大豆异黄酮抗骨质疏松症的实验研究及作用机制的探讨

目的 观察大豆异黄酮的抗骨质疏松作用,并探讨其作用机制。方法 (1)切除10月龄大鼠双侧卵巢,3个月后分为己烯雌酚组、模型组、大豆异黄酮高、中、低荆量组,并灌胃,每日1次,连续3个月;用切除双侧卵巢旁部分脂肪的假手术同龄大鼠作对照(对照组)。测定各组动物的骨形态学指标、血清雌二醇(E2)、血清骨源性碱性磷酸酶(BALP)、血清钙磷、骨钙磷、尿钙磷以及子宫系数。(2)用含药血清培养成骨细胞,以MTT法检测成骨细胞增殖情况。结果 (1)大豆异黄酮能明显增加去卵巢大鼠骨小梁平均宽度、单位面积骨小粱骨细胞数、骨皮质平均厚度、股骨头平均吸光度和股骨干平均吸光度;明显升高血清E水平：明显降低血清BALP值;明显增加血清钙、骨钙含量：明显增加子宫系数。(2)大豆异黄酮能明显促进成骨细胞增殖。结论 大豆异黄酮具有明显的抗骨质疏松作用,其作用机制可能与其拟雌激素作用、提高体内雌激素水平、提高机体对钙的吸收与利用、促进成骨细胞DNA合成和生长增殖有关。     

骨元肽结肠溶胶囊对去卵巢大鼠骨质疏松症的防治作用

目的探讨骨元肽结肠溶胶囊对去卵巢大鼠引起骨质疏松症的防治作用。方法采用切除大鼠双侧卵巢的方法建立大鼠骨质疏松症模型,以雌二醇(E2)作阳性对照,用放射免疫法检测大鼠血清中E2、骨钙素(BGP)、转化生长因子(TGF-β1);用生物化学的方法检测大鼠血清中碱性磷酸酶(ALK)、钙(Ca)、磷(P)和24h尿Ca、P,同时采用骨矿密度检测仪测定骨密度(BMD)。结果骨元肽结肠溶胶囊组可明显提高血清中E2、TGF-β,增加BMD,降低血清中ALK、BGP和24h尿Ca、P含量。结论骨元肽结肠溶胶囊具有提高雌激素作用,可有效抑制骨吸收,降低骨转化率,加强成骨细胞活性,增加骨密度。对去卵巢引起的大鼠骨质疏松症具有明显的防治作用。     

瘦素对绝经后骨质疏松大鼠骨髓基质细胞生物学活性影响的实验研究

目的 观察人瘦素(hLEP)对去卵巢大鼠骨髓基质细胞(BMSCs)生物学活性的影响.方法 将3月龄雌性SD大鼠双侧卵巢去除后构建绝经后骨质疏松模型.术后12周检测大鼠股骨骨密度以确定建模成功.以全骨髓法培养BMSCs,添加hELP (100 ng/mL)予以刺激,并与对照组比较.观察体外hLEP对去卵巢大鼠BMSCs细胞形态、生长情况以及碱性磷酸酶(ALP)变化的影响.结果 成功构建SD大鼠骨质疏松模型.成功分离培养了去卵巢大鼠BMSCs.经hLEP刺激7d后的BMSCs形态无明显改变,相对于对照组,实验组细胞保持较好的增殖能力,其ALP活性的表达有显著提高(P＜0.05).结论 去卵巢大鼠BMSCs经hLEP刺激后其成骨分化能力有显著增强.     

