仙灵骨葆胶囊对大鼠骨质疏松性骨折愈合过程中骨形态发生蛋白2和血小板衍生生长因子表达的影响

The Effect of Xianling Gubao Capsule on Bone Morphogenetic Protein 2 and Expression of Platelet-derived Growth Factor in Healing Process of Rat with Osteoporotic Fracture

目的观察仙灵骨胶囊葆对大鼠骨质疏松性骨折(OF)愈合过程中骨形态发生蛋白2(BMP-2)和血小板衍生生长因子(PDGF)表达的影响并探讨其机制。方法雄性SD大鼠96只,先将其中72只采用手术切除双侧睾丸的同时颈部皮下注射D-半乳糖(100 mg/kg)以建立大鼠骨质疏松模型,8周后再手术折断右侧股骨干中段制备骨质疏松性骨折模型,成模后随机分为模型组、睾酮组和干预组(各24只)。另24只不手术造模,设为假手术组对照。干预组给予仙灵骨葆胶囊[250 mg/(kg·d)]灌胃治疗18周,睾酮组给予十一酸睾酮[2 mg/(kg·d)]灌胃作为阳性对照,假手术组和模型组灌等量0.9%氯化钠注射液18周。分别于治疗第5、9、18周取大鼠静脉血采用酶联免疫吸附法测定大鼠静脉血PDGF、前列腺环素(PGI2)和前列腺素E2(PGE2)含量,取骨痂和关节软骨组织采用免疫组织化学染色法分别检测大鼠BMP-2及基质金属蛋白酶抑制因子-1(TIMP-1)的表达,采用骨密度仪测量大鼠骨密度(BMD)。结果模型组第5、9、18周BMD降低,PDGF、PGI2和PGE2均升高,与假手术组比较差异有统计学意义(P<0.05)。睾酮组第5、9、18周BMD均明显提高,PGI2和PGE2在第5、9周明显升高,第18周时PGI2和PGE2则明显降低,与模型组比较差异有统计学意义(P<0.05)。干预组第18周时BMD明显提高,与模型组比较差异有统计学意义(P<0.05),但BMD提高程度无睾酮组明显(P<0.05);PDGF在第5、9周明显升高,BMP-2和TIMP-1在第9、18周均高表达,与假手术组、模型组和睾酮组比较差异有统计学意义(P<0.05),但PDGF在第18周时明显降低,与睾酮组与模型组比较差异有统计学意义(P<0.05);PGI2和PGE2在第5、9周明显升高,但两者升高程度无睾酮组明显,与假手术组、模型组和睾酮组比较差异有统计学意义(P<0.05),第18周时两者则明显降低,与模型组比较差异有统计学意义(P<0.05)。结论仙灵骨葆胶囊提高雄性去势骨质疏松性骨折大鼠BMD的作用与十一酸睾酮相似,但这一作用主要通过调节骨质疏松性骨折愈合不同阶段BMP-2、TIMP-1和PDGF的表达与PGI2和PGE2合成来影响骨折的修复。

Objective： To investigate the effects of Xianling Gubao Capsule on the expression of bone morphogenetic protein 2（BMP-2） and platelet derived growth factor（PDGF） during the healing process of osteoporotic fracture（OF）. Methods： 96 male Sprague-Dawley rats were randomly divided into two groups. The first group of72 SD rats,which were treated by bilateral excision of the testis and the other by subcutaneous injection of D-galactose（100 mg/kg）. The middle right femur fracture was made to prepare osteoporotic fracture models. After modeling,the rats were randomly divided into the model group,testosterone group and intervention group（24 rats in each）. Another 24 with non-surgical modeling,was set as the control group sham operation. The intervention group was given Xianling Gubao Capsule [250 mg/（kg·d）] intragastrically for 18 weeks and the testosterone group was administered with testosterone undecanoate [20 mg/（kg·d）] as the positive control. The sham operation group and the model group received equal volume of normal saline for 18 weeks as negative control. The venous blood samples of rats were collected at the 5 th,9 th and 18 th week of treatment respectively. The levels of PDGF,PGI2 and PGE2 in venous blood of rats were measured by enzyme-linked immunosorbent assay. The callus and articular cartilage The expression of BMP-2 and TIMP-1 in rats was detected by immunohistochemical staining,and the bone mineral density（BMD） was measured by bone densitometry. Results： The BMD decreased at the5 th,9 th and 18 th week in the model group,while the levels of PDGF,PGI2 and PGE2 were increased in the model group compared with the sham operation group（P〈0.05）. The serum levels of PGD2 and PGE2 in the testosterone group were significantly increased at the 5 th,9 th and 18 th weeks;PGI2 and PGE2 were significantly increased at the 5 th and 9 th week;PGI2 and PGE2 at the 18 th week were significantly lower than those in the model group（P〈0.05）. Compared with the model group（P〈0.05）,BMD in the intervention group was significantly increased at the 18 th week（P〈0.05）,but there was no significant difference between the test group and the testosterone group（P〈0.05） The expression of BMP-2 and TIMP-1 were all significantly higher in the 9 th and18 th weeks than those in the sham operation group,the model group and the testosterone group（P〈0.05）,but the level of PDGF in the 18 th week was significantly lower than that in the testosterone group and the model group（P〈0.05）. PGI2 and PGE2 were significantly increased at 5 and 9 weeks,but both of them were higher in testosterone-free group than in the sham operation group,the model group and testosterone group（P〈0.05）. Both were significantly lower,compared with the model group（P〈0.05）. Conclusion： Xianling Gubao Capsule enhances the function of BMD in male osteoporotic fracture rats,which is similar to testosterone,but this effect is mainly through the regulation of different stages of osteoporotic fracture healing