运动加中药骨康对去卵巢大鼠骨质疏松治疗作用的研究
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摘要
目的:观察运动加中药骨康对去卵巢大鼠骨的影响,初步阐明运动加中药骨康治疗骨质疏松的作用机制。为组成一种有效对抗该病症的适宜“复合治疗方案”,提供必要的理论依据。
方法:根据文献资料本研究总结了近年来运动和中药在防治骨质疏松症中的应用概况,并在此基础上进行了相关的实验研究.实验方法:清洁级、6月龄、雌性SD大鼠72只随机分成正常对照组(SHAM)、去卵巢手术组(OVX)、雌激素对照组(OVX+ES)、运动组(OVX+ET),中药组(OVX+TCD),运动+中药组(OVX+ET+TCD)6组,每组置2只,正常对照组行假手术,其余各组摘除双侧卵巢。手术后正常饲养3月,经双能X线(DAX)检测证实已形成骨质疏松后,开始3个月的治疗。正常对照组、去卵巢手术组、运动组以蒸馏水10ml/kg灌胃,1次/天;中药组和运动+中药组均按4.8g/kg骨康(相当于临床剂量的10倍),灌胃给药,每天重次;雌激素对照组给予尼尔雌醇1mg/k8灌胃,1次/周,其余每天给予蒸馏水10ml/kg灌胃。运动组和运动+中药组按要求进行跑步运动。采用PT98型电动跑台进行运动训练,运动强度逐渐增大,两周内达到设计负荷(跑速为20～22米/分,坡度为5度,每天运动1小时。每周训练5天)。于大鼠处死前第13天和第14天,及处死前第3天和第4天分别皮下注射盐酸四环素25mg/kg和钙黄绿素5mg/kg,进行荧光标记。动物处死前1天,用双能X线进行活体骨密度和骨矿含量的检查。治疗结束后采用断头处死动物,取左股骨和左胫骨,做离体骨密度和骨矿含量的检查;取右胫骨和第2腰椎进行骨组织形态计量学检测;取右股骨以及第3腰椎做生物力学指标的测定;取子宫、左侧股直肌做组织切片检查;取右侧股直肌进行蛋白含量、抗氧化能力相关指标的检测。
结果:各测试指标的变化如下:①体重:去卵巢手术组的大鼠体重最重,运动+中药组的大鼠的体重在各实验组中最接近正常对照组,②骨密度:运动+中药组的大鼠全身和腰椎部的骨密度(BMD)提高非常明显.是各组中最高的,较卵巢摘除后大鼠分别增加了8.33%和12.44%(P<0.01);运动+中药组的大鼠股骨和胫骨部的骨密度,分别比去卵巢手术组提高了1.53%(P>0.05)和5.59%(P<0.05),其股骨增长幅度小于雌激素对照组和运动组(P>0.05),而胫骨增高幅度弱于单纯运动和单纯中药治疗(P>0.05)。③骨矿含量:各种治疗对去卵巢大鼠全身、腰椎、股骨的骨矿含量的影响不明显。经单纯运动、单纯中药骨康以及运动+中药骨康治疗后的去卵巢大鼠的胫骨部位的骨矿含量(BMC)有显著提高(P<0.05)。④骨组织形态计量学静态指标:运动+中药组和去卵巢手术组比较,胫骨的骨小梁面积(Tb.Ar)增大了103.94%(P<0.01),
Objective: Observing the effect of exercise training plus Chinese traditional medicine GuKang on the bones of ovariectomized rat, elementary clarifying the mechanism of using sports and Chinese traditional medicine GuKang to treat osteoporosis, aim to provide the theory foundation of forming an efficient "compound treatment scheme" .Methods: According to the literature information, this study summarized the treatment of using sports and Chinese traditional medicine to treat osteoporosis and did some relevant research based on it. Experimental methods: All 72 SD rats were cleaned and female and 6-month-old. The rats were randomly divided into 6 groups, each group had 12 rats: normal supplement group (SHAM), ovariectomized group(OVX), estrogen supplement group(OVX+ES), exercise training group (OVX+ET), traditional Chinese medicine group (OVX+TCD), exercise training plus traditional Chinese medicine group(OVX+ET+TCD). Carried ovariectomy in every group except the SHAM group. Three months later, made sure osteoporosis had appeared by dual energy X-ray absorptionmetry (DXA), then took another three months' treatment. Gave distilled water to the SHAM group, the OVX group and the OVX+ET group, once per day; gave GuKang 4.8g/kg (equal to ten times of normal clinic level) to the OVX+TCD group and the OVX+ET+TCD group, once per day; gave nylestriol 1mg/kg to the OVX+ES group, once per week, gave distilled water in the other days. Running as require in those two groups with exercise training. Using the PT98 running machine of electricity to train rats, the intensity of exercise training was increased gradually, to reach the designed demands (the speed of running was 20~22m/min, the slope was 5°, 1 hour per day, 5 days per week). Took hypodermic injection
with Tetracycline Hydrochloride 25mg/kg and Calcein 5mg/kg on 13 and 14 days, 3 and 4 days before execution, double in vivace fluorochrome labeling were administered to all rats. On the day before execution, used DXA to check the bone mineral density (BMD) and bone mineral contant (BMC). After execution, took out the left femur and tibia to check their BMD and BMC. Take the right tibia and the second lumbar vertebrae to check their bone histomorphometric. Took the right femur and the third lumbar vertebrae to check the biomechanics. Sliced up and checked the uterus, left