断的抗绝经后骨质疏松症作用进行系统深入的研究,发掘、分离得到重要活性成分—续断总皂苷;在体外原代培养成骨细胞模型上阐明续断防治绝经后骨质疏松症的物质基础及其机制。为续断的深入研究提供理论依据,为天然活性产物防治骨丢失、骨质疏松症提供新线索。
研究方法
1.本课题采用3月龄Sprague-Dawley大鼠,以模拟手术作为对照,行双侧卵巢切除术(OVX)建立大鼠绝经后骨质疏松症模型,以雌二醇为阳性对照药物,考察续断提取物对去卵巢引起的大鼠绝经后骨质疏松症的防治效果。OVX术后4周将动物随机分组,分别给予续断提取物100,300,500mg/kg/d和17β-雌二醇25μg/kg/d进行灌胃治疗。给药16周收集大鼠24h尿液,麻醉动物采集血清,处死动物取子宫、心脏、肝脏、脾、肺、肾、胸腺、脑和左右股骨等样本,脏器称重计算脏器系数,对子宫进行病理组织学检查。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MTS材料测试系统对大鼠股骨生物力学特性进行分析,采用骨密度仪及MicroCT对股骨的BMD、BMC及干骺端骨小梁微结构进行分析。
2. 60%乙醇回流提取续断总皂苷,D101型大孔树脂对续断总皂苷进行分离纯化,以续断皂苷Ⅵ作为对照品,采用比色法进行分析。
3.采用酶消化法分离得到大鼠原代成骨细胞,并从形态、组织化学及功能学等方面对细胞进行鉴定。
4.采用MTT、茜素红染色等方法,观察续断总皂苷对大鼠原代成骨细胞的增殖、分化及矿化过程的影响;采用RT-PCR的方法对续断总皂苷对大鼠原代成骨细胞OPG和RANKLmRNA表达的影响进行分析。
实验结果
1.续断提取物可剂量依赖性地抑制OVX引起的大鼠体重增加;降低OVX所致的大鼠尿Ca、P的升高;降低骨转换指标ALP、OCN和DPD的含量,减慢骨转换率;续断提取物还可以增加大鼠股骨BMD,改善其生物力学特性中的骨应力和弹性模量等内在特性;同时可以改善大鼠股骨干骺端骨小梁的微结构,包括增加骨组织容量,增加骨小梁的数量、厚度以及连接密度,降低骨小梁间隙和SMI指数。长期应用续断提取物进行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.成功分离纯化得到续断总皂苷,纯度为76.5%。
3.成功分离得到大鼠原代成骨细胞,鉴定结果显示,该细胞具有成骨细胞的形态学、生物化学和功能学特性。
4.续断总皂苷可剂量依赖性地促进大鼠原代成骨细胞的增殖,促进率可达180%;可剂量依赖性地提高大鼠原代成骨细胞ALP活性,最多可达2.5倍;还可增加大鼠原代成骨细胞矿化结节的形成。
5.续断皂苷可剂量依赖性地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8倍以上。
结论
1.续断对大鼠OVX所致绝经后骨质疏松症具有明显的防治作用,能对抗去势引起的大鼠体重增加,减少骨丢失、保持骨矿密度、增加骨强度、防止骨折发生,与雌激素相比较具有无子宫刺激的优点。
2.续断总皂苷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。这是续断防治绝经后骨质疏松症的物质基础,也是其抗骨质疏松症的细胞学机制。
3.续断总皂苷剂量依赖性地提高大鼠原代成骨细胞OPG/RANKLmRNA表达水平的比值,表明续断总皂苷可能通过调节OPG/RANKL表达对破骨过程产生影响。
4.本研究揭示了中药续断防治绝经后骨质疏松症的作用及其机制,为续断的临床应用提供了实验依据;发现续断皂苷防治绝经后骨质疏松作用,证明续断皂苷抗骨质疏松作用可能与其促进成骨细胞增殖、分化与成熟,并与通过调节OPG/RANKL表达对破骨过程产生影响有关。本研究不仅揭示了续断防治绝经后骨质疏松的物质基础及其机制,而且发现了续断皂苷新的作用及其潜在的作用靶点;不仅为续断及其皂苷的应用提供了实验依据,而且为发掘新的抗骨质疏松药物提供了线索。
Background
Osteoporosisisakindofsystematicskeletondiseaseinwhichbonemass isreduced, bone microstructure is degenerated, bones become fragile and morelikely to break. Postmenopausal osteoporosis is the main type of primarycultured osteoporosis. Osteoporosis has become a major public health threat allthe country of the world. Up to now, in our country, 90 million chineses or 7%of the people have osteoporosis. Almost 50% women over 50 years old wereaffected by the disease. The up-to-date data of the National OsteoporosisFoundationshows that in theUSAtoday,10 millionindividuals are estimatedtoalready have the disease and almost 34 million more are estimated to have lowbone mass, placing them at increased risk for osteoporosis. Osteoporosis oftencauses serious complications and lowers a patient’s quality of life. The rate ofosteoporosis-related fracture is very high, fractures due to osteoporosis causevery serious results even cause the death of the patient. Osteoporosis-relatedfractures cost alargeamount ofeconomicand healthresources. For a longtime,hormone replacement therapy (HRT) by estrogen and progestogen has been themain route for the prevention and treatment of postmenopausal osteoporosis. But several large scale clinical trials showed that long-term application of HRTincreased the risks of cardiovascular events and cancers of reprodutive system.Some trials