杜仲汤对去卵巢大鼠骨质疏松症的影响及机制研究

Study on the Effect and Mechanism of Eucommiae Cortex Decoction on Osteoporosis in Ovariecto⁃mized Rats

目的探讨杜仲汤对去卵巢大鼠骨质疏松症的影响及机制。方法将42只雌性SD大鼠随机分为假手术组、模型组、阳性药组(戊酸雌二醇,0.09 mg·kg^(-1))、谷胱甘肽组(36 mg·kg^(-1))以及杜仲汤低、中、高剂量组(0.54、1.08、2.16 g·kg^(-1))。去卵巢模型大鼠复制成功后,按照设定剂量灌胃给药,灌胃体积为10 mL·kg^(-1),每日1次,连续给药200 d。采用生化试剂盒检测大鼠血清中谷胱甘肽过氧化物酶(GSH)含量;ELISA法检测大鼠血清中抗酒石酸酸性磷酸酶(TRACP)、骨保护素(OPG)含量;骨密度仪检测大鼠股骨骨密度;HE染色法观察大鼠胫骨组织病理变化;Western Blot法检测骨组织中骨形态发生蛋白2(BMP-2)、OPG、一型胶原(COL1)蛋白表达水平;RT-PCR法检测骨组织中BMP-2、Runt相关转录因子2(RUNX2)mRNA表达水平。结果与假手术组比较,模型组大鼠体质量增长值显著升高(P<0.01);血清GSH、OPG的水平显著降低(P<0.05,P<0.01),TRACP水平显著升高(P<0.01);骨密度明显降低(P<0.05);骨小梁结构不完整,出现明显断裂,小梁数稀疏且明显变薄;骨组织中BMP-2、OPG、COL1蛋白表达水平均显著下降(P<0.01),BMP-2、RUNX2mRNA表达水平明显降低(P<0.05)。与模型组比较,杜仲汤低、中、高剂量组大鼠体质量增长值均显著降低(P<0.05,P<0.01),血清GSH水平显著升高(P<0.01),骨小梁结构明显改善,骨小梁变粗,数量增加,骨组织中BMP-2、RUNX2 mRNA表达水平均显著升高(P<0.01);杜仲汤中剂量组大鼠的血清OPG水平显著升高(P<0.01);杜仲汤低剂量组大鼠的血清TRACP水平显著降低(P<0.01),骨组织中COL1蛋白表达水平明显升高(P<0.05);杜仲汤低、中剂量组大鼠的骨密度显著升高(P<0.01),骨组织中OPG蛋白表达水平明显升高(P<0.01);杜仲汤中、高剂量组大鼠骨组织中BMP-2蛋白表达水平明显升高(P<0.05)。结论杜仲汤能提高去卵巢大鼠的骨密度,保护骨小梁完整性,对去卵巢大鼠骨质疏松症有一定防治作用,其机制可能与提高血清GSH、OPG水平,降低TRACP水平,上调BMP-2、OPG、COL1蛋白以及BMP-2、RUNX2mRNA表达,从而促进骨形成有关。

To investigate the effect and mechanism of Eucommiae Cortex Decoction on osteoporosis in ovariectomized rats.Methods Forty-two female SD rats were randomly divided into sham operation group,model group,positive drug group(Estradiol Valerate,0.09 mg·kg^(-1)),glutathione group(36 mg·kg^(-1)),and low-,medium-and high-dose of Eucommiae Cortex Decoction groups(0.54,1.08,2.16 g·kg^(-1)).After the successful replication of ovariectomized model rats,the rats were given intragastric administration at a set dose of 10 mL·kg^(-1),once a day for 200 days.The content of glutathione peroxidase(GSH)in serum of rats was detected by biochemical kit.The contents of tartrate-resistant acid phosphatase(TRACP)and osteoprotegerin(OPG)in serum of rats were detected by ELISA.Bone mineral density of rat femur was detected by bone densitometer.The pathological changes of tibial bone tissue in rats were observed by HE staining.The mRNA expression levels of bone morphogenetic protein 2(BMP-2),OPG and type I collagen(COL1)in bone tissue were detected by Western Blot.The mRNA expression levels of BMP-2 and Runt-related transcription factor 2(RUNX2)in bone tissue were detected by RT-PCR.Results Compared with the sham operation group,the body mass growth values of rats in the model group was significantly increased(P<0.01).The levels of serum GSH and OPG were significantly decreased(P<0.05,P<0.01),and the level of TRACP was significantly increased(P<0.01).Bone mineral density was significantly decreased(P<0.05).The structure of trabecular bone was incomplete,with obvious fracture,and the number of trabecular bone was sparse and significantly thinned.The protein expression levels of BMP-2,OPG and COL1 in bone tissue were significantly decreased(P<0.01).The mRNA expressions of BMP-2 and RUNX2 were decreased(P<0.05).Compared with the model group,the body mass growth values of rats in the low-,medium-and high-dose groups of Eucommiae Cortex Decoction were significantly decreased(P<0.05,P<0.01),the serum GSH level was significantly increased(P<0.01),the trabecular structure was significantly improved,the trabecular bone was thickened,the number was increased,and the mRNA expression levels of BMP-2 and RUNX2 in bone tissue were significantly increased(P<0.01).The serum OPG level of rats in the medium-dose group of Eucommiae Cortex Decoction was significantly increased(P<0.01).The serum TRACP level of rats in the low-dose group was significantly decreased(P<0.01),and the protein expression level of COL1 in bone tissue was significantly increased(P<0.05).The bone mineral density of rats in the low-and medium-dose groups of Eucommiae Cortex Decoction was significantly increased(P<0.05,P<0.01),and the protein expression level of OPG in bone tissue was significantly increased(P<0.01).The protein expression level of BMP-2 in bone tissue of rats in the medium-and high-dose groups of Eucommiae Cortex Decoction was significantly increased(P<0.05).Conclusion Eucommiae Cortex Decoction can improve the bone mineral density of ovariectomized rats,protect the integrity of trabecular bone,and has a certain preventive and therapeutic effect on osteoporosis in ovariectomized rats.The mechanism may be related to the increase of serum GSH and OPG levels,the decrease of TRACP level,the up-regulation of BMP-2,OPG,COL1 proteins and mRNA expressions of BMP-2,RUNX2,thus promoting bone formation.