【摘要】 该研究基于骨形态发生蛋白-2(bone morphogenetic protein-2, BMP-2)/SMAD4信号通路探讨龙牙楤木皂苷Ⅳ(tarasaponin Ⅳ, TRS)对踝关节骨折(AF)大鼠骨重建及破骨细胞形成的影响。构建AF模型大鼠,并将其随机分为AF组、TRS低浓度组(TRS-L组)、TRS中浓度组(TRS-M组)、TRS高浓度组(TRS-H组)、TRS-H+BMP特异抑制剂(Noggin)组,并设立未进行骨折的正常大鼠为假手术组(Sham组)。酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测各组大鼠血清中骨重建标志物[Runt相关转录因子-2(Runt-related transcription factor 2, Runx2)、BMP-2、碱性磷酸酶(alkaline phosphatase, ALP)、核因子κB受体活化因子配体(receptor activator of nuclear factor kappa B ligand, RANKL)]的水平;微计算机断层扫描(Micro-CT)检测各组大鼠骨小梁微结构变化[骨密度(bone mineral density, BMD)、骨体积分数(bone volume fraction, BV/TV)、骨小梁数(number of trabeculae, Tb.N)、骨小梁厚度(thickness of trabeculae, Tb.Th)];苏木素–伊红(hematoxylin-eosin,HE)和抗酒石酸磷酸酶(tartrate resistant acid phosphatase, TRAP)染色法分别检测各组大鼠骨痂组织形态学病理变化及破骨细胞的数量; Western blot检测各组大鼠Runx2、ALP、RANKL、BMP-2和SMAD4蛋白表达水平。与Sham组相比, AF组大鼠Runx2、BMP-2、ALP、SMAD4表达水平及BMD、BV/TV、Tb.N、Tb.Th水平显著降低, RANKL表达水平和破骨细胞数量显著升高,踝骨刚度和最大载荷明显降低(P<0.05);与AF组相比, TRS-L、TRS-M、TRS-H组大鼠Runx2、BMP-2、ALP、SMAD4表达水平及BMD、BV/TV、Tb.N、Tb.Th水平逐渐升高, RANKL表达水平和破骨细胞数量逐渐降低,踝骨刚度和最大载荷逐渐升高,且各指标变化程度与TRS浓度呈正比(P<0.05);与TRS-H组相比, TRS-H+Noggin组大鼠Runx2、BMP-2、ALP、SMAD4表达水平及BMD、BV/TV、Tb.N、Tb.Th水平显著降低, RANKL表达水平和破骨细胞数量显著升高,踝骨刚度和最大载荷明显降低(P<0.05)。TRS可能通过激活BMP-2/SMAD4信号通路,促进AF大鼠骨重建并抑制破骨细胞的形成。更多还原

【Abstract】 This study investigates the effects of TRS(tarasaponin Ⅳ) on bone reconstruction and osteoclast formation in rats with AF(ankle fractures) via the BMP-2(bone morphogenetic protein-2)/SMAD4 signaling pathway. AF models were established in rats, which were then randomly divided into the AF group, TRS-L(low-concentration TRS) group, TRS-M(medium-concentration TRS) group, TRS-H(high-concentration TRS) group, and TRS-H+Noggin(BMP-specific inhibitor) group. Additionally, a sham operation group(Sham group) consisting of normal rats without fractures was included. ELISA(enzyme linked immunosorbent assay) was used to detect bone remodeling markers [Runx2(Runt-related transcription factor 2), BMP-2, ALP(alkaline phosphatase), RANKL(receptor activator of nuclear factor kappa B ligand)] in serum of rats in each group. Micro-CT was used to detect the microstructural changes of bone trabeculae of rats in each group [BMD(bone mineral density), BV/TV(bone volume fraction), Tb.N(number of trabeculae), and Tb.Th(thickness of trabeculae)]. The HE(hematoxylin-eosin) and TRAP(tartrate resistant acid phosphatase) staining methods were used to detect the morphological and pathological changes of callus tissue and the number of osteoclasts of rats in each group. Western blot was used to detect the Runx2, ALP, RANKL, BMP-2, and SMAD4 proteins in each group. Compared with the Sham group, the AF group showed great decreased in Runx2, BMP-2, ALP, SMAD4, BMD, BV/TV, Tb.N, Tb.Th, great increases in RANKL and osteoclast count, and conspicuous decreased in ankle stiffness and maximum load(P<0.05). Compared with the AF group, the TRS-L, TRS-M, and TRS-H groups showed gradual increases in Runx2, BMP-2, ALP, SMAD4, BMD, BV/TV, Tb.N, Tb.Th in rats, gradual decreased in RANKL and osteoclast count, and gradual increases in ankle stiffness and maximum load, and the degree of change in each indicator was directly proportional to TRS concentration(P<0.05). Compared with the TRS-H group, the TRS-H+Noggin group showed conspicuous decreased in Runx2, BMP-2, ALP, SMAD4, BMD, BV/TV, Tb.N, Tb.Th, conspicuous increased in RANKL and osteoclast count, and great decreases in ankle stiffness and maximum load(P<0.05). TRS may promote bone remodeling and inhibit osteoclast formation in AF rats by activating BMP-2/SMAD4 signaling pathway.更多还原