糖皮质激素性骨质疏松症大鼠“骨肉不相亲”病理机制及中医不同治法的比较研究

【作者】 杨芳；

【导师】 郑洪新；

【作者基本信息】 辽宁中医药大学 ， 中医基础理论， 2011， 博士

【摘要】 目的:本研究采用分子生物学的方法,以骨质疏松症大鼠的骨骼、骨骼肌中钙代谢、Ⅰ型胶原、能量代谢的相关酶学表达等为研究核心,阐述骨骼肌收缩与骨骼协调性对骨质疏松症形成的影响,并且观察补肾方法、健脾方法、活血化瘀方法对骨质疏松症的防治效果和作用机制的异同。该研究为临床提高骨质疏松症的防治效果、选择最佳的治疗方案提供科学的实验依据。材料与方法:本实验采用地塞米松肌肉注射方法建立骨质疏松症动物模型,以补肾方法、健脾方法、活血化瘀方法对模型大鼠进行防治,同时选用骨疏康颗粒剂作为阳性对照药,正常大鼠作为标准对照,模型大鼠作为空白对照组,造模及给药时间为9周。9周后处死动物进行取材及实验指标检测:①应用双能X线骨密度仪检测离体股骨的骨密度（BMD）;②用生化方法检测血清抗酒石酸酸性磷酸酶（TRAP）、碱性磷酸酶（ALP）、血钙和血磷;③用ELISA方法检测骨骼肌的肌钙蛋白和Ca²⁺-Mg²⁺-ATP酶等指标;④用PCR法及Western印迹法检测各组大鼠骨组织、骨骼肌的骨钙素、Ⅰ型胶原、Na⁺-K⁺-ATP酶的mRNA和蛋白表达。结果:1.大鼠离体股骨上1/3骨密度检测结果显示:与正常组比较,模空组大鼠离体股骨上1/3骨密度极显著降低（P<0.01）;与模空组比较,各治疗组大鼠股骨上1/3骨密度均有不同程度的升高,其中以补肾中药组升高最为显著（P<0.01）。2.大鼠骨代谢生化指标结果显示:①与正常组比较,模空组大鼠血清ALP含量有升高趋势,但不具有统计学意义;与模空组比较,各治疗组大鼠的血清ALP含量均升高,其中以活血组升高最为明显,具有统计学意义（P<0.05）。②与正常组比较,模空组大鼠血清TRAR含量升高极为显著（P<0.01）;与模空组比较,各治疗组大鼠血清TRAR含量均明显降低,其中以补肾组和骨疏康组降低最为明显,与其他各组比较具有极显著差异（P<0.01）。③与正常组比较,模空组大鼠血钙含量明显升高,但不具有统计学意义;与模空组比较,各治疗组大鼠血钙含量均明显升高,其中以补肾组和骨疏康组升高最为明显,与其他各组比较具有极显著差异（P<0.01）。④各组大鼠血磷含量变化不明显,治疗组中骨疏康组血磷含量升高,与正常组比较具有显著差异（P<0.01）。3.大鼠骨组织骨钙素、骨骼肌钙蛋白指标结果显示:①与正常组相比,模空组大鼠骨组织骨钙素mRNA表达水平明显下降（P<0.05）,与模空组相比,各治疗组大鼠骨钙素mRNA表达水平均有不同程度的升高,其中补肾组升高趋势最为显著（P<0.01）。与正常组比较,模空组大鼠骨钙素的蛋白表达明显降低,但统计比较无显著差异;与模空组比较,各治疗组大鼠骨钙素的蛋白表达均明显升高,其中补肾组升高趋势最显著（P<0.01）。②与正常组比较,各组大鼠骨骼肌的肌钙蛋白显著降低（P<0.01）;与模空组比较,补肾组大鼠肌钙蛋白均明显升高（P<0.01）;补肾组大鼠骨骼肌的肌钙蛋白升高最为明显,明显高于其他各组,具有显著性差异（P<0.01）;活血组大鼠骨骼肌的肌钙蛋白升高程度最低,与正常组、模空组、补肾组比较具有显著统计学差异（P<0.01）。4.大鼠骨组织、骨骼肌Ⅰ型胶原指标结果显示:①与正常组相比,模空组大鼠骨组织Ⅰ型胶原mRNA表达水平明显下降（P<0.05）,与模空组相比,各治疗组大鼠的Ⅰ型胶原mRNA表达水平均有不同程度的升高,其中补肾组升高趋势最为显著（P<0.01）,其次为健脾组（P<0.05）,活血组升高程度略低,与补肾组相比有显著差异（P<0.05）;骨疏康组升高程度最低,与补肾组相比有极显著差异（P<0.01）。与正常组比较,各组大鼠骨组织Ⅰ型胶原的蛋白表达均明显下降（P<0.01）;与模空组比较,各治疗组大鼠Ⅰ型胶原的蛋白表达均明显升高,其中补肾组、骨疏康组升高趋势最显著（P<0.01）。②与正常组相比,模空组大鼠骨骼肌Ⅰ型胶原mRNA表达水平明显下降（P<0.01）,与模空组相比,各治疗组大鼠的骨骼肌Ⅰ型胶原mRNA表达水平均有不同程度的升高,其中补肾组、健脾组升高趋势最为显著,与模空组比较均有极明显差异（P<0.01）。与正常组比较,各组大鼠骨骼肌Ⅰ型胶原的蛋白表达均明显下降（P<0.01）;与模空组比较,各治疗组大鼠骨骼肌Ⅰ型胶原的蛋白表达均明显升高（P<0.01）,其中补肾组升高趋势最明显。5.大鼠骨组织、骨骼肌能量代谢指标结果显示:①与正常组相比,模空组大鼠骨组织Na⁺-K⁺-ATP酶mRNA表达水平明显下降（P<0.05）,与模空组相比,各治疗组大鼠Na⁺-K⁺-ATP酶mRNA表达水平均有不同程度的升高,其中补肾组升高趋势最为显著,与模空组比较有极明显差异（P<0.01）,其次为健脾组（P<0.01）,是正常组的2.43倍;活血组升高程度最低,与补肾组、健脾组相比均有极显著差异（P<0.01）。②与正常组相比,模空组大鼠骨骼肌中Na⁺-K⁺-ATP酶mRNA表达水平明显下降（P<0.01）,与模空组相比,各治疗组大鼠的骨骼肌Na⁺-K⁺-ATP酶mRNA表达水平均有不同程度的升高,其中健脾组升高趋势最为显著,与模空组比较有极明显差异（P<0.01）,是正常组的1.00倍,其次为补肾组（P<0.01）,是正常组的0.68倍;活血组与骨疏康组升高程度最低。③与正常组比较,其他各组大鼠骨骼肌的Ca²⁺-Mg²⁺-ATP酶显著降低（P<0.01）;与模空组比较,各治疗组大鼠骨骼肌的Ca²⁺-Mg²⁺-ATP酶均明显升高（P<0.01）;补肾组大鼠骨骼肌的Ca²⁺-Mg²⁺-ATP酶升高最为明显,明显高于骨疏康组、活血组、健脾组,差异具有统计学意义（P<0.01）结论:1.运用地塞米松2.5mg/kg,一周两次,连续9周,后肢臀部肌肉注射,可以成功诱导骨质疏松症大鼠模型。2.地塞米松可导致大鼠骨组织骨钙素、Ⅰ型胶原、Na⁺-K⁺-ATP酶基因和蛋白表达水平下降;骨骼肌中肌钙蛋白、Ca²⁺-Mg²⁺-ATP酶、Ⅰ型胶原、Na⁺-K⁺-ATP酶基因和蛋白表达水平下降。提示骨组织、骨骼肌能量代谢与协调性下降,可能是糖皮质激素诱导的骨质疏松症发生的重要机制之一。3.补肾中药通过有效提高骨质疏松症大鼠骨组织、骨骼肌的钙代谢、Ⅰ型胶原和能量代谢相关基因的表达水平,提高骨质疏松症大鼠骨组织、骨骼肌的能量代谢,从而促进骨形成,抑制骨吸收,对糖皮质激素诱导的骨质疏松症有明显的防治作用。4.健脾中药通过提高骨骼肌的钙代谢、Ⅰ型胶原和能量代谢相关基因的表达水平,提高骨质疏松症大鼠骨骼肌的能量代谢,进而有效的保护骨骼,提高骨骼与肌肉的协调性,促进骨形成,对糖皮质激素诱导的骨质疏松症有一定的防治作用。5.通过骨质疏松症大鼠骨骼、骨骼肌钙代谢、Ⅰ型胶原和能量代谢相关基因的表达变化影响,比较补肾中药、健脾中药、活血化瘀中药的防治效果,认为补肾方法〉健脾方法〉活血化瘀方法。说明补肾方法是防治骨质疏松症的基本治法,健脾方法是辅助补肾方法治疗骨质疏松症的一个重要治法。更多还原

