补肾活血方对去卵巢大鼠骨代谢及骨密度的影响

Effect of Bushen Huoxue Fang(补肾活血方) on bone metabolism and bone mineral density in the ovariectomized rats

目的:观察补肾活血方对去卵巢大鼠骨代谢和骨密度的影响。方法:将40只SD雌性大鼠随机分成假手术组、模型组、补肾活血方组和尼尔雌醇组,每组10只。假手术组行开腹术但不摘除卵巢,其他3组行双侧卵巢切除术。术后假手术组和模型组大鼠以蒸馏水灌胃(每次3 m L,每天1次),补肾活血方组大鼠以补肾活血方汤剂灌胃(每次15 g·kg^(-1),每天1次),尼尔雌醇组大鼠以研细的尼尔雌醇片和生理盐水配制成的混悬液灌胃(每次0.6 g·kg^(-1),每周1次)。灌胃12周后从大鼠心脏取血,离心后吸取上层血清,置入冰箱保存待检。处死大鼠后,切取右侧股骨中段做标本,同时取大鼠腰椎,置入冰箱保存待检。采用ELISA法测定血清Ⅰ型前胶原氨基端原肽(procollagen typeⅠN-terminal propeptide,PINP)、β-胶原降解产物(βisomer of C-terminal telopeptide of typeⅠcollagen,β-CTX)含量,并用双能X线骨密度测量仪检测腰椎骨密度。将冰冻切片做HE染色后,在显微镜下观察骨组织病理学改变。结果:1血清PINP、β-CTX含量。药物干预后各组大鼠血清PINP含量比较,差异有统计学意义(F=914.448,P=0.000)。组间两两比较,假手术组血清PINP含量低于模型组[(0.109±0.008)μg·m L^(-1),(0.252±0.006)μg·m L^(-1),q=-51.807,P=0.000];假手术组血清PINP含量与补肾活血方组[(0.163±0.006)μg·m L^(-1)]、尼尔雌醇组[(0.169±0.005)μg·m L^(-1)]比较,差异均无统计学意义[q=0.559,P=0.208;q=0.850,P=0.253];模型组血清PINP含量高于补肾活血方组和尼尔雌醇组(q=32.248,P=0.000;q=29.957,P=0.000);补肾活血方组与尼尔雌醇组比较,差异无统计学意义(q=-0.290,P=0.317)。药物干预后各组大鼠血清β-CTX含量比较,差异有统计学意义(F=963.955,P=0.000)。组间两两比较,假手术组血清β-CTX含量低于模型组[(0.432±0.007)ng·m L^(-1),(0.766±0.005)ng·m L^(-1),q=-48.601,P=0.000];假手术组血清β-CTX含量与补肾活血方组[(0.482±0.006)ng·m L^(-1)]、尼尔雌醇组[(0.494±0.008)ng·m L^(-1)]比较,差异均无统计学意义(q=-0.172,P=0.215;q=0.980,P=0.322);模型组血清β-CTX含量高于补肾活血方组和尼尔雌醇组(q=41.429,P=0.000;q=39.622,P=0.000);补肾活血方组血清β-CTX含量与尼尔雌醇组比较,差异无统计学意义(q=-0.808,P=0.790)。2腰椎骨密度。药物干预后各组大鼠腰椎骨密度比较,差异有统计学意义(F=419.969,P=0.000)。组间两两比较,假手术组腰椎骨密度高于模型组[(0.170±0.004)g·cm-2,(0.097±0.005)g·cm-2,q=35.198,P=0.000];假手术组腰椎骨密度与补肾活血方组[(0.127±0.005)g·cm-2]、尼尔雌醇组[(0.126±0.004)g·cm-2]比较,差异均无统计学意义(q=2.444,P=0.157;q=1.167,P=0.230);模型组腰椎骨密度低于补肾活血方组和尼尔雌醇组(q=^(-1)4.754,P=0.000;q=^(-1)4.031,P=0.000);补肾活血方组腰椎骨密度与尼尔雌醇组比较,差异无统计学意义(q=0.723,P=0.474)。3骨组织形态。假手术组股骨骨小梁粗壮、饱满,壁厚,形态结构完整,排列紧密有序,呈网状,密度、面积正常,骨小梁间隙较小。模型组股骨骨小梁变细、变薄,有扭曲或断裂,骨小梁间隙增大。补肾活血方组和尼尔雌醇组股骨骨小梁较模型组明显增粗,排列尚整齐并连接成网,部分区域骨小梁间隙略增大。结论:补肾活血方能降低去卵巢大鼠血清PINP、β-CTX含量,提高骨密度,改善骨组织状况,但其具体作用机制尚不明确,有待进一步研究。

