辛伐他汀对去卵巢大鼠骨代谢影响的实验研究
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摘要
目的：骨质疏松症是以低骨量及骨组织微结构退变为特征的一种全身性骨骼疾病，伴有骨脆性增加，易发生骨折。随着社会人口的老龄化，骨质疏松已成为全球性普遍关注的问题。目前防治骨质疏松症的药物主要有骨吸收抑制剂（包括雌激素、选择性雌激素受体调节剂、降钙素、二膦酸盐、钙剂、维生素D及其衍生物等）和骨形成促进剂（包括氟化物、小剂量的甲状旁腺激素等）两大类。前者主要抑制骨的重吸收，对骨骼形成没有明显的促进作用，难以使已发生骨质疏松组织恢复正常；后者氟化物虽然可以促进新骨形成，却可以导致骨质强度降低，其他如PTH等尚处于研究阶段，因此寻找安全有效的药物是未来骨质疏松症治疗的主要内容。近期发现他汀类降血脂药物具有促进骨形成的作用，从而成为骨质疏松症药物治疗研究的热点。本实验通过去除雌性大鼠双侧卵巢建立绝经后骨质疏松动物模型，并进行药物干预。研究辛伐他汀（Simvastatin）及小剂量辛伐他汀与小剂量尼尔雌醇联合应用对去卵巢大鼠骨代谢的影响，从而进一步阐述他汀类药物治疗骨质疏松症的实用性及联合用药的可行性。
方法：选择健康雌性Wistar大鼠45只，3个月龄，体重(200±30)g，随机分为A、B、C、D、E五组。A组大鼠仅行开关术，其余各组行双侧卵巢切除术。术后1周A、B两组均灌服生理盐水1.5ml·只-1·d-1，C、D、E三组大鼠分别给
予辛伐他汀5mg·kg-1·d-1、尼尔雌醇0.01 mg·kg-1·d-1、辛伐他汀3mg·kg-1·d-1和尼尔雌醇0.005mg·kg-1·d-1，三组药物均以生理盐水混合液的形式灌服，共10周。所有大鼠均自由饮水和进食。实验结束后，观测指标：首先，测量实验前后大鼠的体重。第二，检测血尿骨代谢指标。代谢笼收集24小时尿液，采用甲基百里香酚蓝比色法测定尿钙（Ca），苦味酸法测定尿肌酐（Cr），消化法测定尿羟脯氨酸（HOP）。断头处死大鼠留取全血后，取血清采用放射免疫法测定骨钙素（BGP）及碱性磷酸酶（ALP）。第三，测量骨密度。分离大鼠左侧股骨和胫骨，运用双能x线骨密度仪小动物软件包测定其骨密度。第四，观察股骨干骺端形态学改变。分离大鼠左侧股骨，常规石蜡包埋HE染色后光镜下观察骨小梁的形态。股骨干骺端经过处理后扫描电镜下观察骨小梁的超微结构。分析各组大鼠体重、骨代谢指标和骨密度的改变及关系，运用SPSS 10.0 FOR WINDOWS 软件包处理。计量资料以x±s表示，并应用方差分析进行分析，α=0.05。
结果：实验前各组大鼠体重无差异（P>0.05），实验给药10周后B组大鼠体重较A组显著增加，差异有显著性（P<0.01）；骨代谢中反映骨形成的指标血清BGP：B组与A组相比明显降低，差异有显著性（P<0.05），C、D、E三组与B组相比均明显升高，差异有显著性（P<0.05）。血清ALP：B组与A组相比有所升高，差异有显著性（P<0.05），C、D、E三组与B组相比均降低，差异有显著性（P<0.05）。反映骨吸收的指标尿Ca/Cr、HOP/Cr比值：B组与A组相比均呈不同程度的升高，C、D、E三组与B组相比有所降低，但均无统计学意义（P>0.05）；骨密度的测量：股骨骨密度B组较A
组明显降低，差异有显著性（P<0.05），而C、D、E三组较B组则有所升高，差异有显著性（P<0.05）。胫骨骨密度B、C、D、E四组较A组下降，但无显著性差异（P>.05）；以上各观察指标C、D、E三组两两之间比较差异无显著性（P>0.05）。镜下观察：光镜下B组骨小梁明显变薄、变细，甚至断裂，C组、D组、E组三组骨小梁排列紧密，形态结构接近正常。扫描电镜下三组骨胶原纤维排列比较整齐，接近正常结构。
结论：成熟雌性大鼠去除卵巢后骨代谢活跃，呈高转换型。表现为骨吸收和骨形成均加速，并且由于骨吸收的过程较短，成骨过程较长而造成骨量丢失，从而导致骨密度降低，正常的组织结构遭到破坏。辛伐他汀、尼尔雌醇均可以减缓高转换型的骨代谢活动，表现为骨代谢指标数值的降低，骨密度值的升高，本实验认为：辛伐他汀对去卵巢大鼠的高转换型骨代谢有抑制作用，并且其积极的骨形成作用优于尼尔雌醇对骨吸收的抑制作用。此外，小剂量的辛伐他汀和尼尔雌醇联合应用具有单药治疗的临床效果，还可以减少两药的不良反应及降低用药成本。
Objective:Osteoporosis is characterized by a decrease in bone mass as well as a deterioration of the bone architecture resulting in an increased risk of fracture.With the increasing life expectancy in society,osteoporosis is a major and growing health problem worldwide.Now data suggests that drugs to treat osteoporosis mainly include bone resorption inhibitors (estrogen,selective estrogen receptor modulators,calcitonin, bisphosphonates,calcium,vitamin D and its analogues,etc)and bone anabolic agents(fluoride,PTH,etc).The former mainly inhibit bone resorption,and they can’t stimulate bone formation markedly to make osteopic tissue restore normal one.While the latter for example fluoride can stimulate the formation of new bone,but also can reduce the strength of bone,others remain on the stage of study.So it is important to discover safe and effective drug to treat osteoporosis in the future.Recently data suggests that statins lipid-lowering drugs can stimulate bone formation,which becomes focus of medicine treatment of osteoporosis.This study observes the effect of simvastatin on bone metabolism in ovariectomized rats.
