雷奈酸锶对激素诱导骨质疏松大鼠的防治作用及其对骨组织OPG/RANKL mRNA和蛋白表达的实验研究
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摘要
研究背景
骨质疏松症(osteoporosis,OP)是一种以骨量减少,骨微结构破坏,骨强度降低,从而导致骨的脆性增加和易于发生骨折为特征的疾病。糖皮质激素(glucocorticoid,GC)因具有强大的抗炎、抗免疫和抗休克作用,被广泛应用于器官抑制、自身免疫疾病、炎症及肿瘤治疗等领域。但其导致的主要副作用之一—糖皮质激素性骨质疏松症(glucocorticoid-induced osteoporosis,GIOP),已成为普遍存在的临床问题。GIOP是继发性骨质疏松的首要原因,也是骨质疏松症的第3位原因。GIOP发病机制及其防治也越来越受到关注。长期大剂量使用GC不仅可抑制成骨细胞(osteoblast,OB)增殖,促进其凋亡,减少骨骼中活性成骨细胞成分,导致骨形成下降,还能增加破骨细胞(osteoclast,OC)的数量和活性,促进骨吸收,造成骨量丢失,骨质疏松。研究表明,每日接受7.5mg或者更高剂量的GC治疗3个月以上,超过50%的病人会发生严重的骨丢失,而且GC不仅降低了骨密度(bone mineral density,BMD),更重要的是它能破坏骨超微结构,造成骨强度下降导致骨折的发生。
核因子-κB受体活化因子(receptor activator of nuclear factor-κB,RANK)及其配体(receptor activator of nuclear factor-κB ligand,RANKL)和骨保护素(osteoprotegerin,OPG)是近年来发现的调控破骨细胞分化激活的细胞因子,其在骨代谢中起重要作用。成骨细胞及骨髓基质细胞表达RANKL,与破骨细胞前体细胞或破骨细胞表面上的RANK结合后,促进破骨细胞的分化和激活,并抑制破骨细胞的凋亡。由成骨细胞分泌的OPG可以与RANKL结合,竞争性抑制RANKL与RANK之间的结合,从而抑制成熟破骨细胞的生成。该系统对多种骨丢失相关疾病(骨质疏松症、类风湿关节炎、强直性脊柱炎、肿瘤的骨转移等)都具有重要意义,是极具发展前途的基因治疗靶位。
双膦酸盐是目前预防和治疗GIOP的药理学金标准,阿仑膦酸钠(alendronate,Aln)属第三代双膦酸盐,己证实其具有强大的骨吸收抑制作用,且不影响骨矿化,已成功应用于GIOP和老年性骨质疏松。雷奈酸锶(strontium ranelate,SrR)是新一代抗骨质疏松药物,具有促进骨形成和抑制骨吸收的双重作用,且具有良好的安全性和有效性,有望为骨质疏松的预防和临床治疗翻开新的一页。
目的
本研究旨在通过对激素诱导骨质疏松大鼠给予雷奈酸锶(SrR)干预下,运用骨组织形态计量学、骨显微形态学、骨密度、骨生物力学和骨生化代谢指标测定等手段来综合评价SrR对激素诱导大鼠骨质疏松症的防治作用,同时运用分子生物学技术,从基因及蛋白表达水平,研究OPG/RANKL在激素诱导骨质疏松大鼠中的表达变化,阐明其相应的分子生物学基础；SrR进行干预后,观察上述蛋白的表达变化,对其相关的作用机制进行初步探讨,为临床防治骨质疏松症的合理用药提供科学的实验依据。
材料和方法
1、模型制作：将12只3.5月龄SPF级雄性SD大鼠适应性喂养1周后,随机等分为2组：Nrm组：自由活动；Met组：皮下注射甲强龙(Methylprednisolone, Met)5mg/kg/d,每周5次；
2、在成功建模实验的基础上,将32只3.5月龄SPF级雄性SD大鼠适应性喂养1周后,随机等分为4组：Nrm组：自由活动；Met组：皮下注射甲强龙(Met)5 mg/kg/d,每周5次；Aln组：在Met组基础上给予阿仑膦酸钠1.0mg/kg/day灌胃；SrR组：在Met组基础上给予雷奈酸锶900mg/kg/day灌胃。9周后根据实验需要留取相应标本并处死实验动物。留取血标本作ALP、OC和TRACP检测；取双侧股骨、胫骨及第4、5腰椎(L4、L5),并剔除附着的软组织。右侧股骨用于骨密度测定和三点弯曲试验(行骨密度测定后),左股侧骨用于半定量Real-time-PCR检测OPG/RANKL mRNA的表达和Western-blotting检测OPG/RANKL的蛋白表达,右侧胫骨行骨形态计量学分析,左侧胫骨近端用于扫描电镜观察,L4、L5分别进行凹入试验和压缩试验。
统计方法
所有数据采用均数±标准差(x±SD)表示,采用SPSS 13.0统计软件分析,Nrm组与Met组各项指标采用组间两独立样本t检验,判断制模是否成功。治疗各组间比较采用单因素方差分析(one-way ANOVA),方差齐时组间差异采用SNK法,方差不齐时采用WELCH近似方差分析,P<0.05作为有显著性差异的检验标准。
结果
1、激素诱导大鼠骨质疏松模型的建立：本实验所建立的骨质疏松模型,Nrm组和Met组间大鼠体质量有显著性差异(P<0.001)；骨代谢生化指标中,Met组大鼠血清骨形成标志物ALP、OC含量较Nrm组降低(P<0.01),血清骨吸收标志物TRACP较Nrm组升高(P<0.01)；大鼠右侧股骨和L5的BMD,Met组较Nrm组降低(P<0.001)；反映皮质骨力学性质指标的股骨三点弯曲试验和反映松质骨力学性质指标的L4凹入试验的最大载荷、刚度,Met组均较Nrm组降低(P<0.01)。骨形态计量学显示,松质骨静态参数中%Tb.Ar、Tb.Th、Tb.N,Met组较Nrm组显著降低(P<0.01)。而Tb.Sp,Met组较Nrm组增加(P<0.01)。松质骨动态参数中代表骨形成的%L.Pm.MAR.BFRs和%O.Pm,Met组较Nrm组下降(P<0.01)；代表骨吸收的%Er.Pm,Met组较Nrm组增加(P<0.01)。上述结果表明已成功建立了激素诱导大鼠骨质疏松的动物模型。
2、雷奈酸锶对激素诱导大鼠骨质疏松症防治作用的实验研究：
(1)右侧股骨和L5 BMD测定：四组大鼠间股骨和L5的BMD均有差异(P<0.05),Met组低于Nrm组(P<0.001)；Aln组和SrR组均高于Met组(P<0.001)；SrR组高于Aln组且具有显著性差异(P<0.05)
(2)骨生物力学测定：主要反映皮质骨力学特性的股骨三点弯曲试验中,四组大鼠间最大载荷、刚度均有差异(P<0.05)。Met组最大载荷、刚度均低于Nrm组(P<0.001);Aln组最大载荷、刚度与Met组比较均无显著差异(P>0.05)；SrR组最大载荷、刚度均高于Met组(P<0.001)；SrR组最大载荷、刚度均高于Aln组,差异具有统计学意义(P<0.05)。反映松质骨力学特性的腰椎压缩试验中,四组大鼠间最大载荷和刚度均有差异(P<0.05)。Met组最大载荷和刚度较Nrm组均降低(P<0.001),Aln组最大载荷和刚度较Met组增加(P<0.05)；SrR组最大载荷和刚度较Met组增加(P<0.05);Aln组与SrR组比较各指标无显著性差异(P>0.05)。反映松质骨力学特性的腰椎凹入试验中,四组大鼠间最大载荷和刚度均有差异(P<0.05)。Met组最大载荷和刚度较Nrm组均降低(P<0.001);Aln组最大载荷和刚度较Met组增加(P<0.05)；SrR组最大载荷和刚度较Met组增加(P<0.05);Aln组与SrR组比较各指标无显著性差异(P>0.05)。
(3)骨转换指标检测：四组大鼠间血清ALP.OC和TRACP均有差异(P<0.05)。Met组ALP、OC均低于Nrm组(P<0.001);Aln组ALP.OC与Met组比较无显著性差异(P>0.05)；SrR组ALP.OC均高于Met组(P<0.05);Met组血清TRACP较Nrm组升高(P<0.001),Aln组和SrR组TRACP均低于Met组(P<0.05),Aln与SrR组各指标间均无显著性差异(P>0.05)。
(4)胫骨扫描电镜观察：Nrm组大鼠胫骨近端松质骨丰富,骨小梁数目较多,小梁壁厚、均匀,间隙较小,相互连接成密集的立体网状结构。高倍镜下骨胶原纤维走向清晰,排列整齐。Met组较Nrm组骨髓腔扩大,骨小梁变细,数量减少,排列稀疏,小梁间距增宽,立体网状结构被破坏,变细的骨小梁较多见,高倍镜下骨胶原纤维排列杂乱、结构松散。Aln组和SrR组胫骨骨小梁数目较Met组增多,网状结构尚在。高倍镜下骨胶原纤维排列较Met组规整、紧密,走向较清晰。