straight muscle of thigh. Checked the content of albumen and antioxidation of the right straight muscle of thigh.Results: Changes of datum: (1) The body weight: The body weight of rat in the OVX group was most heavy and the body weight of rat in the OVX+ET+TCD group was most close to the SHAM group compared with the other groups. (2)The BMD : The BMD in the OVX+ET+TCD group increased by 8.33% and 12.44%(P<0.01) respectively in whole body and lumbar vertebrae, compared with the OVX group; BMD in the OVX+ET+TCD group increased by 1.53% (P>0.05) and 5.59% (P<0. 05) respectively in femur and tibia compared with the OVX group, the increase of femur was less than the OVX+ES group and the OVX+ET group (P>0. 05), the increase of tibia was less than the OVX+TCD group and the OVX+ET group (P>0. 05) . (3) The BMC: In whole body, lumbar and femur, the changes of BMC of ovariectomed rats through all treatments were not obvious. In tibia the BMC of ovariectomzed rat was increased remarkably by therapy of exercise training, traditional Chinese medicine GuKang and exercise training plus traditional Chinese medicine GuKang(P<0.05). (4) The index of motionless state of bone histomorphometric: Tibia' s trabecular area (Tb. Ar) increased by 103.94% (P<0. 01), the percent trabecular area (%Tb. Ar) increased by 110. 49% (P<0. 01), in the OVX+ET+TCD group compared with the OVX group , the range of increase was greater than the other treatment groups. The %Tb. Ar of lumbar vertebrae of the OVX+ET+TCD group increased by 17.00% (P>0. 05) compared with the OVX group, only less than the OVX+TCD group; Tibia' s trabecular perimeter (Tb. Pm) of the OVX+ET+TCD group increased by 80.28% (P<0.01) compared with the OVX group, only less than the OVX+ES group; Tibia' s trabecular width (Tb. Th) was the most width within all the groups, compared with the OVX group, Tb. Th of tibia increased by 19.63% (P>0.05) and the Tb.Th of lumbar vertebrae increased by 11. 30%(P>0.05); In the OVX+ET+TCD group, trabecular number (Tb. N)
of tibia increased by 83.99%(P<0.01) compared with the OVX group, only less than the OVX+ES group, and the Tb. N of lumbar vertebrae was the most compared with other treatment groups, increased by 7.11% (P>0. 05) compared with the OVX group; Trabecular sparation (Tb. SP) of tibia and lumbar vertebrae in the OVX+ET+TCD group decreased by 60.96%(P<0. 01)and 10.38% (P>0. 05) respectively, compared with the OVX group , was the best group within all treatment groups, was the only group that with the least difference with the SHAM group (P>0. 05) . (5) The index of motion state of bone histomorphometric: Percent label perimeter (%L.Pm) in the OVX+ET+TCD group, tibia and lumbar vertebrae increased by 100.38% and 59. 42%(P<0.05) respectively compared with the OVX+ES group, also greater than the SHAM group, the range increased in tibia was greater than the OVX+TCD group but less than the OVX+ET group, the range increased in lumbar vertebrae was greater than the OVX+ET group but less than the OVX+TCD group; The mineral apposition rate (MAR) of tibia and lumbar vertebrae in the OVX+ET+TCD group increased by 65.14% and 20. 28%(P<0. 05) respectively compared with the OVX+ES group, the range increased in tibia was greater than the OVX+TCD group, close to the SHAM group but less than the OVX+ET group, lumbar vertebrae MAR was the highest compared with the other treatment groups; Lumbar vertebrae' s bone formation rate (BFR) in the OVX+ET+TCD group was the highest compared with other treatment groups; bone formation rate /BS (BFR/BS), bone formation rate /BV (BFR/BV) , bone formation rate /TV (BFR/TV) of lumbar vertebrae in the OVX+ET+TCD group increased by 86.38% , 82.63% and 112.27%(P<0. 