目的 探讨大黄素调控核因子 E2相关因子 2（Nrf2）表达对脂多糖（LPS）诱导 RAW264.7细胞破骨分化的 作用及机制。方法 采用 0.1 μg·mL-1 LPS 诱导 RAW264.7 细胞破骨分化，并用大黄素（1、3、9 μmol·L-1）进行 干预，培养72 h。采用CCK-8法检测RAW264.7细胞的生存率；抗酒石酸酸性磷酸酶（TRAP）染色法检测破骨细 胞数目；比色法测定破骨细胞丙二醛（MDA）水平、TRAP及超氧化物歧化酶（SOD）活性；荧光探针检测破骨细 胞活性氧（ROS）和谷胱甘肽（GSH）水平；免疫荧光法检测破骨细胞 F-actin 环的形成和 Nrf2 核转位情况；RTqPCR法检测破骨细胞相关基因（c-Fos、NFATc1、Nrf2、c-Src）的表达；Western Blot法检测破骨细胞Nrf2蛋白 表达水平。结果 1~9 μmol·L-1浓度的大黄素均无明显细胞毒性作用，故以 9 μmol·L-1为最高给药浓度。空白 组未观察到破骨细胞，而 LPS组的破骨细胞数量明显增多且最多，出现边界清晰、厚且完整的 F-actin环。与 空白组比较，LPS 组破骨细胞的 ROS、MDA 水平明显升高（P＜0.01），GSH、SOD 水平明显下降（P＜0.01）； Nrf2总蛋白、核蛋白表达均明显下调（P＜0.01）；Nrf2在细胞核中表达下调，核浆比明显降低（P <0.01）。与LPS 组比较，大黄素各浓度组的破骨细胞数量明显减少（P＜0.05，P＜0.01），TRAP 活性明显降低（P＜0.05，P＜ 0.01）；F-actin 环变薄变小，缺失甚至消失；c-Fos、NFATc1 mRNA 表达明显下调（P＜0.05，P＜0.01），Nrf2 mRNA 表达明显上调（P＜0.05，P＜0.01）；ROS、MDA 水平明显下降（P＜0.05，P＜0.01），GSH、SOD 水平明 显升高（P＜0.05，P＜0.01）；Nrf2 核蛋白表达均明显上调（P＜0.05，P＜0.01）。大黄素中、高浓度组细胞的 c-Src mRNA表达明显下调（P＜0.01）；Nrf2总蛋白表达明显上调（P＜0.01）；Nrf2在细胞核中表达上调，核浆比 明显升高（P＜0.01）。结论 大黄素能抑制 LPS 诱导 RAW264.7 细胞的破骨分化能力，可能与调控 Nrf2 表达， 抑制氧化应激有关。

Objective To explore the effect and mechanism of emodin on lipopolysaccharide （LPS）-induced osteoclastic differentiation in RAW264.7 cells based on nuclear factor E2-related factor 2（Nrf2）expression. Methods Osteoclastic differentiation of RAW264.7 cells was induced by 0.1 μg·mL-1 LPS and interfered by emodin（1， 3， 9 μmol·L-1）for 72 h. The cell viability of RAW264.7 cells was determined by CCK-8 assay. Tartrate-resistant acid phosphatase（TRAP）staining was used to determine the number of osteoclast. Malondialdehyde（MDA）level，TRAP and superoxide dismutase（SOD）activity of osteoclast were measured by colorimetry. The levels of reactive oxygen species（ROS）and glutathione（GSH）were detected by fluorescence analysis. The formation of F-actin loop and nuclear translocation of Nrf2 were observed by immunofluorescence. The expression levels of osteoclast-related genes （c-Fos， NFATc1， c-Src and Nrf2）were detected by RT-qPCR， and the protein expression of Nrf2 was analyzed by Western Blot. Results Emodin at concentrations of 1~9 μmol·L-1 has no cytotoxic effects. Therefore the highest administration concentration is 9 μmol·L-1. No osteoclasts were observed in the blank group，the number of osteoclasts were significantly increased in LPS group. A well-defined， thick and intact F-actin loop appeared. Compared with blank group，the levels of ROS and MDA were significantly increased（P＜0.01）and the levels of GSH and SOD were decreased（P＜0.01）. The expression of total protein and the nuclear protein of Nrf2 were decreased in LPS group （P＜0.01）. Nrf2 expression was down-regulated in the nucleus，and the nuclear-cytoplasmic ratio was significantly reduced（P＜0.01）. Compared with LPS group， the number of osteoclasts were significantly decreased in emodin groups at different concentrations（P＜0.05，P＜0.01）. TRAP activity was obviously decreased（P＜0.05，P＜0.01）. F-actin loop was found thin，small，missing or even disappearing. The mRNA expression of c-Fos and NFATc1 were significantly down-regulated（P＜0.05，P＜0.01），while Nrf2 mRNA expression significantly up-regulated（P＜0.05， P＜0.01）. The levels of ROS and MDA were obviously decreased（P＜0.05，P＜0.01），the levels of GSH and SOD were significantly increased（P＜0.05，P＜0.01）. The expression of the nuclear protein of Nrf2 was significantly upregulated（P＜0.05，P＜0.01）. The mRNA expression of c-Src in medium- and high- concentration groups of emodin was significantly down-regulated（P＜0.01）. The total protein expression of Nrf2 was obviously increased（P＜0.01）. Nrf2 expression was up-regulated in the nucleus，and the nuclear-cytoplasmic ratio was significantly increased（P＜ 0.01）. Conclusion Emodin can inhibit the osteogenic differentiation ability of LPS-induced RAW264.7 cells，which may be related to the regulation of Nrf2 expression and the inhibition of oxidative stress.

R285.5

广东省中医药局科研项目（20251217）；广州市科学技术局创新环境项目（2060702）