(5)胫骨形态计量学的分析：
①对松质骨骨形态计量学指标的影响：四组大鼠间松质骨静态学指标均有差异(P<0.05)。Met组的静态学指标%Tb.Ar、Tb.Sp和Tb.N较Nrm组均降低(P<0.01)；Aln组各指标较Met组显著增加(P<0.05);SrR组较Met组显著增加(P<0.05);Aln组与SrR组比较各指标间无显著性差异(P>0.05)。静态学指标Tb.Sp,Met组较Nrm组显著增加(P<0.001);Aln组较Met组显著降低(P<0.01)；SrR组较Met组显著降低(P<0.01);Aln组与SrR组比较各指标无显著性差异(P>0.05)。四组大鼠间松质骨动态学指标均有差异(P<0.05)。Met组动态学指标MS/BS、MAR和BFRs较Nrm组均降低(P<0.001); Aln组各指标与Met组比较无显著性差异(P>0.05)；SrR组较Met组增加(P<0.05);SrR组MS/BS较Aln组升高(P<0.05)。动态学指标ES/BS,Met组较Nrm组增加(P<0.001);Aln组和SrR组均较Met组降低(P<0.01)；Aln组与SrR组比较各指标无显著性差异(P>0.05)。
②对皮质骨骨形态计量学指标的影响：四组大鼠间皮质骨形态计量学静态参数及骨外膜动态参数均有差异(P<0.05)。Met组计量学指标%Ct.Ar、MS/BS、MAR、BFRs较Nrm组均降低(P<0.01),Aln组各指标与Met组比较无显著性差异(P>0.05)；SrR组各指标较Met组增加(P<0.05);Aln组与SrR组比较各指标间均无显著性差异(P>0.05)。静态指标%Ma.Ar,Met组较Nrm组增高(P<0.01),Aln组和SrR组与Met组比较均无显著性差异(P>0.05);Aln组与SrR组比较两者无显著性差异(P>0.05)。四组大鼠间胫骨皮质骨骨内膜动态参数均有差异(P<0.05)。Met组计量学指标MS/BS、MAR和BFRs与Nrm组比较均无显著性差异(P>0.05),Aln组MAR和BFRs较Met组均降低(P<0.05)；SrR组MS/BS和MAR较Met组增加(P<0.05)。Aln组与SrR组MS/BS、MAR和BFRs比较均有显著性差异(P<0.01)。骨吸收指标ES/BS,Met组较Nrm组增高(P<0.01),Aln组和SrR组较Met组均降低(P<0.01).Aln组与SrR组比较两者无显著性差异(P>0.05)。
(6)RT-PCR和Western-blot检测大鼠骨组织中OPG和RANKL的mRNA及蛋白表达：
①对OPG mRNA表达的影响：大鼠骨组织可分别于423bp(OPG)、146bp(β-actin)见到条带,与设计引物相符。半定量RT-PCR显示：各组大鼠的OPG mRNA表达水平均有差异(P<0.001)。与Nrm组比较,Met组OPG mRNA表达水平显著降低(P<0.001),SrR组较Met组显著上调OPG mRNA表达水平(P<0.001)。
②对RANKL mRNA表达的影响：大鼠骨组织可分别于140bp(RANKL)、146bp (β-actin)见到条带,与设计引物相符。半定量RT-PCR显示：各组大鼠的RANKL mRNA表达水平均有差异(P<0.001)。与Nrm组比较,Met组RANKL mRNA表达水平显著降低(P<0.001),SrR组较Met组显著上调RANKL mRNA的表达(P<0.001)。
③OPG Western印迹杂交结果：大鼠骨组织可检测到OPG的蛋白表达,Western blot灰度值结果显示：各组大鼠OPG的蛋白表达水平均有差异(P<0.001)。与Nrm组比较,Met组OPG蛋白表达水平显著降低(P<0.001),SrR组OPG蛋白表达水平较Met组显著升高(P<0.001)。
④RANKL Western印迹杂交结果：大鼠骨组织可检测到RANKL的蛋白表达,Western blot结果分析显示：各组大鼠RANKL的蛋白表达水平均有差异(P<0.001)。与Nrm组比较,Met组RANKL蛋白表达水平显著升高(P<0.001),SrR组RANKL蛋白表达水平较Met组显著降低(P<0.001)。
结论
1、糖皮质激素(Methylprednisolone)5mg/kg/d,一周5次,干预9周后,能显著诱导大鼠骨量丢失、微结构退变,骨质量降低,呈现低转换率骨质疏松,且具有骨形成显著降低,而骨吸收显著增加的特点。表明已成功建立了激素诱导大鼠骨质疏松的动物模型。
2、给予大鼠Aln 1.0mg/kg/d和SrR 900mg/kg/d灌胃,在提高激素诱导骨质疏松大鼠的骨密度,改善骨超微结构和骨强度,抑制骨吸收,降低骨转换率,延缓骨丢失方面起到一定的干预作用：另外,SrR在改善骨的矿化和促进骨形成方面效果优于Aln。
3、正常大鼠骨组织OPG和RANKL在基因及蛋白水平均有表达；在糖皮质激素诱导骨质疏松大鼠的骨组织中,OPG mRNA和蛋白表达水平均显著下降,RANKL mRNA和蛋白表达水平均显著升高；SrR能够显著上调大鼠骨组织OPGmRNA和蛋白表达水平,同时下调RANKL mRNA和蛋白表达水平,影响OPG/RANKL系统,从而起到防治骨质疏松的作用,可能是其改善激素诱导骨质疏松的重要作用机理之一。
Background
Osteoporosis(OP) is considered as dysostosis disorder due to the damage of microstructure of bony tissues, the persistent decreasing of bony matrixes and mineral bones,thinness of sclerotin, the decrease of the number of trabecular bones, and the increase of bony fragility and the risk of fracture. Glucocorticoids(GC) are valuable and indispensable therapeutic agents in the management of many diseases such as asthma, rheumatic diseases, and connective tissue diseases.GC impair the proliferation, differentiation, and function of osteoblasts and induce the apoptosis of mature osteoblasts and osteocytes, also favor in osteoclastogenesis and as a consequence increase bone resorption. Half a percent of the population takes oral GC, making GC-induced osteoporosis(GIOP) is the most common and serious side effect for patients receiving GC.GIOP is characterized by disturbed bone remodelling associated with reduced bone mass and abnormally low quality of the bone tissue, resulting in loss of both cortical and cancellous bone, thus increasing the risk of fracture.
