目的 探讨特女贞苷对绝经后骨质疏松症（PMOP）的防治作用及其分子作用机制。方法 体外培养成骨细胞与核因子κB受体活化因子配体（RANKL）诱导的RAW264.7细胞（破骨细胞），加入0（对照组）、5、10μmol·L^-1的特女贞苷溶液处理72 h，茜素红染色观察特女贞苷对成骨细胞成熟和分化的影响；实时荧光定量PCR（qRT-PCR）和Western blotting检测成骨细胞Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）、Osterix、骨保护素（OPG）/RANKL mRNA和蛋白表达；qRT-PCR检测破骨细胞中基质金属蛋白酶9（MMP9）、c-FOS、活化T细胞核因子c1（NFATc1）、组织蛋白酶K（CTSK）mRNA水平，Western blotting检测破骨细胞中c-FOS、NFATc1、肿瘤坏死因子受体相关因子6（TRAF6）、p-磷脂酰肌醇3-激酶（PI3K）/PI3K、p-蛋白激酶B（Akt）/Akt蛋白表达；体内实验建立去卵巢大鼠PMOP模型，给予特女贞苷低、高剂量(1、2 mg·kg^-1)干预8周，Masson染色观察胫骨结构；Micro-CT检测胫骨骨小梁数量（TB.N）、骨小梁相对体积（BV/TV）、骨小梁厚度（TB.Th）、骨小梁分离指数（TB.Sp）;ELISA法检测外周血中骨特异性碱性磷酸酶（BALP）、抗酒石酸酸性磷酸酶5b（TRAP-5b）、CTSK、β-骨胶原交联（β-CTX）、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6水平；qRT-PCR检测骨组织中Runx2、Osterix、OPG/RANKL、NFATc1、c-FOS、TRAF6 mRNA水平；Western blotting检测骨组织中NFATc1、c-FOS、TRAF6蛋白表达。结果 成骨细胞：与对照组比较，特女贞苷5、10μmol·L^-1组矿化度显著增加（P<0.05）,Runx2、ALP、Osterix、OPG/RANKL mRNA和蛋白水平显著升高（P<0.05、0.01、0.001）。破骨细胞：与对照组比较，特女贞苷组的MMP9、c-FOS、NFATc1、CTSK mRNA水平显著下降（P<0.05、0.01）,c-FOS、NFATc1、TRAF6、p-PI3K/PI3K、p-Akt/Akt蛋白表达显著下调（P<0.05、0.01）。动物实验：与模型组比较，特女贞苷低、高剂量组骨小梁面积明显上升（P<0.01）;TB.Th、TB.N和BV/TV值显著提高（P<0.05、0.01）,TB.Sp值显著降低（P<0.01）；外周血中BALP水平显著上升（P<0.01）,TRAP-5b水平显著下降（P<0.01、0.001）,CTSK、β-CTX水平显著降低（P<0.01）,TNF-α、IL-6水平显著降低（P<0.05、0.01、0.001）；骨组织中Runx2、Osterix、OPG/RANKL mRNA水平显著上升（P<0.001）,NFATc1、c-FOS、TRAF6 mRNA水平显著下降（P<0.01、0.001）;NFATc1、c-FOS、TRAF6蛋白含量显著下降（P<0.05、0.01）。结论 特女贞苷通过调控RANKL/RANK及PI3K/Akt信号通路，发挥促成骨、抑破骨的双重作用。

