右归丸调控BMP-2/Smad信号通路促进绝经后骨质疏松症大鼠骨形成

Mechanism of Youguiwan Regulating BMP-2/Smad Signaling Pathway to Promote Bone Formation in Postmenopausal Rats with Osteoporosis

目的:观察右归丸对去卵巢骨质疏松大鼠的骨代谢及骨形态发生蛋白-2(BMP-2)/Smad信号通路的影响,研究右归丸防治骨质疏松症的作用机制。方法:采用双侧卵巢摘除法,制备绝经后骨质疏松症大鼠模型,将40只SD雌性大鼠随机分为5组,分别为假手术组、模型组、阿仑膦酸钠组(0.1 mg·kg^(-1))、右归丸高、低剂量组(5.36、2.68 g·kg^(-1))。造模7 d后给药,连续12周,每日1次。给药结束后,采用微计算机断层扫描技术(micro-CT)观察大鼠股骨组织结构变化,包括骨密度(BMD)、骨体积/总体积(BV/TV)、骨小梁数(Tb.N)、骨小梁厚度(Tb.Th)、骨表面/骨体积(BS/BV)和骨小梁分离度(Tb.Sp)。番红-固绿染色观察成骨情况。酶联免疫吸附测定法(ELISA)检测血清中骨代谢标志物水平,包括骨碱性磷酸酶(BALP)、骨钙素(BGP)、Ⅰ型前胶原氨基端原肽(PINP)和抗酒石酸酸性磷酸酶-5b(TRACP-5b)。实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测大鼠股骨中Runt相关转录因子2(Runx2)、BMP-2和Smad1 mRNA及蛋白表达水平。结果:与假手术组比较,模型组大鼠骨小梁变稀疏,BMD、BV/TV、Tb.N及Tb.Th下降(P<0.05,P<0.01),BS/BV(P<0.05)及Tb.Sp上升;血清中BGP、BALP、PINP和TRACP-5b含量显著升高(P<0.01);大鼠股骨中Runx2、BMP-2和Smad1的mRNA及蛋白表达明显降低(P<0.05,P<0.01);与模型组比较,右归丸高剂量组与右归丸低剂量组骨小梁数目增加,骨微结构得到改善,BMD、BV/TV、Tb.N及Tb.Th均明显增加(P<0.05,P<0.01),BS/BV及Tb.Sp有上升趋势;骨代谢标志物含量下降(P<0.05,P<0.01),骨组织中Runx2、BMP-2和Smad1 mRNA和蛋白水平明显升高(P<0.05,P<0.01)。结论:右归丸对绝经后骨质疏松症具有一定的防治作用,其作用机制可能与调控BMP-2/Smad信号通路促进骨形成有关。

Objective:To observe the effects of Youguiwan on bone metabolism and bone morphogenetic protein-2(BMP-2)/Smad signaling pathway in ovaries-removed rats with osteoporosis and study the mechanism of Youguiwan in the prevention and treatment of osteoporosis.Method:A postmenopausal rat model of osteoporosis was prepared by bilateral ovariectomy.The 40 female SD rats were randomly divided into five groups,including sham operation group,model group,alendronate sodium group(0.1 mg·kg^(-1)),and highdose and low-dose(5.36 and 2.68 g·kg^(-1))groups of Youguiwan.The drug was given seven days after modeling,once a day for 12 weeks.After the treatment,the changes in femur tissue structure were observed by micro-CT,including bone mineral density(BMD),bone volume/total volume(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th),bone surface/bone volume(BS/BV),and trabecular separation(Tb.Sp).Ossification was observed by saffrane-solid green staining,and serum levels of bone metabolism markers,including bone alkaline phosphatase(BALP),osteocalcin(BGP),typeⅠprocollagen amino terminal propeptide(PINP),and tartrate-resistant acid phosphatase 5b(TRACP-5b),were determined by enzyme-linked immunosorbent assay(ELISA).The protein and mRNA expression levels of Runx2,BMP-2,and Smad1 in rat femur were detected by Western blot and Real-time fluorescence quantitative polymerase chain reaction(Realtime PCR).Result:Compared with the sham operation group,bone trabecula in the model group was sparse.BMD,BV/TV,Tb.N,and Tb.Th were decreased(P<0.05,P<0.01).BS/BV(P<0.05)and Tb.Sp were increased.The content of BGP,BALP,PINP,and TRACP-5b in serum was significantly increased(P<0.01).The mRNA and protein expressions of Runx2,BMP-2,and Smad1 in rat femur were significantly decreased(P<0.05,P<0.01).Compared with the model group,the number of bone trabeculae in the high-dose and low-dose groups of Youguiwan was increased,and the bone microstructure was improved.BMD,BV/TV,Tb.N,and Tb.Th were increased significantly(P<0.05,P<0.01),and BS/BV and Tb.Sp were increased.The content of bone metabolic markers decreased(P<0.05,P<0.01).Conclusion:Youguiwan has certain preventive and therapeutic effects on postmenopausal osteoporosis,and its mechanism may be related to promoting bone formation by regulating the BMP-2/Smad signaling pathway.