Receptor activator of nuclear factor-KB(RANK),receptor activator of nuclear factor-KB ligand(RANKL) and osteoprotegerin(OPG) are the cytokines which regulate the differentiation and activation of osteoclast. RANKL/RANK/OPG system plays a vital role in bone metabolism. RANKL binds to its receptor, RANK, can induce differentiation and activation of osteoclast and inhibit steoclast apoptosis. This process can be blocked by OPG, which can decrease bone destruction. The RANKL/RANK/OPG system is implicated in many bone loss diseases.This system could be performed as a therapeutic target.
Bisphosphonates are considered to be the pharmacologic gold standard of care for the prevention and treatment of GIOP. Aln is a member of the third generation of modified pyrophosphates, which is a potent antiresorptive agents; both have been approved and are in use for treatment of various disorders characterized by increased osteoclast-mediated bone resorption. GIOP and male osteoporosis are also treated with Aln successfully. SrR is a novel anti-osteoporotic treatment with a dual mode of action, both increasing bone formation and decreasing bone resorption, which rebalances bone turnover in favor of bone formation and increases bone strength. Therefore, SrR might be applicable as a bone therapeutic agent to treat secondary osteoporosis in the clinic.
Objective
To study the effects of administration Strontium ranelate(SrR) on the cancellous and cortical bone in glucocorticoid(GC)-treated rats by means of bone micromorphology, DEXA, biomechanics and histomorphometry, bone turnover marker, and observe the expression of OPG/RANKL mRNA and protein activity in the left femur, as well as to provide futher experimental evidence for exploring the mechanism of SrR.
Methods
In successful model experiment foundation, thirty-two 3.5-month male Sprague-Dawley rats were randomized into four groups:age-matched normal control(Nrm), methylprednisolone(Met) (5.0mg/kg, sc, per day for 5 days/week), Met plus Aln orally(1.0 mg/kg/d) and Met plus SrR orally(900 mg/kg/d).The study period was 9 weeks. Blood samples were obtained before sacrifice to determine the serum ALP, OC and TRACP.DEXA was evaluated in the right femoral diaphysis and fifth lumbar vertebrae(L5), after measuring BMD, the right femur was prepared for shaft three-point bending test, L4 and L5 were prepared for indentation and compression test. Histomorphometry was performed in the right proximal tibial metaphysic and tibial diaphysis, RT-PCR and Western-blot were detected in the left femur, SEM was performed in the left tibia.
Statistical methods
All data were reported as mean±standard error, SPSS13.0 software was used for these analyses.Group Nrm and group Met comparison were run using independent-samples T test, and when a significant difference was revealed(P<0.05);group Aln and group SrR assays performed were analyzed by one-way ANOVA,when significance was revealed(P<0.05).
Results
1.Osteoporosis model established by simulated weightlessness:All rats gained substantial body weight, significant differences were found in two groups(P<0.001). Serum ALP and OC were significantly lower in Met than in Nrm(P<0.01),serum TRACP was significantly higher in Met than in Nrm(P<0.01);the BMD of the right femur and L5 were significantly lower in Met than in Nrm(P<0.001);the max load and rigidity were significantly lower in Met than in Nrm(P<0.05).Quantitative histomorphometry,%Tb.Ar, Tb.Th and Tb.N were significantly lower in Met than in Nrm(P<0.01).Tb.Sp was significantly higher in Met than in Nrm(P<0.01). %L.Pm、MAR、BFRs and %O.Pm were significantly lower in Met than in Nrm(P< 0.01);%E.Pm was significantly higher in Met than in Nrm(P<0.01).