Methods:45 three-month-old Wistar rats,weighed(200±30)g,were randomly divided into five groups(A,B,C,D,E).Each
group had 9 rats.Group A was made pesudo-ovariectomized surgery and others were made ovariectomized surgery.Group A and Group B, were given 0.9% sodium chloride solution 1.5ml·d-1; Group C,given simvastatin 5mg·kg-1·d-1;Group D, given Nylestriol 0.01mg·kg-1·d-1;Group E,given simvastatin 3mg·kg-1·d-1 and Nylestriol 0.005mg·kg-1·d-1.All of the animals were given the agents through gastric tube for ten weeks.When the study finished,we observed markers:Firstly,at the day before surgery and the 70th day after surgery measured the weight of all the rats.Secondly,some urinary and serum biochemical markers of bone turnover were investigated.We collected twenty-four hours urine of rats by metabolic cage to measure urinary calcium by colorimetric analysis,urinary creatinine by the method of picric acid and urinary hydroxyproline by the method of digestion.Then all the rats were sacrified for the measurement of serum BGP and alkaline phosphatase level.Thirdly,to get left femur and tibia of all the animals for the measurement of bone mineral density(BMD) by the dual energy X-ray absorptiometry.Then to observe the cancellous microstructure of distal femur by light microscope and scanning electron microscope.The association between the weight,biochemical markers of bone turnover and bone mineral density of all groups was demon stated in x±s deviation.The analysis of variance was used to the significance test by SPSS 10.0 FOR WINDOWS software,α=0.05.
Results:There was no obvious difference in weight of all
the animals before surgery.Weight of Group B rats increased significantly compared with that of Group A,when the study finished(P<0.01);Among biochemical markers of bone turnover which reflected bone formation serum BGP:Group B were lower than Group A,Group C,Group D and Group E and the difference was significant(P<0.05);While serum ALP:Group B were higher than other groups(P<0.05).Bone resorption markers the urinary calcium/creatinine ratio and the urinaryhydroxyproline/creatinine ratio:Group B were increased than others,but the difference was not significant(P>0.05).Femoral bone mineral density:Group B were decreased than other groups and the difference was significant(P<0.05).Tibial bone mineral density:Group B,Group C,Group D and Group E were lower than Group A,but the difference was not significant (P>0.05).There was no significant difference among Group C,Group D and Group E of all the results above (P>0.05).Under light microscope and scanning electron microscope,we could see that the bone mass decreased, trabecula lessened and microdamage increased significantly in Group B than other groups.