were ceased for the serious side effects. This limited the applicationof HRT for the prevention and treatment of postmenopausal osteoporosis.Researchers and patients put their eyes on the selective estrogen receptormodulators (SERMs). This kind of drugs can prevent bone loss withoutstimulating effects on the reprodutive system. Also the risks of cardiovascularevents and cancers of reprodutive system are not increased. These drugs evencanbeusedtotreatsomecancers.Buttheystillhavegastrointestinalsideeffectsand can cause thrombus in deep vein. A series of new drugs such asbisphosphonates, salmon calcitonin (CT) and recombinated human parathyroidhormone (rhPTH) show good effects on the prevention or treatment of thepostmenopausal osteoporosis in clinical trials. The shortages such as only targetone site, can not be applicated in combination due to conflict mechanism, andhave very high price limit the application of these drugs. Thus alternative drugsof proven efficacy and more safety and economic should be developed for theprevention and treatment of postmenopausal osteoporosis. Traditional ChineseMedicine (TCM) treat postmenopausal osteoporosis from multiple aspects andhas less side effects. The principle of TCM is consistent with the desire ofpeople to return to the nature. What’s more TCM has more abundant resourcesand lower price compared with chemical drugs, and fit the situation of ourcountry.Objective
The objective of this subject is that observe the effects of Radix Dipsaci,which is often used in the clinical practice in orthopaedics, on OVX-induced postmenopausal osteoporosis comprehensively, systemically and scientifically;isolate and purify the Radix Dipsaci total saponins and observe the effects ofRadix Dipsaci total saponins on osteoblast; discuss the mechanism of theantiosteoporotic effects of Radix Dipsaci on postmenopausal osteoporosis;provide the theoretic witness for the advanced research of Radix Dipsaci andexperimentalevidenceforthenatureproductbasednewdrugdevelopment.
Methods
1. OVX or sham operations were performed on sixty 3-month-old virginSprague-Dawley rats. Observe the effects of Radix Dipsaci extract (RDE) onpostmenopausal osteoporosis by using 17β-estrodiol as positive control. Ratsthat were divided into six groups: sham control group (sham, n=10); OVXcontrol group (OVX, n=10); 17β-estradiol treatment group (E2, n=10); threeRadix Dipsaci extract treatment groups RDE100 (n=10), RDE300 (n=10) andRDE500 (n=10). The treatment began 4 weeks after the surgery and lasted for16 weeks. The rats received RDE 100, 300, 500mg/kg/d or 17β-estrodiol25μg/kg/d respectively. Collect 24h urine, serum, uterus, heart, liver, spleen,lung, kidney, thymus, brain and femur samples. Organs were weighed andcomputed the organ index. Biochemical parameters and bone turnover markerswere analyzed with automatic biochemical analyzer or ELISAassay. Bone masswas analyzed by DEXA, bone biomechanical properties was measured by threepoint bending test and the trabecular bone microarchitecture was evaluated byMicroCT.