含中药骨金散血清对大鼠成骨细胞OPG、RANKL mRNA表达的影响

目的：本研究旨在探讨含中药骨金散血清对成骨细胞的增殖及OPG/RANKL系统的影响。方法：6月龄雌性大鼠48只,随机分为假手术组、模型组、骨金散低剂量组和骨金散高剂量组,每组12只,模型组、骨金散低剂量组和骨金散高剂量组大鼠建立去卵巢大鼠骨质疏松的动物模型,4周后低剂量骨金散组和高剂量骨金散组按1.8g/kg和3.6g/kg灌服骨金散,每日一次;模型组和假手术组均每天一次生理盐水灌胃。连续给药8周后处死大鼠,观察骨金散对去卵巢大鼠股骨骨密度的影响,制备含骨金散血清。体外培养成骨细胞,观察含药血清对成骨细胞的增殖和OPG、RANKL mRNA的表达的影响。结果：与假手术组相比,去卵巢大鼠股骨骨密度明显降低（P〈0.01）,低剂量的骨金散组和高剂量组大鼠股骨骨密度高于去卵巢组（P〈0.01）。低剂量的骨金散组和高剂量组血清促进成骨细胞的增殖（P〈0.05）,升高OPGmRNA表达水平（P〈0.01）,降低RANKL mRNA的表达水平（P〈0.01）,使OPGmRNA/RANKLmRNA的表达比值升高（P〈0.01）。结论：中药骨金散能增加去势大鼠BMD,促进成骨细胞增殖,增加成骨细胞OPGmRNA表达,降低RANKLmRNA表达,发挥抑制破骨细胞的骨吸收作用,这可能与其治疗骨质疏松的机制有关。     

鹰嘴豆芽素A对雌激素缺乏诱发大鼠骨质疏松症的作用

<正>骨质疏松(osteoporosis,OP)是以骨量减少,骨组织微细结构破坏,骨脆性增加为特征的一种系统性、全身性骨骼疾病,是最常见的老年性骨代谢疾病,女性因绝经后雌激素明显缺乏而易发生。鹰嘴豆芽素A(biochanin A)来源于豆科植物鹰嘴豆种子的胚芽部分,红车轴草全草等,已证实具有雌激素样作用。本研究通过去卵巢手术,建立大鼠绝经后骨质疏松动物模型,研究鹰嘴豆芽素A对去卵巢骨质疏松大     

啤酒花活性成分黄腐酚抗骨质疏松作用研究

目的 对黄腐酚在动物及成骨细胞水平上的抗骨质疏松作用进行评价。方法 采用去卵巢小鼠骨质疏松模型进行体内药效学验证。运用Elisa试剂盒及Micro-CT检测方法,对小鼠血清生化指标及股骨骨密度、骨组织形态学进行评价。同时结合成骨细胞增殖、分化和矿化水平分析,以及骨形成相关蛋白的Western blot检测,对黄腐酚抗骨质疏松作用进行系统评价。结果 在体内药效学研究中,黄腐酚可显著提高去卵巢小鼠雌激素水平,降低高骨转换率;改善骨小梁微环境,增强骨密度。在成骨细胞水平上,黄腐酚既可以促进成骨细胞增殖、碱性磷酸酶（ALP）活性以及骨矿化水平,又可以提高骨桥蛋白（OPN）、骨涎蛋白（BSP）和骨形成蛋白（BMP-2）的表达。结论 本研究首次明确了黄腐酚具有抗骨质疏松作用,为开发治疗骨质疏松的药物提供新的资源。     

黄瓜籽多肽的抗骨质疏松作用

目的:研究黄瓜籽多肽的抗骨质疏松作用.方法:利用噻唑蓝(MTT)法、碱性磷酸酶(ALK)比活性测定、茜素红染色矿化骨结节,研究黄瓜籽多肽对原代培养的成骨细胞增殖、分化的影响;体内试验:对SD大鼠实施手术去除双侧卵巢,造成骨质疏松模型,口服3种(10.0,40.0,160.0 mg·kg-1 ·d-1)剂量黄瓜籽多肽,饲...     

基于尿液代谢组学研究豆豉抗骨质疏松作用机制

目的探究豆豉对去卵巢骨质疏松大鼠骨代谢的影响及作用机制。方法将3月龄SPF级SD♀大鼠随机分为假手术组、模型组、雌二醇组及豆豉组,每组8只。手术后1周,通过灌胃将相应药物给予8周。8周后,测量每组的骨矿物质密度(BMD)和子宫指数,利用尿液代谢组学方法鉴定切除卵巢骨质疏松大鼠的生物标记物。结果(1)豆豉可明显增加模型组去卵巢大鼠股骨的BMD、子宫指数,明显降低模型组去卵巢大鼠体质量。(2)确定了与绝经后骨质疏松症密切相关的23个生物标记物及9条代谢通路。豆豉通过调节多种代谢途径,例如肌醇磷酸代谢、初级胆汁酸生物合成代谢和嘌呤代谢,可明显对12个生物标记物产生回调作用。结论豆豉能够有效干预绝经后骨质疏松症的发生和发展,该研究为预防绝经后骨质疏松症药物的研究与开发提供科学的依据。     