05) respectively compared with the OVX+ES group. Tibia' s BFR in the OVX+ET+TCD group was greater than the OVX+ES group, BFR/BS, BFR/BV, BFR/TV increased by 151.66%(P<0.05), 134.80%(P>0.05) and 153.15%(P<0.05) respectively, but less than the OVX+ET group and the OVX+TCD group, with distinctly difference compared with the OVX+ET group(P<0.01).Osteoclast number per ram (Oc. N/mm) in the OVX+ET+TCD group, tibia and lumbar vertebrae decreased by 18. 21%(P>0.05) and 0. 28%(P<0.01) respectively compared with the OVX group. Osteoclast number per mm~2 (0c. N/mm~2) in the OVX+ET+TCD group, tibia and lumbar vertebrae reduced by 32.51%(P<0.05) and 49.08%(P<0.01) respectively compared with the OVX group, the datum of tibia was quite close to the SHAM group (P>0.05), but quite lower in lumbar vertebrae (P<0. 01). (6) The tissue slice of bones : The volume, number and arrangement of trabecular became better in every treatment groups compared with the OVX group, especially
in the OVX+ET+TCD group, but still couldn' t reach the normal level. (7)The biomechanics of bone: Femoral neck structural mechanical strength in the OVX+ET+TCD group was greater than the OVX+ET group, but lower than the other groups; Lumbar vertebrae maximum compressive load and maximum resistant stress in the OVX+ET+TCD group increased by 19.16%(P<0. 01) compared with the OVX group, it' s function was better than other treatment groups, fund distinctive difference compared with the OVX+ET group (P<0. 01), but still hadn' t reached the normal level (P<0.01). (8) The tissue slice of uterus: Uterus shrink and thin, mucous membrane also shrink and thin, gland decreased, internal membrane increased in the OVX group, the OVX+ET group, the OVX+TCD group and the OVX+ET+TCD group; the thickness of internal membrane of uterus in the OVX+ET+TCD group reduced by 23. 73%(P<0. 01) and 12. 99%(P<0. 05) compared with the SHAM group and the OVX+ES group, the weight of uterus in the OVX+ET+TCD group reduced by 43.80%(P<0.01) and 71. 98%(P<0. 01) compared with the SHAM group and the OVX+ES group, these two datum had meaningless in statistics in the OVX+ET+TCD group and the OVX group. (9) Datum of straight muscle of thigh: The content of albumen and Superoxide Dismutase (SOD) activity in the OVX+ET+TCD group were the highest compared with the other groups, increased by 21.50% and 82. 35%(P<0. 01) respectively compared with the OVX group, and the SOD activity had distinctly difference with the other treatment groups (P<0.05). Glutathione Peroxidase (GSH-PX) activity in the OVX+ET+TCD group was higher than the OVX group (P>0.05), but lower than the OVX+TCD group and the OVX+ES group (P<0. 05) . The content of Maleic Dialdehyde (MDA) in the OVX+ET+TCD group was lower than the other groups, reduced by 51. 06%(P<0.01) compared with the OVX group, and with distinctly difference compared with the OVX+TCD group (P<0.01).Conclusion: (1)Thi s study take the 6-month SD rat as the object of experiment, 3 months later, the ovariectomed rat can reproduce the animal model of osteoporosis successfully. (2)The main cause of postmenopausal osteoporosis is lacking estrogen, this study take the nylestriol as positive drug. Estrogen supplement can rectify negative imbalance in bone remodeling process, but have obvious side effect on uterus. (3)The kinesitherapy can take good effects on bone of ovariectomed rat through many mechanisms, and it is no side effect on uterus. The medical supervision should be emphatic in process of
