目的 评价补肾通络方对骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）成骨、成脂分化以及小鼠单核巨噬细胞RAW264.7破骨分化模型的干预作用，并探究其调节骨形成及骨吸收的相关机制。方法 分别给予补肾方、通络方和补肾通络方进行干预，采用茜素红染色法鉴定BMSCs成骨分化后各组钙结节形成情况；采用油红O染色鉴定BMSCs成脂分化后各组脂滴形成情况；采用qRT-PCR法检测BMSCs成骨、成脂分化后各组成骨、成脂分化标志基因mRNA表达，成骨、成脂分化过程和炎症模型中chemerin mRNA表达；采用ELISA法检测BMSCs成骨、成脂分化过程和炎症模型中各组chemerin分泌量。采用抗酒石酸酸性磷酸酶（tartrate-resistant alkaline phosphatase，TRAP）染色鉴定各组RAW264.7细胞破骨分化程度，并测定各组TRAP活性；采用Western blotting法检测RAW264.7细胞破骨分化中各组TRAP蛋白表达情况；采用qRT-PCR法及ELISA法检测RAW264.7细胞破骨分化后各组chemerin、白细胞介素-6（interleukin-6，IL-6）及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）mRNA表达及分泌。结果 与对照组比较，补肾通络方组钙结节显著增多（P<0.01），脂滴显著减少（P<0.01），成骨分化标志基因表达显著升高（P<0.01），成脂分化标志基因表达显著下降（P<0.05），chemerin分泌及基因表达在成骨分化第3天及成脂分化第3、7天显著下降（P<0.05、0.01）。与对照组比较，BMSCs炎症模型chemerin分泌及基因表达显著升高（P<0.01）；与BMSCs炎症模型比较，补肾通络方组chemerin分泌及基因表达显著下降（P<0.01）。与对照组比较，RAW264.7细胞破骨分化中TRAP阳性细胞、活力、蛋白表达以及IL-6、TNF-α分泌与基因表达均显著升高（P<0.05、0.01）；与模型组比较，补肾通络方组TRAP阳性细胞、活力、蛋白表达以及IL-6、TNF-α分泌与基因表达显著降低（P<0.01）。结论 补肾通络方可通过抑制chemerin及炎症因子来调节成骨、成脂分化及破骨细胞分化，从而促进骨形成、抑制骨吸收、改善骨质疏松症。

Objective To evaluate the effect of Bushen Tongluo Formula (补肾通络方) on osteogenesis, adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and RAW264.7 cells osteoclast differentiation model, to explore its related mechanisms for regulating bone formation and bone resorption. Methods Bushen Formula, Tongluo Formula and Bushen Tongluo Formula were given for intervention. Alizarin red staining method was used to identify the formation of calcium nodules in each group after osteogenic differentiation of BMSCs; Oil red O staining was used to identify lipid droplet formation in each group after adipogenic differentiation of BMSCs; qRT-PCR was used to detect mRNA expressions of bone formation and adipogenic differentiation marker genes after osteogenesis and adipogenic differentiation of BMSCs, chemerin mRNA expression during osteogenesis and adipogenic differentiation and inflammation models; ELISA was used to detect chemerin secretion in each group of BMSCs osteogenic, adipogenic differentiation and inflammation models. Tartrate-resistant alkaline phosphatase (TRAP) staining was used to identify the degree of osteoclast differentiation of RAW264.7 cells in each group, and TRAP activity in each group was determined; Western blotting was used to detect TRAP protein expression in osteoclast differentiation of RAW264.7 cells; qRT-PCR and ELISA methods were used to detect chemerin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA expressions and secretion after osteoclast differentiation of RAW264.7 cells in each group. Results Compared with control group, calcium nodules in Bushen Tongluo Formula group were significantly increased (P < 0.01), lipid droplets were reduced (P < 0.01), osteogenic differentiation markers gene expressions were increased (P < 0.01), adipogenic differentiation markers gene expressions were decreased (P < 0.05), chemerin secretion and gene expression were decreased on 3rd day of osteogenic differentiation and 3rd, 7th day of adipogenic differentiation (P < 0.05, 0.01). Compared with control group, chemerin secretion and gene expression in BMSCs inflammation model were increased (P < 0.01); Compared with BMSCs inflammation model, chemerin secretion and gene expression in Bushen Tongluo Formula group were decreased (P < 0.01). Compared with control group, TRAP positive cells, activity and protein expression, IL-6, TNF-α secretions and gene expressions were increased in osteoclast differentiation of RAW264.7 cells (P < 0.05, 0.01); Compared with model group, TRAP positive cells, activity and protein expression, IL-6, TNF-α secretions and gene expressions in Bushen Tongluo Formula group were reduced (P < 0.01). Conclusion Bushen Tongluo Formula can regulate osteogenesis, adipogenic differentiation and osteoclast differentiation by inhibiting chemerin and inflammatory factors, thereby promoting bone formation, inhibiting bone resorption and improving osteoporosis.

R285.5

国家自然科学基金资助项目（81774335）；国家自然科学基金资助项目（81473390）；江苏省研究生科研与实践创新计划项目（KYCX19_1212）；国家重点研发计划项目（2019YFC1709905）