Objective To investigate the preventive and therapeutic effects of specnuezhenide on postmenopausal osteoporosis（PMOP） and its molecular mechanism. Methods Osteoblasts and RAW264.7 cells（osteoclasts） induced by receptor activator of nuclear factor-κB ligand（RANKL） were cultured in vitro. The cells were treated with 0（control group）, 5, and 10 μmol·L^-1 loganin solution for 72 h. Alizarin red staining was used to observe the effects of loganin on osteoblast maturation and differentiation. Realtime fluorescence quantitative PCR（qRT-PCR） and Western blotting were used to detect the mRNA and protein expression of Runtrelated transcription factor 2（Runx2）, alkaline phosphatase（ALP）, Osterix, osteoprotegerin（OPG）/RANKL in osteoblasts. qRT-PCR was used to detect the mRNA levels of matrix metalloproteinase 9（MMP9）, c-FOS, nuclear factor of activated T cells c1（NFATc1）, and cathepsin K（CTSK） in osteoclasts. Western blotting was used to detect the protein expression of c-FOS, NFATc1, tumor necrosis factor receptor-associated factor 6（TRAF6）, p-phosphatidylinositol 3-kinase（PI3K）/PI3K, and p-protein kinase B（Akt）/Akt in osteoclasts. In vivo experiments were conducted to establish a PMOP model in ovariectomized rats. Low and high doses of loganin(1 and 2 mg·kg^-1) were administered for 8 weeks. Masson staining was used to observe the tibial structure. Micro-CT was used to detect the trabecular bone number（TB.N）, trabecular bone volume fraction（BV/TV）, trabecular thickness（TB.Th）, and trabecular separation（TB.Sp） of the tibia. ELISA was used to detect the levels of bone-specific alkaline phosphatase（BALP）, tartrate-resistant acid phosphatase 5b（TRAP-5b）, CTSK, β-C-terminal telopeptide of type I collagen（β-CTX）, tumor necrosis factor（TNF）-α, and interleukin（IL）-6 in peripheral blood. qRT-PCR was used to detect the mRNA levels of Runx2, Osterix, OPG/RANKL, NFATc1, cFOS, and TRAF6 in bone tissue. Western blotting was used to detect the protein expression of NFATc1, c-FOS, and TRAF6 in bone tissue. Results Osteoblasts: Compared with the control group, the mineralization degree in the 5 and 10 μmol·L^-1 loganin groups was significantly increased（P ＜ 0.05）, and the mRNA and protein levels of Runx2, ALP, Osterix, and OPG/RANKL were significantly increased（P ＜ 0.05, 0.01, 0.001）. Osteoclasts: Compared with the control group, the mRNA levels of MMP9, c-FOS, NFATc1, and CTSK in the loganin groups were significantly decreased（P ＜ 0.05, 0.01）, and the protein expression of c-FOS, NFATc1, TRAF6, pPI3K/PI3K, and p-Akt/Akt was significantly downregulated（P ＜ 0.05, 0.01）. Animal experiments: Compared with the model group, the trabecular bone area in the low and high-dose loganin groups was significantly increased（P ＜ 0.01）; TB.Th, TB.N, and BV/TV values were significantly increased（P ＜ 0.05, 0.01）, and TB.Sp values were significantly decreased（P ＜ 0.01）. The levels of BALP in peripheral blood significantly increased（P ＜ 0.01）, while the levels of TRAP-5b significantly decreased（P ＜ 0.01, 0.001）, and the levels of CTSK and β-CTX significantly decreased（P ＜ 0.01）. The levels of TNF-α and IL-6 also significantly decreased（P ＜ 0.05, 0.01, 0.001）. In bone tissue, the mRNA levels of Runx2, Osterix, and OPG/RANKL significantly increased（P ＜ 0.001）, while the mRNA levels of NFATc1, c-FOS, and TRAF6 significantly decreased（P ＜ 0.01, 0.001）. The protein contents of NFATc1, c-FOS, and TRAF6 also significantly decreased（P ＜ 0.05, 0.01）. Conclusion Specnue exerts dual effects of promoting osteogenesis and inhibiting osteoclastogenesis by regulating the RANKL/RANK and PI3K/AKT signaling pathways.

R285.5

福建省卫健委中医处科技计划项目(2021zyyj58); 福建省自然科学基金项目(2022J01131153); 福建省教育厅2022年度福建省中青年教师教育科研项目(科技类)(JAT220136)