骨质疏松胶囊对大鼠骨质疏松症骨密度的影响

目的观察骨质疏松胶囊对实验性骨质疏松大鼠骨密度的影响。方法摘除大鼠双侧卵巢,术后4周,制造骨质疏松症模型,以骨质疏松胶囊1.0、2.0、4.0 g/kg 3个剂量灌胃去卵巢骨质疏松大鼠,连续12周,观察本品对去卵巢骨质疏松大鼠骨密度、血液生化指标的影响。结果骨质疏松胶囊明显改善去卵巢骨质疏松大鼠一般状况:中、高剂量骨质疏松胶囊显著降低骨质疏松大鼠体质量(P<0.05);明显增加骨质疏松大鼠骨密度(P<0.05);降低骨质疏松大鼠血清抗酒石酸酸性度酶5b及血清骨碱性磷酸酶活性(P<0.05,P<0.01)。结论骨质疏松胶囊具有增加去卵巢骨质疏松大鼠骨密度;抑制骨吸收、促进骨形成的作用。     

阿仑膦酸钠抗骨质疏松作用实验

目的:观察和分析阿仑膦酸钠对切除卵巢所致实验性骨质疏松大鼠的防治作用。方法:观察阿仑膦酸钠(1.8,3.6mg.kg-1.d-1)对切除卵巢所致实验性骨质疏松大鼠股骨头和股骨干部位的骨矿密度、股骨头的钙磷含量的影响。结果:与模型对照组相比,阿仑膦酸钠可明显增加切除卵巢所致实验性骨质疏松大鼠的股骨头和股骨干部位的骨矿密度,以及股骨头的钙磷含量,与模型对照组相比有显著差异。结论:阿仑膦酸钠可使切除卵巢所致实验性骨质疏松大鼠骨钙磷含量提高,骨矿密度增加,有明显的防治骨质疏松作用。     

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

Aim:

To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.

Methods:

MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.

Results:

Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation.

Conclusion:

Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.     

地黄活性成分梓醇对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响

目的检测地黄活性成分梓醇对成骨细胞株MC3T3-E1细胞增殖、分化和矿化的影响。方法制备不同浓度地黄活性成分梓醇提取液。以小鼠成骨细胞株MC3T3-E1作为药物筛选的细胞模型;用MTT法测定不同浓度的梓醇溶液的促细胞增殖作用;采用ALP活性和骨钙素定量检测分别观察不同浓度的梓醇溶液的促细胞分化作用;以Vonkos-sa钙化染色法了解不同浓度的梓醇溶液的促细胞钙化作用。结果梓醇在1×10-7～1×10-9mol·L-1浓度范围内培养24及48h促进成骨细胞株MC3T3-E1细胞增殖。梓醇在浓度1×10-5～1×10-6mol·L-148及72h提高成骨细胞株MC3T3-E1细胞内碱性磷酸酶的活性。梓醇在浓度1×10-5～1×10-6mol·L-1培养8及12d时能明显促进成骨细胞MC3T3-E1骨钙素合成和分泌。梓醇在浓度1×10-5～1×10-6mol·L-1培养19d时成骨细胞株MC3T3-E1细胞的矿化结节(mineralized bone nodular structure,MBNS)数目增多。结论梓醇可以提高成骨细胞株MC3T3-E1增殖和分化能力,梓醇可能是地黄治疗骨质疏松作用的活性成分之一。     