方法:根据文献资料本研究总结了近年来运动和中药在防治骨质疏松症中的应用概况,并在此基础上进行了相关的实验研究.实验方法:清洁级、6月龄、雌性SD大鼠72只随机分成正常对照组(SHAM)、去卵巢手术组(OVX)、雌激素对照组(OVX+ES)、运动组(OVX+ET),中药组(OVX+TCD),运动+中药组(OVX+ET+TCD)6组,每组置2只,正常对照组行假手术,其余各组摘除双侧卵巢。手术后正常饲养3月,经双能X线(DAX)检测证实已形成骨质疏松后,开始3个月的治疗。正常对照组、去卵巢手术组、运动组以蒸馏水10ml/kg灌胃,1次/天;中药组和运动+中药组均按4.8g/kg骨康(相当于临床剂量的10倍),灌胃给药,每天重次;雌激素对照组给予尼尔雌醇1mg/k8灌胃,1次/周,其余每天给予蒸馏水10ml/kg灌胃。运动组和运动+中药组按要求进行跑步运动。采用PT98型电动跑台进行运动训练,运动强度逐渐增大,两周内达到设计负荷(跑速为20～22米/分,坡度为5度,每天运动1小时。每周训练5天)。于大鼠处死前第13天和第14天,及处死前第3天和第4天分别皮下注射盐酸四环素25mg/kg和钙黄绿素5mg/kg,进行荧光标记。动物处死前1天,用双能X线进行活体骨密度和骨矿含量的检查。治疗结束后采用断头处死动物,取左股骨和左胫骨,做离体骨密度和骨矿含量的检查;取右胫骨和第2腰椎进行骨组织形态计量学检测;取右股骨以及第3腰椎做生物力学指标的测定;取子宫、左侧股直肌做组织切片检查;取右侧股直肌进行蛋白含量、抗氧化能力相关指标的检测。
结果:各测试指标的变化如下:①体重:去卵巢手术组的大鼠体重最重,运动+中药组的大鼠的体重在各实验组中最接近正常对照组,②骨密度:运动+中药组的大鼠全身和腰椎部的骨密度(BMD)提高非常明显.是各组中最高的,较卵巢摘除后大鼠分别增加了8.33%和12.44%(P<0.01);运动+中药组的大鼠股骨和胫骨部的骨密度,分别比去卵巢手术组提高了1.53%(P>0.05)和5.59%(P<0.05),其股骨增长幅度小于雌激素对照组和运动组(P>0.05),而胫骨增高幅度弱于单纯运动和单纯中药治疗(P>0.05)。③骨矿含量:各种治疗对去卵巢大鼠全身、腰椎、股骨的骨矿含量的影响不明显。经单纯运动、单纯中药骨康以及运动+中药骨康治疗后的去卵巢大鼠的胫骨部位的骨矿含量(BMC)有显著提高(P<0.05)。④骨组织形态计量学静态指标:运动+中药组和去卵巢手术组比较,胫骨的骨小梁面积(Tb.Ar)增大了103.94%(P<0.01),
Objective: Observing the effect of exercise training plus Chinese traditional medicine GuKang on the bones of ovariectomized rat, elementary clarifying the mechanism of using sports and Chinese traditional medicine GuKang to treat osteoporosis, aim to provide the theory foundation of forming an efficient "compound treatment scheme" .Methods: According to the literature information, this study summarized the treatment of using sports and Chinese traditional medicine to treat osteoporosis and did some relevant research based on it. Experimental methods: All 72 SD rats were cleaned and female and 6-month-old. The rats were randomly divided into 6 groups, each group had 12 rats: normal supplement group (SHAM), ovariectomized group(OVX), estrogen supplement group(OVX+ES), exercise training group (OVX+ET), traditional Chinese medicine group (OVX+TCD), exercise training plus traditional Chinese medicine group(OVX+ET+TCD). Carried ovariectomy in every group except the SHAM group. Three months later, made sure osteoporosis had appeared by dual energy X-ray absorptionmetry (DXA), then took another three months' treatment. Gave distilled water to the SHAM group, the OVX group and the OVX+ET group, once per day; gave GuKang 4.8g/kg (equal to ten times of normal clinic level) to the OVX+TCD group and the OVX+ET+TCD group, once per day; gave nylestriol 1mg/kg to the OVX+ES group, once per week, gave distilled water in the other days. Running as require in those two groups with exercise training. Using the PT98 running machine of electricity to train rats, the intensity of exercise training was increased gradually, to reach the designed demands (the speed of running was 20~22m/min, the slope was 5°, 1 hour per day, 5 days per week). Took hypodermic injection
with Tetracycline Hydrochloride 25mg/kg and Calcein 5mg/kg on 13 and 14 days, 3 and 4 days before execution, double in vivace fluorochrome labeling were administered to all rats. On the day before execution, used DXA to check the bone mineral density (BMD) and bone mineral contant (BMC). After execution, took out the left femur and tibia to check their BMD and BMC. Take the right tibia and the second lumbar vertebrae to check their bone histomorphometric. Took the right femur and the third lumbar vertebrae to check the biomechanics. Sliced up and checked the uterus, left straight muscle of thigh. Checked the content of albumen and antioxidation of the right straight muscle of thigh.Results: Changes of datum: (1) The body weight: The body weight of rat in the OVX group was most heavy and the body weight of rat in the OVX+ET+TCD group was most close to the SHAM group compared with the other groups. (2)The BMD : The BMD in the OVX+ET+TCD group increased by 8.33% and 12.44%(P<0.01) respectively in whole body and lumbar vertebrae, compared with the OVX group; BMD in the OVX+ET+TCD group increased by 1.53% (P>0.05) and 5.59% (P<0. 