Hu Letao, Luo Meimei, Zhu Xingyu, Wang Zheng, Yan Cuiping, Wu Li, Zhu Hui, Yang Zijing, Xin Yatong, Feng Shuo, Wu Yu, Li Weidong
School of Pharmacy, Nanjing University of Chinese Medicine, Jiangsu, Nanjing, 210023, China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Jiangsu, Nanjing, 210023, China.
Department of Pharmacy, General Hospital of Eastern Theatre Command, Jiangsu, Nanjing, 210018, China.
Phytomedicine. 2025 Nov 25;148:157319. doi: 10.1016/j.phymed.2025.157319. Epub 2025 Sep 23.
Postmenopausal osteoporosis (PMOP) is a bone metabolic disorder caused by estrogen (E2) deficiency. The traditional Chinese medicine Psoraleae Fructus (P) is often used in combination with walnut kernels (J, Juglans regia L.) to treat osteoporosis. However, whether the combination of walnut kernels and Psoraleae Fructus (PJ) is more effective than Psoraleae Fructus alone remains unclear, and the material basis and mechanism of the synergistic effects of this combination are not fully understood.
This study aimed to elucidate the synergistic mechanisms of PJ in the treatment of PMOP and to identify the active components and their targets.
The compounds in PJ were analyzed using UPLC-MS/MS. Bilateral ovariectomy (OVX) was performed to establish a rat model of PMOP. Femoral pathological changes were evaluated by serum ELISA, micro-CT, H&E and TRAP staining. UPLC-MS/MS analysis was also performed to screen for active components in the serum, focusing on key monomers. Transcriptome sequencing was conducted to identify core pathways. An MC3T3-E1 ferroptosis model was established by erastin treatment. A combination index was used to evaluate the synergistic effects of the monomers. In addition to measuring cell viability, the effects of the key monomers on ferroptosis and osteoblastic differentiation were verified using cryogenic transmission electron microscopy, confocal laser scanning microscopy, and flow cytometry. Molecular docking, molecular dynamics (MD) simulations, microscale thermophoresis (MST), immunofluorescence, and western blotting were performed to validate the targets of this activity.
In vivo experimental results indicated that PJ significantly improved OVX-induced osteoporosis more prominently than P alone. The UPLC-MS/MS results showed that the serum concentrations of eight potentially active components increased significantly after compatibility with J. Transcriptome analysis revealed significant enrichment of ferroptosis in the OVX group, whereas in vivo experiments demonstrated that PJ inhibited ferroptosis by regulating the Nrf2/GPX4/SLC7A11 pathway. In vitro experiments identified norbakuchinic acid (NA) and α-linolenic acid (ALA) as the main pharmacodynamic components of PJ and confirmed that both components synergistically inhibited ferroptosis while promoting osteogenic mineralization. This effect was dependent on Nrf2/GPX4/SLC7A11 pathway activation. MST and MD simulations revealed that both NA and ALA were directly bind to Keap1, thereby promoting Nrf2 nuclear translocation and triggering downstream biological responses. Furthermore, when Nrf2 expression was inhibited in vitro by ML385, the inhibitory effects of NA and ALA on ferroptosis were suppressed.
This study provided the first systematic evidence that walnut kernels can enhance the therapeutic efficacy of Psoraleae Fructus against PMOP by improving the bioavailability of active constituents and forming a synergistic network of NA and ALA. Furthermore, it has been determined that the Nrf2/GPX4/SLC7A11 pathway serves as the principal mechanism through which NA and ALA synergistically inhibit osteoblast ferroptosis.
绝经后骨质疏松症（PMOP）是一种由雌激素（E2）缺乏引起的骨代谢紊乱疾病。中药补骨脂（P）常与核桃仁（J，胡桃科胡桃属植物）配伍用于治疗骨质疏松症。然而，核桃仁与补骨脂的配伍（PJ）是否比单用补骨脂更有效尚不清楚，且该配伍协同作用的物质基础和机制也未完全明确。
本研究旨在阐明PJ治疗PMOP的协同机制，并确定其活性成分及其靶点。
采用超高效液相色谱 - 串联质谱法（UPLC-MS/MS）分析PJ中的化合物。通过双侧卵巢切除术（OVX）建立PMOP大鼠模型。采用血清酶联免疫吸附测定（ELISA）、显微计算机断层扫描（micro-CT）、苏木精 - 伊红（H&E）染色和抗酒石酸酸性磷酸酶（TRAP）染色评估股骨病理变化。还进行UPLC-MS/MS分析以筛选血清中的活性成分，重点关注关键单体。进行转录组测序以确定核心通路。通过用埃拉斯汀处理建立MC3T3-E1铁死亡模型。使用联合指数评估单体的协同作用。除了测量细胞活力外，还使用低温透射电子显微镜、共聚焦激光扫描显微镜和流式细胞术验证关键单体对铁死亡和成骨细胞分化的影响。进行分子对接、分子动力学（MD）模拟、微量热泳动（MST）、免疫荧光和蛋白质印迹法以验证该活性的靶点。
体内实验结果表明，PJ比单用P更显著地改善了OVX诱导的骨质疏松症。UPLC-MS/MS结果显示，与J配伍后，8种潜在活性成分的血清浓度显著增加。转录组分析显示OVX组中铁死亡显著富集，而体内实验表明PJ通过调节Nrf2/GPX4/SLC7A11通路抑制铁死亡。体外实验确定去甲库拉索酸（NA）和α-亚麻酸（ALA）为PJ的主要药效成分，并证实这两种成分协同抑制铁死亡，同时促进成骨矿化。这种作用依赖于Nrf2/GPX4/SLC7A11通路的激活。MST和MD模拟显示NA和ALA均直接与Keap1结合，从而促进Nrf2核转位并触发下游生物学反应。此外，当用ML385在体外抑制Nrf2表达时，NA和ALA对铁死亡的抑制作用受到抑制。
本研究提供了首个系统性证据，表明核桃仁可通过提高活性成分的生物利用度并形成NA和ALA的协同网络，增强补骨脂对PMOP的治疗效果。此外，已确定Nrf2/GPX4/SLC7A11通路是NA和ALA协同抑制成骨细胞铁死亡的主要机制。