【Abstract】 Purpose:This study took the expression of collagenⅠand the enzymes related to calcium and energy metabolism in bones and skeletal muscles of rats with osteoporosis as research core by molecular biology approach to expound the effect of the coordination of skeletal muscles contraction and bones on the formation of osteoporosis and observe the preventing and treating effect of reinforcing the kidney method,strengthening the spleen method and promoting blood circulation to remove blood stasis method on osteoporosis and their mechanisms.This study provides scientific experimental evidence for increasing the preventing and treating effect on osteoporosis and choosing the best therapeutic schedule.Materials and methods:This experiment established the osteoporosis animal models by intramuscular injection of dexamethasone, prevented and treated the model rats with reinforcing the kidney method,strengthening the spleen method and promoting blood circulation to remove blood stasis method,chose GUSHUKANG granula as positive control drug,took the normal rats as standard comparison and the model rats as blank comparison and the time of establishing models and administrating was 9 weeks. After 9 weeks,we put all the rats to death and get materials to detect the following indicators:①We detected the vitro femur bone mineral density（BMD） by using the dual energy X-ray absorptiometry;②We detected the tartrate-resistant acid phosphatase（TRAP）,alkaline phosphatase（ALP）, calcium and phosphorus in blood serum and troponin and Ca²⁺-Mg²⁺-ATP enzyme of skeletal muscles by biochemistry method;③We detected the mRNA and protein expression of bone gla protein, collagenⅠand Na⁺-K⁺-ATP enzyme in the bone tissue and skeletal muscles of each group rats by PCR and Western blot.Results:1. Rats’vitro upper 1/3 femur bone mineral density （BMD）:compared with normal group,blank model group decreased significantly（P <0.01）;compared with blank model group,all the treating groups increased at different degrees,reinforcing the kidney herbs group increased the most significantly（P<0.01）.2. Rats’biochemistry indicators of bone metabolism:①compared with normal group,blank model group rats’ALP in blood serum trended to increase,but the difference had not statistical significance; compared with blank model group,all the treating groups rats’ALP in blood serum increased, promoting blood circulation group increased the most significantly（P<0.05）.②compared with normal group, blank model group rats’TRAP in blood serum increased significantly（P<0.01）;compared with blank model group, all the treating groups rats’TRAP in blood serum decreased significantly, reinforcing the kidney group and GUSHUKANG group decreased the most significantly, compared with other groups, the difference had statistical significance（P < 0.01）.③compared with normal group, blank model group rats’calcium in blood serum increased,but the difference had not statistical significance; compared with blank model group,all the treating groups rats’calcium in blood serum increased significantly, reinforcing the kidney group and GUSHUKANG group increased the most significantly, compared with other groups, the difference had statistical significance（P<0.01）.④Phosphorus in blood serum had no obvious variation. compared with normal group,GUSHUKANG group rats’phosphorus in blood serum increased significantly（P<0.01）.3. Rats’calcium metabolism indicators in bones and skeletal muscles:①compared with normal group,blank model group rats’bone gla protein mRNA expression in bones decreased significantly（P<0.05）; compared with blank model group,all the treating groups rats’bone gla protein mRNA expression increased at different degrees,reinforcing the kidney group increased the most significantly（P<0.01）. Compared with normal group, blank model group rats’bone gla protein protein expression in bones decreased ,but the difference had not statistical significance; compared with blank model group, all the treating groups rats’bone gla protein protein expression increased,reinforcing the kidney group increased the most significantly（P<0.01）.