α-玉米赤霉醇对成骨细胞增殖、分化以及OPG和RANKL mRNA表达的影响

目的:探讨α-玉米赤霉醇(α-zearalanol,α-ZAL)对小鼠成骨样细胞MC3T3-E1增殖、分化及护骨素(osteoprotegerin,OPG)和NF-кB受体活化配体(receptor activator of nuclear factor-кB ligand,RANKL)mRNA表达的影响。方法:传代培养小鼠成骨样细胞株MC3T3-E1,采用不同浓度的α-ZAL作用于细胞72 h后,MTT法检测细胞增殖活性,PNPP法检测碱性磷酸酶(alkaline phosphatase,ALP)活性,RT-PCR法检测ALP,OPG及RANKL mRNA的表达水平。结果:10-6～10-12mol·L-1的α-ZAL可显著抑制成骨细胞的增殖(P<0.05);显著增加ALP活性(P<0.05),但不同剂量间存在作用时间差异;并且可显著上调成骨细胞内OPG/RANKL mRNA的比值(P<0.05)。结论:α-ZAL可抑制成骨细胞的增殖、促进其分化,并可通过上调OPG/RANKL mRNA表达比值抑制破骨细胞的形成,有望作为骨质疏松症的治疗药物。     

High cholesterol diet increases osteoporosis risk via inhibiting bone formation in rats

Aim:

To investigate the effects of high cholesterol diet on the development of osteoporosis and the underlying mechanisms in rats.

Methods:

Female Sprague-Dawley rats were randomly separated into 3 groups: (1) the high cholesterol fed rats were fed a high cholesterol diet containing 77% normal diet food, 3% cholesterol and 20% lard for 3 months; (2) ovariectomised (OVX) rats were bilaterally ovariectomised and fed a standard diet; and (3) the control rats were fed the standard diet. Bone mineral density (BMD) of the rats was measured using dual-energy X-ray absorptiometry. Serum levels of oestradiol (E2), osteocalcin (BGP) and carboxy-terminal collagen crosslinks (CTX) were measured using ELISA. Gene expression profile was determined with microarray. Mouse osteoblast cells (MC3T3-E1) were used for in vitro study. Proliferation, differentiation and oxidative stress of the osteoblasts were investigated using MTT, qRT-PCR and biochemical methods.

Results:

In high cholesterol fed rats, the femur BMD and serum BGP level were significantly reduced, while the CTX level was significantly increased. DNA microarray analysis showed that 2290 genes were down-regulated and 992 genes were up-regulated in this group of rats. Of these genes, 1626 were also down-regulated and 1466 were up-regulated in OVX rats. In total, 370 genes were up-regulated in both groups, and 976 genes were down-regulated. Some of the down-regulated genes were found to code for proteins involved in the transforming growth factor beta (TGF-β)/bone morphogenic protein (BMP) and Wnt signaling pathways. The up-regulated genes were found to code for IL-6 and Ager with bone-resorption functions. Treatment of MC3T3-E1 cells with cholesterol (12.5-50 μg/mL) inhibited the cell proliferation and differentiation in vitro in a concentration-dependent manner. The treatment also concentration-dependently reduced the expression of BMP2 and Cbfa1, and increased the oxidative injury in MC3T3-E1 cells.

Conclusion:

The results suggest a close correlation between hypercholesterolaemia and osteoporosis. High cholesterol diet increases the risk of osteoporosis, possible via inhibiting the differentiation and proliferation of osteoblasts.     

A synergetic role of 1,25-dihydroxyvitamin D(3) in 17β-estradial induced-proliferation and differentiation of osteoblastic MC3T3-E1 cells

