肌骨宁对去势大鼠骨质疏松及肌萎缩的保护作用研究

Study on Protective Effect of Jiguning on Osteoporosis and Muscular Atrophy in Castrated Rats

目的:探讨去势对大鼠骨骼和肌肉的影响,以及肌骨宁对去势大鼠骨质疏松及肌萎缩的保护作用。方法:将60只6月龄SPF级SD大鼠随机分为正常组、假手术组、模型组、肌骨宁组(2.083 g·kg^(-1))和雌激素组(0.104 mg·kg^(-1)),每组12只。除正常组及假手术组外,其余大鼠采用去势法切除双侧卵巢建立去势大鼠模型,假手术组切除卵巢附近等量脂肪组织。造模3个月后,开始药物干预,正常组、假手术组、模型组大鼠每天灌胃10 mL·kg^(-1)生理盐水,连续灌胃3个月后,使用大小鼠抓力仪每周定时测量大鼠前爪抓力;分析天平称取子宫、肝脏和肾脏的质量;双能X线骨密度仪检测大鼠全身骨密度(bone mineral density,BMD)、股骨BMD、瘦肉量、脂肪量、骨矿量和四肢瘦肉量,计算大鼠全身骨矿含量(bone mineral content,BMC)、脂肪含量百分比(fat content,FAT)、骨骼肌质量指数(skeletal mass index,SMI)和相对骨骼肌质量指数(RSMI);micro CT检测骨体积分数(bone volume fraction,BVF,BV/TV)、骨表面密度(bone surface density,BS/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁数(trabecular number,Tb.N)和骨小梁分离度(trabecular separation,Tb.Sp);ELISA法检测大鼠血清碱性磷酸酶(alkaline phosphatase,AKP)、I型前胶原氨基端肽(type I procollagen amino terminal peptide,PINP)、I型胶原交联羧基末端肽(type I collagen cross-linked carboxy terminal peptide,β-CTx)、胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)、雌二醇(estradiol,E_(2))含量;HE染色观察股骨远端和腓肠肌的病理变化。结果:与正常组比较,模型组大鼠子宫质量降低,BMC下降,FAT增加,SMI、RSMI、BMD及股骨BMD、BV/TV、BS/BV、BS/TV、Tb.Th和Tb.N下降,股骨Tb.Sp升高,AKP、IGF-1和E_(2)水平下降,β-CTx水平增加;与模型组比较,肌骨宁组BMC升高,FAT降低,SMI、RSMI、BMD及股骨BMD、BV/TV、BS/BV、BS/TV、Tb.Th和Tb.N升高,股骨Tb.Sp降低,AKP、IGF-1和E_(2)水平增加,β-CTx水平下降,差异均具有统计学意义(P<0.05)。HE染色显示,肌骨宁组大鼠股骨骨小梁数量明显增加,厚度增加,分离度减小,骨髓脂肪化减轻;腓肠肌肌纤维排列紧密,肌间隙减小,肌纤维增粗,肌细胞核增多增大。结论:肌骨宁可有效防治骨质疏松和肌萎缩,“肌骨同治”,强肌壮骨,为骨质疏松合并肌萎缩患者提供了中医方案。

Objective:To investigate the effect of castration on bone and muscle in rats and the protective effect of Jiguning(肌骨宁,JGN)on osteoporosis and muscular atrophy in castrated rats.Methods:A total of 60 SPF-grade SD rats aged 6 months were randomly divided into the normal group,the sham operation group,the model group,the JGN group(2.083 g·kg^(-1))and the estrogen group(0.104 mg·kg^(-1)),with 12 rats in each group.Except the normal group and the sham operation group,the remaining rats were treated with castration method to remove bilateral ovaries to establish castration rat models,and the same amount of adipose tissue near the ovaries was removed in the sham operation group.After 3 months of modeling,drug intervention was started.Rats in the normal group,the sham operation group and the model group were given 10ml/kg normal saline every day,and after continuous gavage for 3 months,the grasping force of the rats'forepaws was measured with a mouse gripping instrument every week.The analytical balance was used to weigh the uterus,liver and kidneys.The whole body bone mineral density(BMD),femur BMD,lean meat mass,fat mass,bone mineral mass and limb lean mass of rats were measured by dual-energy X-ray bone densitometer,and the whole body bone mineral content(BMC)of rats,FAT content(FAT),skeletal mass index(SMI)and RSMI were calculated.By using micro CT,bone volume fraction(BVF,BV/TV),bone surface density(BS/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N)and trabecular separation(Tb.Sp)were analyzed.By using ELISA,the contents of alkaline phosphatase(AKP),type I procollagen amino terminal peptide(PINP),type I collagen cross-linked carboxy terminal peptide(β-CTx),insulin-like growth factor 1(IGF-1)and estradiol(E,)were detected by ELISA.The pathological changes of distal femur and gastrocnemius were observed by HE staining.Results:Compared with the normal group,the wet weight of uterus in the model group decreased,BMC decreased,FAT increased,SMI,RSMI,BMD and femoral BMD,BV/TV,BS/TV,Tb.Th and Tb.N decreased,femoral Tb.Sp increased,AKP,IGF-1 and E,levels decreased,β-CTx levels increased.Compared with the model group,in the JGN group,BMC increased,FAT decreased,SMI,RSMI,BMD and femoral BMD,BV/TV,BS/BV,BS/TV,Tb.Th and Tb.N increased,femoral Tb.Sp decreased,AKP,IGF-1 and E,levels increased,whileβ-CTx levels decreases,and all the differences were statistically significant(P<0.05).HE staining showed that in the JGN group,the number of femoral bone trabeculae increased significantly,the thickness increased,the separation degree decreased,and the bone marrow fat decreased;Gastrocnemius muscle fibers were closely arranged,muscle space was reduced,muscle fibers were thickened,and muscle nuclei increased and enlarged.Conclusion:JGN can effectively prevent and cure osteoporosis and muscle atrophy."simultaneous treatment of muscle and bone"can strengthen muscles and bones,which provides a TCM program for patients with osteoporosis combined with muscle atrophy.