②Compared with normal group, troponin of the rats’skeletal muscle of the other groups decreased significantly（P<0.01）; compared with blank model group, troponin of the rats’skeletal muscle of all the treating groups increased significantly（P<0.01）; troponin of the rats’skeletal muscle of reinforcing the kidney group increased the most significantly,higher than other groups and the difference had statistical significance（P<0.01）;the increasing degree of troponin of the rats’skeletal muscle of promoting blood circulation group was the lowest and had statistical significance（P<0.01）, comparing with normal group, blank model group and reinforcing the kidney group.4. Rats’collagenⅠin bones and skeletal muscles:①compared with normal group,blank model group rats’collagenⅠmRNA expression in bones decreased significantly（P<0.05）; compared with blank model group,all the treating groups rats’collagenⅠmRNA expression increased at different degrees,reinforcing the kidney group increased the most significantly（P<0.01）, secondly, strengthening the spleen group（P<0.05）, thirdly, promoting blood circulation group, had statistically significant difference, comparing with reinforcing the kidney group（P<0.05） ;the increasing degree of GUSHUKANG group was the lowest and had statistically significant difference （P < 0.01）, comparing with reinforcing the kidney group. Compared with normal group, collagenⅠprotein expression of the rats’bone of the other groups decreased significantly（P<0.01）; compared with blank model group, collagenⅠprotein expression of the rats’bone of all the treating groups increased, reinforcing the kidney group and GUSHUKANG group increased the most significantly（P<0.01）.②compared with normal group,blank model group rats’collagenⅠmRNA expression in skeletal muscles decreased significantly（P<0.01）; compared with blank model group,all the treating groups rats’collagenⅠmRNA expression increased at different degrees,reinforcing the kidney group and strengthening the spleen group increased the most significantly（P<0.01）. Compared with normal group, collagenⅠprotein expression of the rats’skeletal muscles of the other groups decreased significantly（P<0.01）; compared with blank model group, collagenⅠprotein expression of the rats’skeletal muscles of all the treating groups increased significantly（P<0.01）, reinforcing the kidney group increased the most significantly.5. Rats’energy metabolism indicators in bones and skeletal muscles:①compared with normal group,blank model group rats’Na⁺-K⁺-ATP enzyme mRNA expression in bones decreased significantly（P<0.05）; compared with blank model group,all the treating groups rats’Na⁺-K⁺-ATP enzyme mRNA expression increased at different degrees,reinforcing the kidney group increased the most significantly（P<0.01）, secondly, strengthening the spleen group（P<0.01）,2.43 times of normal group; the increasing degree of promoting blood circulation group was the lowest and had statistical significance（P<0.01）, comparing with reinforcing the kidney group and strengthening the spleen group.