2.Effects of SrR on osteoporosis in GC-treated rats:
(1)In the right femur and L5 BMD were found significant differences in all groups(P <0.05).Met was significantly lower than Nrm group(P<0.001);Aln and SrR were significantly higher than Met group(P<0.001).It should be noted that SrR led to significantly higher in the right femur and L5 BMD compared with the Aln group(P <0.05).
(2) The cortical bone biomechanical property such as the max load and rigidity of femoral shaft three-point bending test, four groups were found significant differences (P<0.05).Met was significantly lower than in Nrm group(P<0.001);there were no significant difference between Aln and Met groups(P>0.05);SrR was significantly higher than in Met group(P<0.001).It should be noted that SrR led to significantly higher compared with Aln group(P<0.05).The cancellous bone biomechanical property like the max load and rigidity of vertebral body(L4) indentation were found significant differences in four groups(P<0.05).Met was significantly lower than in Nrm group(P<0.01);Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05);there were no significant difference between Aln and SrR groups(P>0.05).The max load and rigidity of vertebral body(L5) in the compression test were found significant differences in four groups(P<0.05).Met was significantly lower than in Nrm(P<0.01);Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05); there were no significant difference between Aln and SrR groups(P>0.05).
(3)Bone turnover marker:Serum ALP, OC and TRACP,significant differences were found in four groups(P<0.05).Serum OC and ALP were significantly lower in Met than in Nrm group(P<0.001),serum TRACP was significantly higher in Met than in Nrm group(P<0.001);there were no significant difference between Aln and Met groups(P>0.05); SrR was significantly higher than in Met group(P<0.05),but lower in Nrm group(P<0.05);serum TRACP was significantly lower in Aln and SrR than in Met group(P<0.05), but higher in Nrm group(P<0.05);there were no significant difference between Aln and SrR groups(P>0.05).
(4) In SEM observation, trabecular bone of the left tibia was found with normal network structure and collagenous fibers with regular arrangement in Nrm group; trabecular bone of the left tibia was manifested with fewness, fragility, microcrack and collagen fibers displaced by absorption lacuna in Met group;trabecular bone getting more, network structure getting better and collagenous fiber were much more regular in Aln and SrR groups.
(5) Quantitative histomorphometry
①Histomorphometric indices in the proximal tibia metaphysic: Static histomorphometric indices(%Tb.Ar、Tb.Sp、Tb.N and Tb.Sp) in cancellous bone, differences were found in four groups(P<0.05).Met significantly decreased %Tb.Ar、Tb.Sp and Tb.N compared with the Nrm group(P<0.01).Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05);there were no significant differences between Aln and SrR groups(P>0.05).Tb.Sp, Met was significantly higher than in Nrm group(P<0.001), Aln was significantly lower than in Met group(P<0.01);SrR was significantly lower than in Met groups<0.01);there were no significant difference between Aln and SrR groups(P>0.05).Dynamic histomorphometric indices(MS/BS、MAR、BFRs and ES/BS),differences were found in four groups(P<0.05).Met significantly decreased MS/BS、MAR and BFR/BS compared with the Nrm group(P<0.001).there were no significant differences between Aln and Met groups(P>0.05);SrR was significantly higher than in Met group(P<0.05).It should be noted that SrR led to higher in MS/BS compared with the Aln group(P<0.05).ES/BS,Met was significantly higher than in Nrm group(P<0.001),Aln was significantly lower than in Met group(P< 0.01);SrR was significantly lower than in Met group(P<0.01);there were no significant difference between Aln and SrR groups(P>0.05).
②Histomorphometric indices in the tibial diaphysis: Static histomorphometric indices(%Ct.Ar and%Ma.Ar) in cortical bone and (MS/BS, MAR and BFRs)in periosteum, differences were found in four groups(P<0.05).Met significantly decreased Ct.Ar、MS/BS、MAR and BFRs compared with the Nrm group(P<0.01).there were no significant difference between Aln and Met groups(P >0.05); SrR was significantly higher than in Met group(P<0.05); there were no significant differences between Aln and SrR groups(P>0.05).%Ma.Ar, Met was significantly higher than in Nrm groups<0.01);there were no significant difference between Aln and Met groups(P>0.05), and no significant difference between SrR and Met groups(P>0.05), and no significant difference between SrR and Aln groups(P>0.05).Dynamic histomorphometric indices (MS/BS,MAR, BFRs and ES/BS)in periosteum, significant differences were found in four groups(P<0.05). Dynamic histomorphometric indices (MS/BS,MAR and BFRs), there were no significant differences between Met and Nrm groups(P>0.05).For MAR and BFRs, Aln was lower than in Met group(P<0.05); for MAR and MS/BS,SrR was higher than in Met group(P<0.05).Furthermore, SrR led to a significantly higher in MS/BS, MAR, BFR/BS compared with the Aln group group(P<0.01).ES/BS,Met was significantly higher than in Nrm group(P<0.01),Aln was significantly lower than in Met group(P<0.01);SrR was significantly lower than in Met group(P<0.01);there were no significant differences between Aln and SrR groups(P>0.05).
(6) Effects of Strontium ranelate on OPG/RANKL related changes in protein activity in GC-treated rats
①RT-PCR assay:the expression levels of OPG mRNA and RANKL mRNA, differences were found in three groups(P<0.001),Met significantly decreased OPG mRNA and RANKL mRNA compared with the Nrm group(P<0.001).SrR was significantly higher than in Met group(P<0.001).
②Western blot detection:OPG and RANKL protein expression, differences were found in three groups(P<0.001),Met significantly decreased OPG and RANKL protein expression compared with the Nrm group(P<0.001).SrR was significantly higher than in Met group(P<0.001).
Conclusion
1.The effect of long-term(9 weeks) GC administration, employing a dose equivalent to 5.0mg/kg, sc, per day for 5 days/week, the model of GC-induced osteoporosis characterized by a severe increasing bone formation and decreasing bone resorption, a decrease in BMD, and bone strength. The model was established successfully.
2.SrR at the dose of 900mg/kg/d has more effective than Aln 1.0mg/kg/d in stimulate an anabolic bone response, and improve the resistance of bone over load to fracture in GC-treated rats.SrR has mildly effective in stimulate an anabolic bone response than Aln in prevention GC-induced osteopenia, suggesting the possibility that the SrR may be applicable to use as a bone therapeutic agent to treat secondary osteoporosis in clinic.
3.The level of gene and protein expression of OPG/RANKL in the normal rat bone. The GC administration decreased bone OPG gene and protein expression, increased RANKL gene and protein expression; SrR increased bone OPG gene and protein expression as well as decreased RANKL gene and protein expression, one of the mechanism maybe that the SrR could regulate OPG and RANKL in GC-induced osteoporosis.