Objective：To observe the effect of Bushen Huoxue Fang（补肾活血方, BSHXF）on bone metabolism and bone mineral den- sity（BMD） in the ovariectomized rats. Methods： Forty female SD rats were randomly divided into sham- operated group, model group, BSHXF group and nylestriol group, 10 cases in each group. The rats in the sham - operated group underwent simple laparotomy while the others underwent bilateral ovariectomy. The rats in the sham - operated group and model group were intragastric administrated with distilled water （3 ml at a time, once a day）. The rats in BSHXF group were intragastric administrated with BSHXF decoction （ 15 g/kg at a time, once a day）and the rats in nylestriol group were intragastric administrated with the suspension of nylestriol tablets and normal saline （0.6 g/kg at a time, once a week）. After 12 -week intragastric administration, the blood samples were obtained from the heart of rats. The upper serum was sucked from the blood samples after centrifuging and was put into the refrigerator for further inspection. The rats were sacrificed and their right middle femurs were fetched out for making specimens and their lumbar vertebraes were fetched out and put into the refrigerator for further inspection. The serum content of procollagen type I N - terminal propeptide（PINP） and β isomer of C - terminal telopeptide of type I collagen（ β -CTX）were measured by using enzyme -linked immunosorbent assay（ ELISA）, and the BMD of lumbar spine were also detected by using dual - energy X - rays BMD radiomete. The pathological changes of bone tissues were observed under the microscope after the frozen sections were received HE staining. Results：There was statistical difference in the serum content of PINP between the 4 groups after the drug intervention（ F = 914. 448 ,P = 0.000）. Further pairwise comparison showed that the serum content of PINP of sham -opera- ted group was lower than that of model group（0. 109 ＋/-0. 008 vs 0. 252 ＋/-0.006 μg/mL,q = -51. 807,P =0.000）. There was no sta- tistical difference in the serum content of PINP between sham -operated group and BSHXF group（0. 163 ＋/-0. 006 μg/mL）and between sham - operated group and nylestriol group（0. 169 ＋/- 0.005 μg/mL） （ q = 0.559, P = 0. 208 ; q = 0. 850, P = 0. 253 ）. The serum content of PINP was higher in model group compared to BSHXF group and nylestriol group （ q = 32. 248, P = 0.000 ; q = 29. 957, P = 0. 000）. There was no statistical difference in the serum content of PINP between BSHXF group and nylestriol group （ q = - 0. 290, P = 0.317 ）. There was statistical difference in the serum content of 13 - CTX between the 4 groups after the drug intervention （ F = 963. 955, P = 0. 000）. Further pairwise comparison showed that the serum content of β - CTX of sham - operated group was lower than that of model group （0.432 ＋/- 0. 007 vs 0. 766 ＋/- 0.005 ng/mL, q = - 48. 601, P = 0.000）. There was no statistical difference in the serum content ofβ - CTX between sham -operated group and BSHXF group （0. 482 ＋/-0. 006 ng/mL）and between sham -operated group and nylestriol group （0.494 ＋/- 0.008 ng/mL） （q = -0. 172,P =0. 215;q =0. 980,P =0. 322）. The serum content of β - CTX was higher in model group compared to BSHXF group and nylestriol group （ q = 41. 429, P = 0. 000 ; q = 39. 622, P = 0. 000）. There was no statistical difference in the serum content of β - CTX between BSHXF group and nylestriol group （ q = - 0. 808, P = 0.790）. There was statistical difference in the BMD of lumbar spine between the 4 groups after the drug intervention（ F = 419. 969, P = 0. 000 ）. Further pairwise comparison showed that the BMD of lum- bar spine of sham -operated group was higher than that of model group （0. 170 ＋/-0. 004 vs 0. 097 ＋/-0.005 g/cm（ 2 ）, q = 35. 198, P = 0. 000）. There was no statistical difference in the BMD of lumbar spine between sham -operated group and BSHXF group （0. 127 ＋/- 0. 005 g/cm（2） ）and between sham - operated group and nylestriol group（0. 126 ＋/-0.004 g/cm（2） ） （q =2. 444 ,P =0. 157 ;q = 1. 167, P = 0. 230 ）. The BMD of lumbar spine was lower in model group compared to BSHXF group and nylestriol group （q = - 14. 754, P = 0. 000 ;q = -14. 031, P = 0. 000）. There was no statistical difference in the BMD of lumbar spine between BSHXF group and nylestriol group（ q = 0. 723 ,P = 0. 474）. The femoral bone trabecula in the sham -operated group was thick and arranged tightly, and their morpho- logical structure was complete and cancellous with normal density and area and small interspaces. The femoral bone trabeeula in the model group was thin, and some of them were distorted or ruptured with enlarged interspace. The femoral bone trabecula in BSHXF group and nylestriol group were thicker than that of model group and arranged tightly and their trabecular interspace was slightly larger in some regions compared to model group. Conclusion ： BSHXF can lower the serum content of PINP and β - CTX and increase the BMD and improve the status of bone tissue in the ovariectomized rats, while its specific mechanism is unclear and need to be further studied.