目的 基于网络药理学及体内外实验探讨补骨脂素治疗绝经后骨质疏松症的作用机制。方法 通过SwissTargetPrediction、PharmMapper、Super-PRED、GEO、DAVID数据库筛选补骨脂素、绝经后骨质疏松症的作用靶点。采用基因集富集分析（GSEA）方法评估基因集功能富集特征。采用AutoDock Vina 1.1.2软件对潜在靶点与补骨脂素进行分子对接。细胞实验培养人骨髓间充质干细胞（BMSCs），进行相关基因的过表达、沉默，进行碱性磷酸酶（ALP）/Bodipy染色及活性检测。通过ELISA方法检测成骨分化标志物骨形态发生蛋白2（BMP2）和成脂分化标志物过氧化物酶体增殖物激活受体γ（PPARγ）的蛋白表达水平。动物实验通过卵巢切除术构建小鼠绝经后骨质疏松症模型，进行骨苏木素–伊红（HE）染色、micro-CT检测。通过标准曲线计算BMP2、PPARγ相对表达量。结果 通过网络药理学数据库分析，发现补骨脂素治疗绝经后骨质疏松症的7个潜在靶基因，包括肌球蛋白ⅤA（MYO5A）、赖氨酸特异性去甲基化酶5C（KDM5C）、组织蛋白酶D（CTSD）、丝裂原激活蛋白激酶激酶2（MAP2K2）、免疫球蛋白重链常数γ1（IGHG1）、肾素结合蛋白（RENBP）、α-生育酚转运蛋白基因（TTPA）。细胞实验结果显示，补骨脂素明显抑制了沉默TTPA造成的BMSCs成骨/成脂分化失衡（P＜0.05、0.01）。相对于p38 MAPK-IN-1抑制剂组，TTPA过表达（OE-TTPA）组使BMP2蛋白表达升高，而PPARγ蛋白表达显著降低（P＜0.01）。动物实验结果显示，补骨脂素组骨小梁数目及厚度明显增加，股骨头内空洞减少，小鼠股骨密度明显升高。与模型组比较，补骨脂素组骨密度（BMD）和骨体积分数（BV/TV）明显升高（P＜0.01）；小鼠股骨成骨相关蛋白BMP2含量显著升高，成脂相关蛋白PPARγ含量显著降低（P＜0.05、0.01）。结论 补骨脂素靶向TTPA介导的p38 MAPK信号通路，调控BMSCs成骨/成脂分化平衡及绝经后骨质疏松症发生。

Objective To explore the mechanism of psoralen in treatment of postmenopausal osteoporosis based on network pharmacology and in vivo and in vitro experiments. Methods The targets of psoralen and postmenopausal osteoporosis were screened through the databases of SwissTargetPrediction, PharmMapper, Super-PRED, GEO and DAVID. The functional enrichment characteristics of gene sets were evaluated by using the GSEA method. Molecular docking of potential targets with psoralen was performed using AutoDock Vina 1.1.2 software. BMSCs were cultured in cell experiments. Overexpression and silencing of related genes were carried out, and ALP/Bodipy staining and activity detection were performed. The protein expression levels of osteogenic differentiation marker BMP2 and adipogenic differentiation marker PPARγ were detected by ELISA method. Animal experiments were conducted to establish a mouse model of postmenopausal osteoporosis through oophorectomy, and bone HE staining and micro-CT detection were performed. The relative expression levels of BMP2 and PPARγ were calculated through the standard curve. Results Through the analysis of the network pharmacology database, seven potential target genes of psoralen in treatment of postmenopausal osteoporosis were discovered, including MYO5A, KDM5C,CTSD, MAP2K2, IGHG1, RENBP, and TTPA. The results of cell experiments showed that psoralen significantly inhibited the osteogenic/adipogenic differentiation imbalance of BMSCs caused by silencing TTPA (P < 0.05, 0.01). Compared with the p38 MAPK-IN-1 inhibitor group, the TTPA overexpression (OE-TTPA) group increased the expression of BMP2 protein, while significantly decreased the expression of PPARγ protein (P < 0.01). The results of animal experiments showed that the number and thickness of trabeculae in the psoralen group significantly increased, the internal cavities of the femoral head decreased, and the femoral density of mice significantly increased. Compared with the model group, the BMD and BV/TV in the psoralen group were significantly increased (P < 0.01); The content of osteogenesis-related protein BMP2 in the femur of mice was significantly increased, while the content of adipogenesis-related protein PPARγ was decreased (P < 0.05, 0.01). Conclusions Psoralen targets the TTPA-mediated p38 MAPK signaling pathway to regulate the osteogenic/adipogenic differentiation balance of BMSCs and the occurrence of postmenopausal osteoporosis.

R285.5;R287.2

潍坊市卫生健康委员会中医药科研项目（WFZYY2023-2-002）