骨质疏松症(osteoporosis,OP)是一种以骨量减少,骨微结构破坏,骨强度降低,从而导致骨的脆性增加和易于发生骨折为特征的疾病。糖皮质激素(glucocorticoid,GC)因具有强大的抗炎、抗免疫和抗休克作用,被广泛应用于器官抑制、自身免疫疾病、炎症及肿瘤治疗等领域。但其导致的主要副作用之一—糖皮质激素性骨质疏松症(glucocorticoid-induced osteoporosis,GIOP),已成为普遍存在的临床问题。GIOP是继发性骨质疏松的首要原因,也是骨质疏松症的第3位原因。GIOP发病机制及其防治也越来越受到关注。长期大剂量使用GC不仅可抑制成骨细胞(osteoblast,OB)增殖,促进其凋亡,减少骨骼中活性成骨细胞成分,导致骨形成下降,还能增加破骨细胞(osteoclast,OC)的数量和活性,促进骨吸收,造成骨量丢失,骨质疏松。研究表明,每日接受7.5mg或者更高剂量的GC治疗3个月以上,超过50%的病人会发生严重的骨丢失,而且GC不仅降低了骨密度(bone mineral density,BMD),更重要的是它能破坏骨超微结构,造成骨强度下降导致骨折的发生。
核因子-κB受体活化因子(receptor activator of nuclear factor-κB,RANK)及其配体(receptor activator of nuclear factor-κB ligand,RANKL)和骨保护素(osteoprotegerin,OPG)是近年来发现的调控破骨细胞分化激活的细胞因子,其在骨代谢中起重要作用。成骨细胞及骨髓基质细胞表达RANKL,与破骨细胞前体细胞或破骨细胞表面上的RANK结合后,促进破骨细胞的分化和激活,并抑制破骨细胞的凋亡。由成骨细胞分泌的OPG可以与RANKL结合,竞争性抑制RANKL与RANK之间的结合,从而抑制成熟破骨细胞的生成。该系统对多种骨丢失相关疾病(骨质疏松症、类风湿关节炎、强直性脊柱炎、肿瘤的骨转移等)都具有重要意义,是极具发展前途的基因治疗靶位。
双膦酸盐是目前预防和治疗GIOP的药理学金标准,阿仑膦酸钠(alendronate,Aln)属第三代双膦酸盐,己证实其具有强大的骨吸收抑制作用,且不影响骨矿化,已成功应用于GIOP和老年性骨质疏松。雷奈酸锶(strontium ranelate,SrR)是新一代抗骨质疏松药物,具有促进骨形成和抑制骨吸收的双重作用,且具有良好的安全性和有效性,有望为骨质疏松的预防和临床治疗翻开新的一页。
目的
本研究旨在通过对激素诱导骨质疏松大鼠给予雷奈酸锶(SrR)干预下,运用骨组织形态计量学、骨显微形态学、骨密度、骨生物力学和骨生化代谢指标测定等手段来综合评价SrR对激素诱导大鼠骨质疏松症的防治作用,同时运用分子生物学技术,从基因及蛋白表达水平,研究OPG/RANKL在激素诱导骨质疏松大鼠中的表达变化,阐明其相应的分子生物学基础；SrR进行干预后,观察上述蛋白的表达变化,对其相关的作用机制进行初步探讨,为临床防治骨质疏松症的合理用药提供科学的实验依据。
材料和方法
1、模型制作：将12只3.5月龄SPF级雄性SD大鼠适应性喂养1周后,随机等分为2组：Nrm组：自由活动；Met组：皮下注射甲强龙(Methylprednisolone, Met)5mg/kg/d,每周5次；
2、在成功建模实验的基础上,将32只3.5月龄SPF级雄性SD大鼠适应性喂养1周后,随机等分为4组：Nrm组：自由活动；Met组：皮下注射甲强龙(Met)5 mg/kg/d,每周5次；Aln组：在Met组基础上给予阿仑膦酸钠1.0mg/kg/day灌胃；SrR组：在Met组基础上给予雷奈酸锶900mg/kg/day灌胃。9周后根据实验需要留取相应标本并处死实验动物。留取血标本作ALP、OC和TRACP检测；取双侧股骨、胫骨及第4、5腰椎(L4、L5),并剔除附着的软组织。右侧股骨用于骨密度测定和三点弯曲试验(行骨密度测定后),左股侧骨用于半定量Real-time-PCR检测OPG/RANKL mRNA的表达和Western-blotting检测OPG/RANKL的蛋白表达,右侧胫骨行骨形态计量学分析,左侧胫骨近端用于扫描电镜观察,L4、L5分别进行凹入试验和压缩试验。
统计方法
所有数据采用均数±标准差(x±SD)表示,采用SPSS 13.0统计软件分析,Nrm组与Met组各项指标采用组间两独立样本t检验,判断制模是否成功。治疗各组间比较采用单因素方差分析(one-way ANOVA),方差齐时组间差异采用SNK法,方差不齐时采用WELCH近似方差分析,P<0.05作为有显著性差异的检验标准。
结果
1、激素诱导大鼠骨质疏松模型的建立：本实验所建立的骨质疏松模型,Nrm组和Met组间大鼠体质量有显著性差异(P<0.001)；骨代谢生化指标中,Met组大鼠血清骨形成标志物ALP、OC含量较Nrm组降低(P<0.01),血清骨吸收标志物TRACP较Nrm组升高(P<0.01)；大鼠右侧股骨和L5的BMD,Met组较Nrm组降低(P<0.001)；反映皮质骨力学性质指标的股骨三点弯曲试验和反映松质骨力学性质指标的L4凹入试验的最大载荷、刚度,Met组均较Nrm组降低(P<0.01)。骨形态计量学显示,松质骨静态参数中%Tb.Ar、Tb.Th、Tb.N,Met组较Nrm组显著降低(P<0.01)。而Tb.Sp,Met组较Nrm组增加(P<0.01)。松质骨动态参数中代表骨形成的%L.Pm.MAR.BFRs和%O.Pm,Met组较Nrm组下降(P<0.01)；代表骨吸收的%Er.Pm,Met组较Nrm组增加(P<0.01)。上述结果表明已成功建立了激素诱导大鼠骨质疏松的动物模型。
2、雷奈酸锶对激素诱导大鼠骨质疏松症防治作用的实验研究：
(1)右侧股骨和L5 BMD测定：四组大鼠间股骨和L5的BMD均有差异(P<0.05),Met组低于Nrm组(P<0.001)；Aln组和SrR组均高于Met组(P<0.001)；SrR组高于Aln组且具有显著性差异(P<0.05)
(2)骨生物力学测定：主要反映皮质骨力学特性的股骨三点弯曲试验中,四组大鼠间最大载荷、刚度均有差异(P<0.05)。Met组最大载荷、刚度均低于Nrm组(P<0.001);Aln组最大载荷、刚度与Met组比较均无显著差异(P>0.05)；SrR组最大载荷、刚度均高于Met组(P<0.001)；SrR组最大载荷、刚度均高于Aln组,差异具有统计学意义(P<0.05)。反映松质骨力学特性的腰椎压缩试验中,四组大鼠间最大载荷和刚度均有差异(P<0.05)。Met组最大载荷和刚度较Nrm组均降低(P<0.001),Aln组最大载荷和刚度较Met组增加(P<0.05)；SrR组最大载荷和刚度较Met组增加(P<0.05);Aln组与SrR组比较各指标无显著性差异(P>0.05)。反映松质骨力学特性的腰椎凹入试验中,四组大鼠间最大载荷和刚度均有差异(P<0.05)。Met组最大载荷和刚度较Nrm组均降低(P<0.001);Aln组最大载荷和刚度较Met组增加(P<0.05)；SrR组最大载荷和刚度较Met组增加(P<0.05);Aln组与SrR组比较各指标无显著性差异(P>0.05)。
(3)骨转换指标检测：四组大鼠间血清ALP.OC和TRACP均有差异(P<0.05)。Met组ALP、OC均低于Nrm组(P<0.001);Aln组ALP.OC与Met组比较无显著性差异(P>0.05)；SrR组ALP.OC均高于Met组(P<0.05);Met组血清TRACP较Nrm组升高(P<0.001),Aln组和SrR组TRACP均低于Met组(P<0.05),Aln与SrR组各指标间均无显著性差异(P>0.05)。
(4)胫骨扫描电镜观察：Nrm组大鼠胫骨近端松质骨丰富,骨小梁数目较多,小梁壁厚、均匀,间隙较小,相互连接成密集的立体网状结构。高倍镜下骨胶原纤维走向清晰,排列整齐。Met组较Nrm组骨髓腔扩大,骨小梁变细,数量减少,排列稀疏,小梁间距增宽,立体网状结构被破坏,变细的骨小梁较多见,高倍镜下骨胶原纤维排列杂乱、结构松散。Aln组和SrR组胫骨骨小梁数目较Met组增多,网状结构尚在。高倍镜下骨胶原纤维排列较Met组规整、紧密,走向较清晰。
(5)胫骨形态计量学的分析：
