目的 探究熟地黄的大麻素2型受体（cannabinoid receptor 2，CB2R）激动活性及其对骨代谢的调控作用。方法 采用双荧光素酶报告基因建立CB2R调节剂筛选体系；分别给予熟地黄提取物或CB2R选择性激动剂HU308或CB2R反向激动剂AM630处理，采用CCK-8法测定成骨细胞和破骨细胞的增殖活性；采用磷酸苯二钠法测定成骨细胞碱性磷酸酶（alkaline phosphatase，ALP）和破骨细胞抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）的活性；采用流式细胞仪测定成骨细胞的细胞周期；采用ALP染色观察成骨细胞分化；采用茜素红染色观察成骨细胞骨矿化结节的形成；采用TRAP染色观察破骨细胞数目；采用罗丹明-鬼笔环肽染色镜观察破骨细胞F-肌动蛋白（F-actin）环的结构和形态；采用Western blotting检测CB2R及骨代谢相关蛋白的表达。结果 500 μg/mL熟地黄显著升高HEK293-CB2R细胞CB2R表达（P＜0.001），抑制forskolin刺激的HEK293-CB2R细胞中环磷酸腺苷（cyclic adenosine monophosphate，cAMP）的产生，并且其对CB2R的激动活性可被AM630逆转（P＜0.001）。500 μg/mL熟地黄显著促进成骨细胞增殖（P＜0.001），升高ALP活性（P＜0.001），促进骨矿化结节形成，上调CB2R及骨形成相关蛋白的表达（P＜0.05、0.01、0.001），抑制p38的表达（P＜0.05）；抑制破骨细胞形成分化，降低TRAP活性（P＜0.001），抑制F-actin环的形成，下调CB2R、p-p38和骨吸收相关蛋白的表达（P＜0.05、0.001）；熟地黄对成骨细胞和破骨细胞的作用可被AM630逆转（P＜0.05、0.01、0.001）。结论 熟地黄具有特异性的CB2R激动活性，并可通过CB2R调控成骨细胞和破骨细胞的功能。

Objective To investigate the agonistic activity of Shudihuang (Rehmanniae Radix Praeparata, RRP) on cannabinoid receptor 2 (CB2R) and its regulatory effects on bone metabolism. Methods The CB2R regulator screening system was established by using double luciferase reporter gene. After treatment with RRP extract or CB2R selective agonist HU308 or CB2R inverse agonist AM630, the proliferation of osteoblasts and osteoclasts was detected by CCK-8. The activities of alkaline phosphatase (ALP) in osteoblasts and tartrate resistant acid phosphatase (TRAP) in osteoclasts were determined by disodium phenylphosphate method. The cell cycle of osteoblasts was determined by flow cytometry. The osteoblastic differentiation was observed by ALP staining. Alizarin red staining was used to observe the formation of bone mineralized nodules in osteoblasts. The number of osteoclasts was observed by TRAP staining. The structure and morphology of F-actin ring in osteoclasts were stained with rhodamine-phalloidin and observed with laser confocal microscopy. The expressions of CB2R and related proteins with bone metabolism were detected by Western blotting. Results 500 μg/mL RRP significantly increased CB2R expression in HEK293-CB2R cells (P < 0.001), inhibited the production of cyclic adenosine monophosphate (cAMP) in HEK293-CB2R cells stimulated by forskolin, and its agonistic activity on CB2R could be reversed by AM630 (P < 0.001). 500 μg/mL RRP significantly promoted the proliferation of osteoblasts (P < 0.001), increased the activity of ALP (P < 0.001), promoted the formation of bone mineralized nodules, and up-regulated the expressions of CB2R and bone formation-related proteins (P < 0.05, 0.01, 0.001), inhibited the expression of p38 (P < 0.05); 500 μg/mL RRP inhibited the formation and differentiation of osteoclasts, decreased TRAP activity (P < 0.001), inhibited the formation of F-actin ring, down-regulated CB2R, p-p38 and bone resorption-related proteins expressions (P < 0.05, 0.001); The effect of RRP on osteoblasts and osteoclasts could be reversed by AM630 (P < 0.05, 0.01, 0.001). Conclusion RRP has specific CB2R agonistic activity, and can regulate the functions of osteoblasts and osteoclasts through CB2R.

国家自然科学基金资助项目(81973534)