②compared with normal group,blank model group rats’Na⁺-K⁺-ATP enzyme mRNA expression in skeletal muscles decreased significantly（P<0.01）; compared with blank model group,all the treating groups rats’Na⁺-K⁺-ATP enzyme mRNA expression increased at different degrees, strengthening the spleen group increased the most significantly（P<0.01）,1.00 times of normal group, secondly, reinforcing the kidney group（P<0.01）,0.68 times of normal group; the increasing degree of promoting blood circulation group and GUSHUKANG group was the lowest.③Compared with normal group, Ca²⁺-Mg²⁺-ATP enzyme of the rats’skeletal muscles of the other groups decreased significantly（P<0.01）; compared with blank model group, Ca²⁺-Mg²⁺-ATP enzyme of the rats’skeletal muscles of all the treating groups increased significantly（P<0.01）, reinforcing the kidney group increased the most significantly,higher than GUSHUKANG group, promoting blood circulation group and strengthening the spleen group significantly and the difference had statistical significance （P<0.01）.Conclusions:1. The method of hind limb intramuscular injection of dexamethasone（2.5mg/kg,twice a week,9 weeks continuously） can induce osteoporosis rat models successfully.2. Dexamethasone can cause gene and protein expression level of bone gla protein, collagenⅠand Na+-K+-ATP enzyme in rats’bones and troponin,Ca²⁺-Mg²⁺-ATP enzyme, collagenⅠand Na+-K+-ATP enzyme of skeletal muscles decreasing.This suggests that maybe the decline of energy metabolism and coordination of bones and skeletal muscles is one aspect of the important pathogenesis of glucocorticoid-induced osteoporosis.3. The reinforcing the kidney herbs have obvious preventing and treating effect on glucocorticoid-induced osteoporosis by increasing the genes expression level of collagenⅠand others related to calcium and energy metabolism in bones and skeletal muscles of rats with osteoporosis effectively and increasing energy metabolism in bones and skeletal muscles of rats with osteoporosis to promote bone formation and inhibit bone resorption.4. The strengthening the spleen herbs have some preventing and treating effect on glucocorticoid-induced osteoporosis by increasing the genes expression level of collagenⅠand others related to calcium and energy metabolism of skeletal muscles to increase energy metabolism of skeletal muscles of rats with osteoporosis to protect bones effectively and increase the coordination of bones and muscles to promote bone formation.5. The comparation of the preventing and treating effect of reinforcing the kidney herbs,strengthening the spleen herbs and promoting blood circulation to remove blood stasis herbs on osteoporosis by the genes expression change of collagenⅠand others related to calcium and energy metabolism in bones and skeletal muscles of rats with osteoporosis shows reinforcing the kidney herbs better than strengthening the spleen herbs and strengthening the spleen herbs better than promoting blood circulation to remove blood stasis herbs.This suggests that reinforcing the kidney method is the main treating method to prevent and treat osteoporosis and strengthening the spleen method is an important treating method to assist reinforcing the kidney method to treat osteoporosis.更多还原