①对松质骨骨形态计量学指标的影响：四组大鼠间松质骨静态学指标均有差异(P<0.05)。Met组的静态学指标%Tb.Ar、Tb.Sp和Tb.N较Nrm组均降低(P<0.01)；Aln组各指标较Met组显著增加(P<0.05);SrR组较Met组显著增加(P<0.05);Aln组与SrR组比较各指标间无显著性差异(P>0.05)。静态学指标Tb.Sp,Met组较Nrm组显著增加(P<0.001);Aln组较Met组显著降低(P<0.01)；SrR组较Met组显著降低(P<0.01);Aln组与SrR组比较各指标无显著性差异(P>0.05)。四组大鼠间松质骨动态学指标均有差异(P<0.05)。Met组动态学指标MS/BS、MAR和BFRs较Nrm组均降低(P<0.001); Aln组各指标与Met组比较无显著性差异(P>0.05)；SrR组较Met组增加(P<0.05);SrR组MS/BS较Aln组升高(P<0.05)。动态学指标ES/BS,Met组较Nrm组增加(P<0.001);Aln组和SrR组均较Met组降低(P<0.01)；Aln组与SrR组比较各指标无显著性差异(P>0.05)。
②对皮质骨骨形态计量学指标的影响：四组大鼠间皮质骨形态计量学静态参数及骨外膜动态参数均有差异(P<0.05)。Met组计量学指标%Ct.Ar、MS/BS、MAR、BFRs较Nrm组均降低(P<0.01),Aln组各指标与Met组比较无显著性差异(P>0.05)；SrR组各指标较Met组增加(P<0.05);Aln组与SrR组比较各指标间均无显著性差异(P>0.05)。静态指标%Ma.Ar,Met组较Nrm组增高(P<0.01),Aln组和SrR组与Met组比较均无显著性差异(P>0.05);Aln组与SrR组比较两者无显著性差异(P>0.05)。四组大鼠间胫骨皮质骨骨内膜动态参数均有差异(P<0.05)。Met组计量学指标MS/BS、MAR和BFRs与Nrm组比较均无显著性差异(P>0.05),Aln组MAR和BFRs较Met组均降低(P<0.05)；SrR组MS/BS和MAR较Met组增加(P<0.05)。Aln组与SrR组MS/BS、MAR和BFRs比较均有显著性差异(P<0.01)。骨吸收指标ES/BS,Met组较Nrm组增高(P<0.01),Aln组和SrR组较Met组均降低(P<0.01).Aln组与SrR组比较两者无显著性差异(P>0.05)。
(6)RT-PCR和Western-blot检测大鼠骨组织中OPG和RANKL的mRNA及蛋白表达：
①对OPG mRNA表达的影响：大鼠骨组织可分别于423bp(OPG)、146bp(β-actin)见到条带,与设计引物相符。半定量RT-PCR显示：各组大鼠的OPG mRNA表达水平均有差异(P<0.001)。与Nrm组比较,Met组OPG mRNA表达水平显著降低(P<0.001),SrR组较Met组显著上调OPG mRNA表达水平(P<0.001)。
②对RANKL mRNA表达的影响：大鼠骨组织可分别于140bp(RANKL)、146bp (β-actin)见到条带,与设计引物相符。半定量RT-PCR显示：各组大鼠的RANKL mRNA表达水平均有差异(P<0.001)。与Nrm组比较,Met组RANKL mRNA表达水平显著降低(P<0.001),SrR组较Met组显著上调RANKL mRNA的表达(P<0.001)。
③OPG Western印迹杂交结果：大鼠骨组织可检测到OPG的蛋白表达,Western blot灰度值结果显示：各组大鼠OPG的蛋白表达水平均有差异(P<0.001)。与Nrm组比较,Met组OPG蛋白表达水平显著降低(P<0.001),SrR组OPG蛋白表达水平较Met组显著升高(P<0.001)。
④RANKL Western印迹杂交结果：大鼠骨组织可检测到RANKL的蛋白表达,Western blot结果分析显示：各组大鼠RANKL的蛋白表达水平均有差异(P<0.001)。与Nrm组比较,Met组RANKL蛋白表达水平显著升高(P<0.001),SrR组RANKL蛋白表达水平较Met组显著降低(P<0.001)。
结论
1、糖皮质激素(Methylprednisolone)5mg/kg/d,一周5次,干预9周后,能显著诱导大鼠骨量丢失、微结构退变,骨质量降低,呈现低转换率骨质疏松,且具有骨形成显著降低,而骨吸收显著增加的特点。表明已成功建立了激素诱导大鼠骨质疏松的动物模型。
2、给予大鼠Aln 1.0mg/kg/d和SrR 900mg/kg/d灌胃,在提高激素诱导骨质疏松大鼠的骨密度,改善骨超微结构和骨强度,抑制骨吸收,降低骨转换率,延缓骨丢失方面起到一定的干预作用：另外,SrR在改善骨的矿化和促进骨形成方面效果优于Aln。
3、正常大鼠骨组织OPG和RANKL在基因及蛋白水平均有表达；在糖皮质激素诱导骨质疏松大鼠的骨组织中,OPG mRNA和蛋白表达水平均显著下降,RANKL mRNA和蛋白表达水平均显著升高；SrR能够显著上调大鼠骨组织OPGmRNA和蛋白表达水平,同时下调RANKL mRNA和蛋白表达水平,影响OPG/RANKL系统,从而起到防治骨质疏松的作用,可能是其改善激素诱导骨质疏松的重要作用机理之一。
Background
Osteoporosis(OP) is considered as dysostosis disorder due to the damage of microstructure of bony tissues, the persistent decreasing of bony matrixes and mineral bones,thinness of sclerotin, the decrease of the number of trabecular bones, and the increase of bony fragility and the risk of fracture. Glucocorticoids(GC) are valuable and indispensable therapeutic agents in the management of many diseases such as asthma, rheumatic diseases, and connective tissue diseases.GC impair the proliferation, differentiation, and function of osteoblasts and induce the apoptosis of mature osteoblasts and osteocytes, also favor in osteoclastogenesis and as a consequence increase bone resorption. Half a percent of the population takes oral GC, making GC-induced osteoporosis(GIOP) is the most common and serious side effect for patients receiving GC.GIOP is characterized by disturbed bone remodelling associated with reduced bone mass and abnormally low quality of the bone tissue, resulting in loss of both cortical and cancellous bone, thus increasing the risk of fracture.
Receptor activator of nuclear factor-KB(RANK),receptor activator of nuclear factor-KB ligand(RANKL) and osteoprotegerin(OPG) are the cytokines which regulate the differentiation and activation of osteoclast. RANKL/RANK/OPG system plays a vital role in bone metabolism. RANKL binds to its receptor, RANK, can induce differentiation and activation of osteoclast and inhibit steoclast apoptosis. This process can be blocked by OPG, which can decrease bone destruction. The RANKL/RANK/OPG system is implicated in many bone loss diseases.This system could be performed as a therapeutic target.