Conclusions:The ovariectomized Wistar rats showed active bone metabolism.Bone formation and bone resorption were all increased,and the latter was shorter than the former,which ca
方法：选择健康雌性Wistar大鼠45只，3个月龄，体重(200±30)g，随机分为A、B、C、D、E五组。A组大鼠仅行开关术，其余各组行双侧卵巢切除术。术后1周A、B两组均灌服生理盐水1.5ml·只-1·d-1，C、D、E三组大鼠分别给
予辛伐他汀5mg·kg-1·d-1、尼尔雌醇0.01 mg·kg-1·d-1、辛伐他汀3mg·kg-1·d-1和尼尔雌醇0.005mg·kg-1·d-1，三组药物均以生理盐水混合液的形式灌服，共10周。所有大鼠均自由饮水和进食。实验结束后，观测指标：首先，测量实验前后大鼠的体重。第二，检测血尿骨代谢指标。代谢笼收集24小时尿液，采用甲基百里香酚蓝比色法测定尿钙（Ca），苦味酸法测定尿肌酐（Cr），消化法测定尿羟脯氨酸（HOP）。断头处死大鼠留取全血后，取血清采用放射免疫法测定骨钙素（BGP）及碱性磷酸酶（ALP）。第三，测量骨密度。分离大鼠左侧股骨和胫骨，运用双能x线骨密度仪小动物软件包测定其骨密度。第四，观察股骨干骺端形态学改变。分离大鼠左侧股骨，常规石蜡包埋HE染色后光镜下观察骨小梁的形态。股骨干骺端经过处理后扫描电镜下观察骨小梁的超微结构。分析各组大鼠体重、骨代谢指标和骨密度的改变及关系，运用SPSS 10.0 FOR WINDOWS 软件包处理。计量资料以x±s表示，并应用方差分析进行分析，α=0.05。
结果：实验前各组大鼠体重无差异（P>0.05），实验给药10周后B组大鼠体重较A组显著增加，差异有显著性（P<0.01）；骨代谢中反映骨形成的指标血清BGP：B组与A组相比明显降低，差异有显著性（P<0.05），C、D、E三组与B组相比均明显升高，差异有显著性（P<0.05）。血清ALP：B组与A组相比有所升高，差异有显著性（P<0.05），C、D、E三组与B组相比均降低，差异有显著性（P<0.05）。反映骨吸收的指标尿Ca/Cr、HOP/Cr比值：B组与A组相比均呈不同程度的升高，C、D、E三组与B组相比有所降低，但均无统计学意义（P>0.05）；骨密度的测量：股骨骨密度B组较A
组明显降低，差异有显著性（P<0.05），而C、D、E三组较B组则有所升高，差异有显著性（P<0.05）。胫骨骨密度B、C、D、E四组较A组下降，但无显著性差异（P>.05）；以上各观察指标C、D、E三组两两之间比较差异无显著性（P>0.05）。镜下观察：光镜下B组骨小梁明显变薄、变细，甚至断裂，C组、D组、E组三组骨小梁排列紧密，形态结构接近正常。扫描电镜下三组骨胶原纤维排列比较整齐，接近正常结构。
结论：成熟雌性大鼠去除卵巢后骨代谢活跃，呈高转换型。表现为骨吸收和骨形成均加速，并且由于骨吸收的过程较短，成骨过程较长而造成骨量丢失，从而导致骨密度降低，正常的组织结构遭到破坏。辛伐他汀、尼尔雌醇均可以减缓高转换型的骨代谢活动，表现为骨代谢指标数值的降低，骨密度值的升高，本实验认为：辛伐他汀对去卵巢大鼠的高转换型骨代谢有抑制作用，并且其积极的骨形成作用优于尼尔雌醇对骨吸收的抑制作用。此外，小剂量的辛伐他汀和尼尔雌醇联合应用具有单药治疗的临床效果，还可以减少两药的不良反应及降低用药成本。
Objective:Osteoporosis is characterized by a decrease in bone mass as well as a deterioration of the bone architecture resulting in an increased risk of fracture.With the increasing life expectancy in society,osteoporosis is a major and growing health problem worldwide.Now data suggests that drugs to treat osteoporosis mainly include bone resorption inhibitors (estrogen,selective estrogen receptor modulators,calcitonin, bisphosphonates,calcium,vitamin D and its analogues,etc)and bone anabolic agents(fluoride,PTH,etc).The former mainly inhibit bone resorption,and they can’t stimulate bone formation markedly to make osteopic tissue restore normal one.While the latter for example fluoride can stimulate the formation of new bone,but also can reduce the strength of bone,others remain on the stage of study.So it is important to discover safe and effective drug to treat osteoporosis in the future.Recently data suggests that statins lipid-lowering drugs can stimulate bone formation,which becomes focus of medicine treatment of osteoporosis.This study observes the effect of simvastatin on bone metabolism in ovariectomized rats.
