固本活血壮骨颗粒对糖皮质激素性骨质疏松症大鼠的作用与机制

The effect and mechanism of Guben Huoxue Zhuanggu Granules on glucocorticoid induced osteoporosis in rats

目的 观察固本活血壮骨颗粒对糖皮质激素性骨质疏松症(glucocorticoid-induced osteoporosis, GIOP)大鼠的作用与机制。方法 3月龄的Wistar大鼠随机分为正常组、模型组、中药组及西药组。采用试剂盒分别检测大鼠血清骨代谢指标钙(calcium, Ca)、磷(phosphorus, P)及碱性磷酸酶(alkaline phosphatase, ALP)含量;采用Micro-CT检测大鼠股骨骨密度(bone mine density, BMD)并分析骨小梁结构系数如骨小梁相对体积(BV/TV)、骨小梁厚度(Tb.Th)、骨表面积组织体积比值(BS/TV)、骨小梁数量(Tb.N)、骨小梁分离度(Tb.Sp);采用苏木精-伊红染色(hematoxylin-eosin staining, HE)观察各组大鼠股骨组织病理形态变化;采用蛋白印迹法(Western blot)检测股骨组织中磷酸化核转录因子κB-p65(phospho-nuclear transcription factor-κB p65,p-NF-κB p65)、活化T细胞核因子蛋白1(recombinant nuclear factor of activated T-cell, NFATc1)、β-连环蛋白(β-catenin)的表达,采用逆转录PCR(reverse transcription-polymerase chain reaction, RT-PCR)检测股骨组织中NFATc1、肿瘤坏死因子受体相关蛋白6(TNF receptor associated factor 6,TRAF6)、β-catenin的mRNA表达。结果 固本活血壮骨颗粒提高GIOP大鼠血清Ca值、血清P值、BMD值、BV/TV值、Tb.Th值、BS/TV值和Tb.N值(P<0.05),降低血清ALP值与Tb.Sp值(P<0.05);改善骨组织中β-catenin、NFATc1、TRAF6、p-NF-κB p65蛋白与mRNA表达(P<0.05)。另外,固本活血壮骨颗粒对GIOP大鼠血清ALP、股骨NFATc1的调节作用比阿法迪三更具优势(P<0.05)。结论 固本活血壮骨颗粒可拮抗GIOP,减少骨质丢失,其作用机制与Wnt/β-catenin通路和TRAF6/NF-κB p65/NFATcl信号通路有关。

Objective To observe the effect and mechanism of the constitution-consolidating,blood-activating,and bone-strengthening granules in rats with glucocorticoid-induced osteoporosis(GIOP).Methods Three-month Wistar rats were randomly divided into the normal group,the model group,the traditional Chinese medicine group,and the Western medicine group.The serum content of calcium(Ca),phosphorus(P),and alkaline phosphatase(ALP)were tested with kits,respectively.Bone mineral density(BMD)and a series of structural parameters of femoral trabeculae including relative volume of bone trabecula(BV/TV),trabecular thickness(Tb.Th),proportion of bone surface to bone volume(BS/TV),trabecular number(Tb.N),and trabecular separation(Tb.Sp)were detected with micro-CT.The pathological morphology of femur was observed by hematoxylin-eosin staining(HE staining);the protein expression of phospho-nuclear transcription factorκB-p65(p-NF-κB p65),recombinant nuclear factor of activated T-cell(NFATc1),andβ-catenin in the femur were test with Western blotting.mRNA expressions of NFATc1,TNF receptor associated factor 6(TRAF6),andβ-catenin in the femur were test with reverse transcription-polymerase chain reaction(RT-PCR).Results Constitution-consolidating,blood-activating and bone-strengthening granules increased the serum contents of Ca and P,BMD,BV/TV,Tb.Th,BS/TV,and Tb.N(P<0.05),and decreased the serum content of ALP and Tb.Sp(P<0.05).It also improved the protein and mRNA expressions ofβ-catenin,NFATcl,TRAF6,and p-NF-κB p65(P<0.05).Moreover,the constitution-consolidating,blood-activating and bone-strengthening granules treatment had more advantage in serum ALP and femoral NFATcl compared with ALPHA D 3 in GIOP rats(P<0.05).Conclusion The constitution-consolidating,blood-activating and bone-strengthening granules inhibit GIOP and reduce the bone loss via Wnt/β-catenin and TRAF6/NF-κB p65/NFATcl signal pathways.