Bisphosphonates are considered to be the pharmacologic gold standard of care for the prevention and treatment of GIOP. Aln is a member of the third generation of modified pyrophosphates, which is a potent antiresorptive agents; both have been approved and are in use for treatment of various disorders characterized by increased osteoclast-mediated bone resorption. GIOP and male osteoporosis are also treated with Aln successfully. SrR is a novel anti-osteoporotic treatment with a dual mode of action, both increasing bone formation and decreasing bone resorption, which rebalances bone turnover in favor of bone formation and increases bone strength. Therefore, SrR might be applicable as a bone therapeutic agent to treat secondary osteoporosis in the clinic.
Objective
To study the effects of administration Strontium ranelate(SrR) on the cancellous and cortical bone in glucocorticoid(GC)-treated rats by means of bone micromorphology, DEXA, biomechanics and histomorphometry, bone turnover marker, and observe the expression of OPG/RANKL mRNA and protein activity in the left femur, as well as to provide futher experimental evidence for exploring the mechanism of SrR.
Methods
In successful model experiment foundation, thirty-two 3.5-month male Sprague-Dawley rats were randomized into four groups:age-matched normal control(Nrm), methylprednisolone(Met) (5.0mg/kg, sc, per day for 5 days/week), Met plus Aln orally(1.0 mg/kg/d) and Met plus SrR orally(900 mg/kg/d).The study period was 9 weeks. Blood samples were obtained before sacrifice to determine the serum ALP, OC and TRACP.DEXA was evaluated in the right femoral diaphysis and fifth lumbar vertebrae(L5), after measuring BMD, the right femur was prepared for shaft three-point bending test, L4 and L5 were prepared for indentation and compression test. Histomorphometry was performed in the right proximal tibial metaphysic and tibial diaphysis, RT-PCR and Western-blot were detected in the left femur, SEM was performed in the left tibia.
Statistical methods
All data were reported as mean±standard error, SPSS13.0 software was used for these analyses.Group Nrm and group Met comparison were run using independent-samples T test, and when a significant difference was revealed(P<0.05);group Aln and group SrR assays performed were analyzed by one-way ANOVA,when significance was revealed(P<0.05).
Results
1.Osteoporosis model established by simulated weightlessness:All rats gained substantial body weight, significant differences were found in two groups(P<0.001). Serum ALP and OC were significantly lower in Met than in Nrm(P<0.01),serum TRACP was significantly higher in Met than in Nrm(P<0.01);the BMD of the right femur and L5 were significantly lower in Met than in Nrm(P<0.001);the max load and rigidity were significantly lower in Met than in Nrm(P<0.05).Quantitative histomorphometry,%Tb.Ar, Tb.Th and Tb.N were significantly lower in Met than in Nrm(P<0.01).Tb.Sp was significantly higher in Met than in Nrm(P<0.01). %L.Pm、MAR、BFRs and %O.Pm were significantly lower in Met than in Nrm(P< 0.01);%E.Pm was significantly higher in Met than in Nrm(P<0.01).
2.Effects of SrR on osteoporosis in GC-treated rats:
(1)In the right femur and L5 BMD were found significant differences in all groups(P <0.05).Met was significantly lower than Nrm group(P<0.001);Aln and SrR were significantly higher than Met group(P<0.001).It should be noted that SrR led to significantly higher in the right femur and L5 BMD compared with the Aln group(P <0.05).