Although the effect of 17β-estradial, a polyphenolic phytoestrogen, on bone cell function has been studied in numerous cell models, the synergetic role of 1, 25-dihydroxyvitamin D(3) on 17β-estradial induced-proliferation and differentiation of osteoblastic cells, and the underlying mechanism are obscure. Here, we investigated the in vitro effect of 17β-estradial on cell proliferation and osteoblastic maturation in MC3T3-E1 cells. 17β-estradial could promote the proliferation and viability of MC3T3-E1 cells, associated with upregulation of cyclin E and proliferation cell nuclear antigen (PCNA) mRNA expression, and downregulation of cyclin-dependent kinase inhibitor 2b (Cdkn2b) mRNA expression. Moreover, 17β-estradial also could stimulate osteoblastic differentiation and bone formation as assessed by alkaline phosphatase (ALP) and Alizarin Red S staining, through induction of the expression of osteoblastic markers, including ALP, osteopontin and type I collagen in MC3T3-E1 cells. However, 1,25-dihydroxyvitamin D(3) treatment alone showed no effect on proliferation and differentiation of MC3T3-E1 cells, but could coordinately augment effects of 17β-estradial on MC3T3-E1 cells. The mechanism conducted demonstrated that 17β-estradial activated ERK1/2 but not JNK and p38, and U0126, an ERK1/2 pathway inhibitor, significantly downregulated vitamin D receptor expression induced by 17β-estradial in MC3T3-E1 cells. Thus, our data demonstrated a synergistical role of 1,25-dihydroxyvitamin D(3) and 17β-estradial in proliferation and differentiation of osteoblasts, and this coordinated regulation might depend on the upregulation of vitamin D receptor in osteoblasts by 17β-estradial. Moreover, during the process of vitamin D receptor upregulation by 17β-estradial, ERK1/2 signaling is involved.     

仙葛颗粒对去卵巢骨质疏松大鼠模型的影响

目的观察仙葛颗粒对去卵巢骨质疏松大鼠模型的影响,探究其防治骨质疏松症的机制。方法将60只雌性大鼠随机分成空白对照组,模型组,仙葛颗粒低、中、高剂量组,尼尔雌醇组共6组。采用卵巢切除的方法制成绝经后骨质疏松动物模型,灌胃给药3个月后测定股骨骨密度及降钙素、骨钙素、雌二醇含量,取股骨下段松质骨进行电镜观察。结果仙葛颗粒能明显提高去卵巢大鼠模型血清降钙素、骨钙素和雌二醇水平,显著增加其骨密度值,改善松质骨的微结构。结论仙葛颗粒对骨质疏松症有防治作用。     

镁锌合金对成骨细胞增殖的影响

目的研究镁锌合金对前成骨细胞MC3T3-E1细胞增殖的影响,探讨镁锌合金成为一种新型骨科内植物材料的可行性。方法在镁锌合金金属片上接种前成骨细胞MC3T3-E1,采用MTT法及碱性磷酸酶（ALP）检测试剂盒等方法检测MC3T3-E1细胞在材料表面的增殖活性,用左旋聚乳酸对照组。结果培养第1,3天后,镁锌合金表面成骨细胞增殖与对照组没有差异（P〉0.05）,第5、7、9后,镁锌合金表面成骨细胞增殖明显高于对照组（P〈0.01）。结论镁锌合金能够促进MC3T3-E1细胞的增殖,可以初步认为具有良好的生物相容性。     

红车轴草异黄酮对去势大鼠骨质疏松影响的实验研究

目的:观察红车轴草异黄酮对绝经后骨质疏松症(PMO)的影响.方法:将雌性大鼠去势复制成PMO动物模型,并随机分为6组:即大、中、小剂量红车轴草异黄酮组、阴性对照组、尼尔雌醇组和假手术组.灌胃给药后,用放射免疫法测定大鼠血清雌三醇(E3)、雌二醇(E2)及骨钙素(BGP)水平,用定量超声法测定股骨超声振幅衰减(BUA),并测定大鼠股骨重量以计算股骨干重指数及灰重/干重.结果:红车轴草异黄酮能使去势大鼠血清E3水平升高,血清BGP水平降低,并提高股骨重量和超声振幅衰减.结论:红车轴草异黄酮通过提高血清雌激素水平,增加成骨细胞活性,降低骨高转换率以及减少骨量丢失等多种途径,能有效防治绝经后骨质疏松症.     

绝经后骨质疏松症复合药物治疗的实验研究

将成年雌性ＳＤ大鼠行双侧卵巢切除去势手术，建立绝经后骨质疏松症模型，术后应用以雌激素、海螵蛸和维生素Ｄ３等合成的复合药物治疗。１２０天后各项测试结果表明，骨密度增加，骨疏松形态改善，骨生物力学性能增强。