菟丝子丸调控BMP2/RUNX2信号通路抗绝经后骨质疏松研究

Study on the regulation of BMP2/RUNX2 signaling pathway by Cuscuizi Pills in the treatment of postmenopausal osteoporosis

关键词:  绝经后骨质疏松症  菟丝子  牛膝  成骨相关因子

Key words:  postmenopausal osteoporosis  cuscuta  hyssop  osteogenesis-related factors

摘要:

[目的] 探讨菟丝子丸（CP）对绝经后骨质疏松症（PMOP）的影响与机制。[方法] 使用双侧卵巢摘除法（OVX）建立绝经后骨质疏松症小鼠模型。将小鼠随机分为假手术组（Sham组）、模型组（OVX组）、雌激素39 μg/kg组（Estrogen组），菟丝子4 g/kg组（T 4 g/kg组）、牛膝4 g/kg组（N 4 g/kg组）、菟丝子丸8 g/kg组（CP 8 g/kg组），每组10只，连续给药12周。双能X射线骨密度仪检测小鼠股骨骨密度（BMD）；YLS-16A小动物骨骼强度测定仪测定胫骨强度；Viva CT40检测股骨骨微结构；苏木精-伊红（HE）染色观察股骨组织病理形态学；酶联免疫吸附（ELISA）法检测血浆中碱性磷酸酶（ALP）、骨特异性碱性磷酸酶（BALP）、骨保护素（OPG）的含量；蛋白免疫印迹法（Western Bolt）检测去势小鼠骨髓中OPG、RUNT相关转录因子2（RUNX2）和骨成型蛋白2（BMP2）蛋白表达。[结果] 与Sham组比较，OVX组小鼠股骨BMD、胫骨骨强度均显著降低（P<0.01），骨小梁大量缺失且骨骼结构稀疏，小鼠血浆中ALP、BALP、OPG水平以及骨髓中OPG、RUNX2和BMP2蛋白表达显著降低（P<0.01），而CP 8 g/kg组显著升高OVX小鼠的股骨BMD及胫骨骨强度（P<0.01），显著增加血浆中成骨相关因子ALP、BALP和OPG等的水平，提高骨组织中RUNX2、OPG、BMP2蛋白表达（P<0.01），且其作用显著高于菟丝子与牛膝单独给药（P<0.05）。[结论] 菟丝子丸显著抑制去势导致的骨丢失，上调成骨相关因子BALP、RUNX2，OPG表达，提示其抗绝经后骨质疏松作用可能是通过BMP2/RUNX2信号通路促进骨形成发挥的。

Abstract:

[Objective] To investigate the effect and mechanism of Cuscuta pills（CP） on postmenopausal osteoporosis（PMOP）. [Methods] A mouse model of postmenopausal osteoporosis was established using bilateral ovarian extraction（OVX）. The mice were randomly divided into sham operation group（Sham），model group（OVX），estrogen 39 μg/kg group（Estrogen），Cuscuta 4 g/kg group（T 4 g/kg），hyssop 4 g/kg group（N 4 g/kg），and Cuscuta pill 8 g/kg group（CP 8 g/kg），which were administered continuously for 12 weeks， with 10 animals in each group. Dual-energy X-ray absorptiometry was used to detect mice’s bone mineral density（BMD）. YLS-16A small animal bone strength tester to measure tibial strength；viva CT40 to detect femoral bone microstructure；hematoxylin-eosin（HE） staining was used to observe the pathological morphology of bone tissue. Enzyme-linked immunosorbent assay（ELISA） was used to detect the contents of alkaline phosphatase（ALP），bone-specific alkaline phosphatase（BALP），and osteoproteectin（OPG） in plasma. Western Bolt was used to detect the expression of OPG，RUNX2 and BMP2 proteins in the bone marrow of castrated mice. [Results] Compared with the Sham group，the BMD and tibial bone strength of the femur in the OVX group were significantly reduced（P<0.01），a large number of trabecular bones were missing and the bone structure was sparse，the levels of ALP，BALP and OPG in plasma and the protein expression of OPG，RUNX2 and BMP2 in bone marrow were significantly reduced（P<0.01），while CP 8g/kg group significantly increased the femoral BMD and tibial bone strength of OVX mice（P<0.01），significantly increased the levels of osteogenesis-related factors ALP，BALP and OPG in plasma，and increased the protein expressions of RUNX2，OPG and BMP2 in bone tissue（P<0.01），and its effect is significantly higher than that of Cuscuta chinensis and hyssop treated alone（P<0.05）. [Conclusion] Cuscuta pills significantly inhibited bone loss caused by castration. They up-regulated the expression of osteogenesis-related factors BALP，RUNX2，and OPG，suggesting that its anti-postmenopausal osteoporotic effect may be played by promoting bone formation through the BMP2/RUNX2 signaling pathway.