05) respectively in femur and tibia compared with the OVX group, the increase of femur was less than the OVX+ES group and the OVX+ET group (P>0. 05), the increase of tibia was less than the OVX+TCD group and the OVX+ET group (P>0. 05) . (3) The BMC: In whole body, lumbar and femur, the changes of BMC of ovariectomed rats through all treatments were not obvious. In tibia the BMC of ovariectomzed rat was increased remarkably by therapy of exercise training, traditional Chinese medicine GuKang and exercise training plus traditional Chinese medicine GuKang(P<0.05). (4) The index of motionless state of bone histomorphometric: Tibia' s trabecular area (Tb. Ar) increased by 103.94% (P<0. 01), the percent trabecular area (%Tb. Ar) increased by 110. 49% (P<0. 01), in the OVX+ET+TCD group compared with the OVX group , the range of increase was greater than the other treatment groups. The %Tb. Ar of lumbar vertebrae of the OVX+ET+TCD group increased by 17.00% (P>0. 05) compared with the OVX group, only less than the OVX+TCD group; Tibia' s trabecular perimeter (Tb. Pm) of the OVX+ET+TCD group increased by 80.28% (P<0.01) compared with the OVX group, only less than the OVX+ES group; Tibia' s trabecular width (Tb. Th) was the most width within all the groups, compared with the OVX group, Tb. Th of tibia increased by 19.63% (P>0.05) and the Tb.Th of lumbar vertebrae increased by 11. 30%(P>0.05); In the OVX+ET+TCD group, trabecular number (Tb. N)
of tibia increased by 83.99%(P<0.01) compared with the OVX group, only less than the OVX+ES group, and the Tb. N of lumbar vertebrae was the most compared with other treatment groups, increased by 7.11% (P>0. 05) compared with the OVX group; Trabecular sparation (Tb. SP) of tibia and lumbar vertebrae in the OVX+ET+TCD group decreased by 60.96%(P<0. 01)and 10.38% (P>0. 05) respectively, compared with the OVX group , was the best group within all treatment groups, was the only group that with the least difference with the SHAM group (P>0. 05) . (5) The index of motion state of bone histomorphometric: Percent label perimeter (%L.Pm) in the OVX+ET+TCD group, tibia and lumbar vertebrae increased by 100.38% and 59. 42%(P<0.05) respectively compared with the OVX+ES group, also greater than the SHAM group, the range increased in tibia was greater than the OVX+TCD group but less than the OVX+ET group, the range increased in lumbar vertebrae was greater than the OVX+ET group but less than the OVX+TCD group; The mineral apposition rate (MAR) of tibia and lumbar vertebrae in the OVX+ET+TCD group increased by 65.14% and 20. 28%(P<0. 05) respectively compared with the OVX+ES group, the range increased in tibia was greater than the OVX+TCD group, close to the SHAM group but less than the OVX+ET group, lumbar vertebrae MAR was the highest compared with the other treatment groups; Lumbar vertebrae' s bone formation rate (BFR) in the OVX+ET+TCD group was the highest compared with other treatment groups; bone formation rate /BS (BFR/BS), bone formation rate /BV (BFR/BV) , bone formation rate /TV (BFR/TV) of lumbar vertebrae in the OVX+ET+TCD group increased by 86.38% , 82.63% and 112.27%(P<0. 