2. Radix Dipsaci total saponins was isolated and purified by 60% ethanolrefluxing and D101 macroporous resin. Total saponins was analyzed bycolorimetricmethodbyusingasperosaponinⅥasstand.
3. Rat primarycultured osteoblasts were obtained byenzyme digestion andidentifiedbymorphological,histochemicalandfunctionalproperties.
4. Observed the effects of Radix Dipsaci total saponins on proliferation,differentiation and calcification of rat primary cultured osteoblasts by usingMTT assay and Alizarin Red S staining. Observed the effects of Radix Dipsacitotal saponins on the expression of OPG and RANKL mRNA in rat primaryculturedosteoblasts.
Results
1. 16 weeks treatment of RDE slowed down the body weight gain inducedby the OVX; reversed the increasing of urinary Ca, P excretion and boneturnover markers such as serum ALP, OCN and urinary DPD induced by theOVX; The treatment could also enhance the bone strength and prevent thedeterioration of trabecular microarchitecture; increased the bone volume,trabecular number, thickness and connected density, decreased the trabecularspace and SMI. The effects mentioned above were not accompanied withstimulating effects on uterus. Long-term using of RDE had not influcence on
otherorgansandtheliverandkidneyfunctions.2. Isolated and purified Radix Dipsaci total saponins successfully, and thepurityoftheRadixDipsacitotalsaponinsis76.5%.
3. Obtained the rat primary cultured osteoblasts successfully, and the cellshadthemorphological,histochemicalandfunctionalpropertiesofosteoblast.
4. Radix Dipsaci total saponins increased the proliferation of rat primaryculturedosteoblasts upto180%at thelargedosageinadose-dependent manner.Radix Dipsaci total saponins also increased ALPactivityof rat primaryculturedosteoblasts up to 2.5 folds, and also increased the amount of the calcification bonenodulesintheratprimaryculturedosteoblasts.
5. Radix Dipsaci total saponins modulate the expression of OPG andRANKL mRNA in the rat primary cultured osteoblasts, the ratio ofOPG/RANKLmRNAwasincreasedupto8folds.
Conclusions
1. Radix Dipsaci had potential antiosteoporotic effects on OVX inducedpostmenopausal osteoporosis, could slow down the body weight gain due toOVX, prevent bone loss, maintain BMD, enhance the bone strength. Comparedwithestrogenithadnostimulatingeffectsonuterus.
2. Radix Dipsaci total saponins increased the proliferation, differentiationand activation of rat primary cultured osteoblasts in a dose-dependent manner.This may be the cytological mechanism of the antiosteoporotic effects on OVXinducedpostmenopausalosteoporosis.
3.Radix Dipsaci total saponins increased theratioofOPG/RANKLmRNAin a dose-dependent manner. It may affect the osteoclast formation andactivationviathispathway.
4.Ourstudyrevealedtheantiosteoporoticeffects andmechanismsofRadixDipsacionpostmenopausalosteoporosis.Discoveredtheantiosteoporoticeffectsof Radix Dipsaci total saponins and proved that the effects were due to thepromoting effects on proliferation, differentiation and calcification ofosteoblasts. Radix Dipsaci total saponins could act on the bone resorption byregulating the OPG/RANKL system. Our study not only revealed the elementsandmechanism ofthe effects ofRadix Dipsaci onpostmenopausal osteoporosis,also found the new effects and potential target site of the Radix Dipsaci totalsaponins. Our study not only provided the experimental evidences for the application of the Radix Dipsaci and its total saponins, also gave a new clue onthedevelopmentoftheantiosteoporoticdrugs.
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