Methods:45 three-month-old Wistar rats,weighed(200±30)g,were randomly divided into five groups(A,B,C,D,E).Each
group had 9 rats.Group A was made pesudo-ovariectomized surgery and others were made ovariectomized surgery.Group A and Group B, were given 0.9% sodium chloride solution 1.5ml·d-1; Group C,given simvastatin 5mg·kg-1·d-1;Group D, given Nylestriol 0.01mg·kg-1·d-1;Group E,given simvastatin 3mg·kg-1·d-1 and Nylestriol 0.005mg·kg-1·d-1.All of the animals were given the agents through gastric tube for ten weeks.When the study finished,we observed markers:Firstly,at the day before surgery and the 70th day after surgery measured the weight of all the rats.Secondly,some urinary and serum biochemical markers of bone turnover were investigated.We collected twenty-four hours urine of rats by metabolic cage to measure urinary calcium by colorimetric analysis,urinary creatinine by the method of picric acid and urinary hydroxyproline by the method of digestion.Then all the rats were sacrified for the measurement of serum BGP and alkaline phosphatase level.Thirdly,to get left femur and tibia of all the animals for the measurement of bone mineral density(BMD) by the dual energy X-ray absorptiometry.Then to observe the cancellous microstructure of distal femur by light microscope and scanning electron microscope.The association between the weight,biochemical markers of bone turnover and bone mineral density of all groups was demon stated in x±s deviation.The analysis of variance was used to the significance test by SPSS 10.0 FOR WINDOWS software,α=0.05.
Results:There was no obvious difference in weight of all
the animals before surgery.Weight of Group B rats increased significantly compared with that of Group A,when the study finished(P<0.01);Among biochemical markers of bone turnover which reflected bone formation serum BGP:Group B were lower than Group A,Group C,Group D and Group E and the difference was significant(P<0.05);While serum ALP:Group B were higher than other groups(P<0.05).Bone resorption markers the urinary calcium/creatinine ratio and the urinaryhydroxyproline/creatinine ratio:Group B were increased than others,but the difference was not significant(P>0.05).Femoral bone mineral density:Group B were decreased than other groups and the difference was significant(P<0.05).Tibial bone mineral density:Group B,Group C,Group D and Group E were lower than Group A,but the difference was not significant (P>0.05).There was no significant difference among Group C,Group D and Group E of all the results above (P>0.05).Under light microscope and scanning electron microscope,we could see that the bone mass decreased, trabecula lessened and microdamage increased significantly in Group B than other groups.
Conclusions:The ovariectomized Wistar rats showed active bone metabolism.Bone formation and bone resorption were all increased,and the latter was shorter than the former,which ca
引文
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Mundy G, Garrett R, Harris S, et al. Stimulation of bone formation on vitro and in rodents by statins .Science, 1999, 286 :1946～1949.
曾天舒，陈璐路，夏文芳.辛伐他汀促进成骨细胞增殖的实验研究[A].中华医学会第一次全国骨质疏松学术会议论文汇编[C].北京：中华医学会，2001，121.
刘忠厚，主编.骨质疏松学.北京：科学出版社，1998，506～511
廖慧娟，廖二元.骨质疏松症动物模型.国外医学内分泌分册，2004，24（1）：60～62.
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