05) respectively compared with the OVX+ES group. Tibia' s BFR in the OVX+ET+TCD group was greater than the OVX+ES group, BFR/BS, BFR/BV, BFR/TV increased by 151.66%(P<0.05), 134.80%(P>0.05) and 153.15%(P<0.05) respectively, but less than the OVX+ET group and the OVX+TCD group, with distinctly difference compared with the OVX+ET group(P<0.01).Osteoclast number per ram (Oc. N/mm) in the OVX+ET+TCD group, tibia and lumbar vertebrae decreased by 18. 21%(P>0.05) and 0. 28%(P<0.01) respectively compared with the OVX group. Osteoclast number per mm~2 (0c. N/mm~2) in the OVX+ET+TCD group, tibia and lumbar vertebrae reduced by 32.51%(P<0.05) and 49.08%(P<0.01) respectively compared with the OVX group, the datum of tibia was quite close to the SHAM group (P>0.05), but quite lower in lumbar vertebrae (P<0. 01). (6) The tissue slice of bones : The volume, number and arrangement of trabecular became better in every treatment groups compared with the OVX group, especially
in the OVX+ET+TCD group, but still couldn' t reach the normal level. (7)The biomechanics of bone: Femoral neck structural mechanical strength in the OVX+ET+TCD group was greater than the OVX+ET group, but lower than the other groups; Lumbar vertebrae maximum compressive load and maximum resistant stress in the OVX+ET+TCD group increased by 19.16%(P<0. 01) compared with the OVX group, it' s function was better than other treatment groups, fund distinctive difference compared with the OVX+ET group (P<0. 01), but still hadn' t reached the normal level (P<0.01). (8) The tissue slice of uterus: Uterus shrink and thin, mucous membrane also shrink and thin, gland decreased, internal membrane increased in the OVX group, the OVX+ET group, the OVX+TCD group and the OVX+ET+TCD group; the thickness of internal membrane of uterus in the OVX+ET+TCD group reduced by 23. 73%(P<0. 01) and 12. 99%(P<0. 05) compared with the SHAM group and the OVX+ES group, the weight of uterus in the OVX+ET+TCD group reduced by 43.80%(P<0.01) and 71. 98%(P<0. 01) compared with the SHAM group and the OVX+ES group, these two datum had meaningless in statistics in the OVX+ET+TCD group and the OVX group. (9) Datum of straight muscle of thigh: The content of albumen and Superoxide Dismutase (SOD) activity in the OVX+ET+TCD group were the highest compared with the other groups, increased by 21.50% and 82. 35%(P<0. 01) respectively compared with the OVX group, and the SOD activity had distinctly difference with the other treatment groups (P<0.05). Glutathione Peroxidase (GSH-PX) activity in the OVX+ET+TCD group was higher than the OVX group (P>0.05), but lower than the OVX+TCD group and the OVX+ES group (P<0. 05) . The content of Maleic Dialdehyde (MDA) in the OVX+ET+TCD group was lower than the other groups, reduced by 51. 06%(P<0.01) compared with the OVX group, and with distinctly difference compared with the OVX+TCD group (P<0.01).Conclusion: (1)Thi s study take the 6-month SD rat as the object of experiment, 3 months later, the ovariectomed rat can reproduce the animal model of osteoporosis successfully. (2)The main cause of postmenopausal osteoporosis is lacking estrogen, this study take the nylestriol as positive drug. Estrogen supplement can rectify negative imbalance in bone remodeling process, but have obvious side effect on uterus. (3)The kinesitherapy can take good effects on bone of ovariectomed rat through many mechanisms, and it is no side effect on uterus. The medical supervision should be emphatic in process of
引文
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