Yang Yuchao, Zhou Haidong, Wang Yining, Yin Ming, Gong Fengying, Wu Shutong, Luo Zhiyin, Bi Zhenyu, Xu Jiahou, Chen Yongfu, Hu Konghe, Ouyang Jun, Tang Jiajun
Department of Orthopedics, Yue Bei People's Hospital, Shaoguan, 512026, China.
Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, China.
Phytother Res. 2025 Aug 19. doi: 10.1002/ptr.8519.
Forsythiae Fructus, a traditional herb known for its anti-inflammatory and antioxidant properties, has demonstrated inconclusive effects on bone metabolism in prior studies. The aim of this study is to investigate whether phillygenin (PHI), an active component of Forsythiae Fructus, promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by modulating the FAK/GSK-3β/β-catenin signaling axis. Experimental procedure: In vivo, an ovariectomized (OVX) mouse model was employed to mimic postmenopausal bone loss. In vitro, osteoclastogenesis was evaluated using tartarate-resistant acid phosphatase (TRAP) enzymatic assays, while osteogenic differentiation of BMSCs was assessed through alkaline phosphatase (ALP) and alizarin red S (ARS) staining. Cell proliferation and viability were examined using the CCK-8 assay. Osteogenic markers, including ALP, RUNX2, osteopontin (OPN), as well as signaling proteins, such as phosphorylated FAK at Y397 (pFAKY397), phosphorylated glycogen synthase kinase-3 beta (pGSK-3β), and β-catenin, were analyzed by western blot and immunofluorescence analyses. Specific inhibitors of the FAK and β-catenin (Y15 and XAV-939) were employed for mechanistic validation. PHI treatment significantly decreased the number and size of osteoclasts in a dose-dependent way. In OVX mice, PHI preserved trabecular microarchitecture, increased bone mineral density, and inhibited osteoclast formation dose-dependently. Low doses of PHI significantly promoted osteogenic differentiation and mineralization of BMSCs without affecting cell proliferation and viability. Mechanistically, PHI upregulated the expression of pFAKY397, pGSK-3β, and β-catenin. Inhibition of the pathway, achieved through the use of Y15 and XAV-939, attenuated PHI-induced osteogenesis. These findings indicate that PHI enhances BMSCs osteogenesis via the FAK/GSK-3β/β-catenin axis, thereby restoring bone metabolic balance. The results highlight PHI's dual regulatory potential in the management of bone disorders, particularly, estrogen deficiency-related osteoporosis, and suggest its potential applications in the therapeutic intervention of bone diseases.
连翘是一种以其抗炎和抗氧化特性而闻名的传统草药，在先前的研究中，其对骨代谢的影响尚无定论。本研究的目的是探讨连翘的活性成分连翘酯素（PHI）是否通过调节黏着斑激酶（FAK）/糖原合酶激酶-3β（GSK-3β）/β-连环蛋白信号轴来促进骨髓间充质干细胞（BMSC）的成骨分化。实验过程：在体内，采用去卵巢（OVX）小鼠模型模拟绝经后骨质流失。在体外，使用抗酒石酸酸性磷酸酶（TRAP）酶法评估破骨细胞生成，同时通过碱性磷酸酶（ALP）和茜素红S（ARS）染色评估BMSC的成骨分化。使用CCK-8法检测细胞增殖和活力。通过蛋白质印迹法和免疫荧光分析检测包括ALP、RUNX2、骨桥蛋白（OPN）在内的成骨标志物以及诸如Y397位点磷酸化的FAK（pFAKY397）、磷酸化的糖原合酶激酶-3β（pGSK-3β）和β-连环蛋白等信号蛋白。使用FAK和β-连环蛋白的特异性抑制剂（Y15和XAV-939）进行机制验证。PHI处理以剂量依赖性方式显著减少破骨细胞的数量和大小。在OVX小鼠中，PHI保留了小梁微结构，增加了骨密度，并剂量依赖性地抑制破骨细胞形成。低剂量的PHI显著促进BMSC的成骨分化和矿化，而不影响细胞增殖和活力。从机制上讲，PHI上调了pFAKY397、pGSK-3β和β-连环蛋白的表达。通过使用Y15和XAV-939抑制该信号通路减弱了PHI诱导的成骨作用。这些发现表明，PHI通过FAK/GSK-3β/β-连环蛋白轴增强BMSC的成骨作用，从而恢复骨代谢平衡。结果突出了PHI在骨疾病管理中的双重调节潜力，特别是在雌激素缺乏相关骨质疏松症方面，并提示其在骨疾病治疗干预中的潜在应用。