(2) The cortical bone biomechanical property such as the max load and rigidity of femoral shaft three-point bending test, four groups were found significant differences (P<0.05).Met was significantly lower than in Nrm group(P<0.001);there were no significant difference between Aln and Met groups(P>0.05);SrR was significantly higher than in Met group(P<0.001).It should be noted that SrR led to significantly higher compared with Aln group(P<0.05).The cancellous bone biomechanical property like the max load and rigidity of vertebral body(L4) indentation were found significant differences in four groups(P<0.05).Met was significantly lower than in Nrm group(P<0.01);Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05);there were no significant difference between Aln and SrR groups(P>0.05).The max load and rigidity of vertebral body(L5) in the compression test were found significant differences in four groups(P<0.05).Met was significantly lower than in Nrm(P<0.01);Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05); there were no significant difference between Aln and SrR groups(P>0.05).
(3)Bone turnover marker:Serum ALP, OC and TRACP,significant differences were found in four groups(P<0.05).Serum OC and ALP were significantly lower in Met than in Nrm group(P<0.001),serum TRACP was significantly higher in Met than in Nrm group(P<0.001);there were no significant difference between Aln and Met groups(P>0.05); SrR was significantly higher than in Met group(P<0.05),but lower in Nrm group(P<0.05);serum TRACP was significantly lower in Aln and SrR than in Met group(P<0.05), but higher in Nrm group(P<0.05);there were no significant difference between Aln and SrR groups(P>0.05).
(4) In SEM observation, trabecular bone of the left tibia was found with normal network structure and collagenous fibers with regular arrangement in Nrm group; trabecular bone of the left tibia was manifested with fewness, fragility, microcrack and collagen fibers displaced by absorption lacuna in Met group;trabecular bone getting more, network structure getting better and collagenous fiber were much more regular in Aln and SrR groups.
(5) Quantitative histomorphometry
①Histomorphometric indices in the proximal tibia metaphysic: Static histomorphometric indices(%Tb.Ar、Tb.Sp、Tb.N and Tb.Sp) in cancellous bone, differences were found in four groups(P<0.05).Met significantly decreased %Tb.Ar、Tb.Sp and Tb.N compared with the Nrm group(P<0.01).Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05);there were no significant differences between Aln and SrR groups(P>0.05).Tb.Sp, Met was significantly higher than in Nrm group(P<0.001), Aln was significantly lower than in Met group(P<0.01);SrR was significantly lower than in Met groups<0.01);there were no significant difference between Aln and SrR groups(P>0.05).Dynamic histomorphometric indices(MS/BS、MAR、BFRs and ES/BS),differences were found in four groups(P<0.05).Met significantly decreased MS/BS、MAR and BFR/BS compared with the Nrm group(P<0.001).there were no significant differences between Aln and Met groups(P>0.05);SrR was significantly higher than in Met group(P<0.05).It should be noted that SrR led to higher in MS/BS compared with the Aln group(P<0.05).ES/BS,Met was significantly higher than in Nrm group(P<0.001),Aln was significantly lower than in Met group(P< 0.01);SrR was significantly lower than in Met group(P<0.01);there were no significant difference between Aln and SrR groups(P>0.05).
②Histomorphometric indices in the tibial diaphysis: Static histomorphometric indices(%Ct.Ar and%Ma.Ar) in cortical bone and (MS/BS, MAR and BFRs)in periosteum, differences were found in four groups(P<0.05).Met significantly decreased Ct.Ar、MS/BS、MAR and BFRs compared with the Nrm group(P<0.01).there were no significant difference between Aln and Met groups(P >0.05); SrR was significantly higher than in Met group(P<0.05); there were no significant differences between Aln and SrR groups(P>0.05).%Ma.Ar, Met was significantly higher than in Nrm groups<0.01);there were no significant difference between Aln and Met groups(P>0.05), and no significant difference between SrR and Met groups(P>0.05), and no significant difference between SrR and Aln groups(P>0.05).Dynamic histomorphometric indices (MS/BS,MAR, BFRs and ES/BS)in periosteum, significant differences were found in four groups(P<0.05). Dynamic histomorphometric indices (MS/BS,MAR and BFRs), there were no significant differences between Met and Nrm groups(P>0.05).For MAR and BFRs, Aln was lower than in Met group(P<0.05); for MAR and MS/BS,SrR was higher than in Met group(P<0.05).Furthermore, SrR led to a significantly higher in MS/BS, MAR, BFR/BS compared with the Aln group group(P<0.01).ES/BS,Met was significantly higher than in Nrm group(P<0.01),Aln was significantly lower than in Met group(P<0.01);SrR was significantly lower than in Met group(P<0.01);there were no significant differences between Aln and SrR groups(P>0.05).
(6) Effects of Strontium ranelate on OPG/RANKL related changes in protein activity in GC-treated rats
①RT-PCR assay:the expression levels of OPG mRNA and RANKL mRNA, differences were found in three groups(P<0.001),Met significantly decreased OPG mRNA and RANKL mRNA compared with the Nrm group(P<0.001).SrR was significantly higher than in Met group(P<0.001).
②Western blot detection:OPG and RANKL protein expression, differences were found in three groups(P<0.001),Met significantly decreased OPG and RANKL protein expression compared with the Nrm group(P<0.001).SrR was significantly higher than in Met group(P<0.001).
Conclusion
1.The effect of long-term(9 weeks) GC administration, employing a dose equivalent to 5.0mg/kg, sc, per day for 5 days/week, the model of GC-induced osteoporosis characterized by a severe increasing bone formation and decreasing bone resorption, a decrease in BMD, and bone strength. The model was established successfully.
2.SrR at the dose of 900mg/kg/d has more effective than Aln 1.0mg/kg/d in stimulate an anabolic bone response, and improve the resistance of bone over load to fracture in GC-treated rats.SrR has mildly effective in stimulate an anabolic bone response than Aln in prevention GC-induced osteopenia, suggesting the possibility that the SrR may be applicable to use as a bone therapeutic agent to treat secondary osteoporosis in clinic.
3.The level of gene and protein expression of OPG/RANKL in the normal rat bone. The GC administration decreased bone OPG gene and protein expression, increased RANKL gene and protein expression; SrR increased bone OPG gene and protein expression as well as decreased RANKL gene and protein expression, one of the mechanism maybe that the SrR could regulate OPG and RANKL in GC-induced osteoporosis.
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