Xu Haoying, Zhou Zijie, Wen Fuli, Sun Hong, Hou Jianming
Shengli Clinical Medical College of Fujian Medical University, Fuzhou 350001, China; Department of Hematology, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou 350212, China.
Shengli Clinical Medical College of Fujian Medical University, Fuzhou 350001, China; Department of Orthopedics, Orthopedic and Sports Medicine Center, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, China.
Bone. 2025 Mar;192:117348. doi: 10.1016/j.bone.2024.117348. Epub 2024 Nov 29.
Excessive osteoclast activity could cause skeletal diseases including osteoporosis. Additionally, autophagy plays an initial role in osteoclast differentiation and function. Ginkgolide B (GB), a key compound in Ginkgo biloba, improves bone mass and suppresses mature osteoclast formation in vitro. This study examines the role of GB role in regulating osteoclast formation via autophagy. Using murine bone marrow-derived macrophages in vitro, we explored GB's effects on autophagy and osteoclast formation. We also assessed bone loss prevention in an ovariectomized (OVX) mouse model using GB combined with an autophagy inhibitor. Tartrate-resistant acid phosphatase staining was used to observe osteoclast formation. Autophagy-related proteins and intracellular microtubule associated protein 1 light chain 3 beta puncta were observed using western blotting and immunofluorescence. The impact of GB on OVX mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. GB directly promoted autophagy in osteoclast precursors (OCPs), but inhibited osteoclast differentiation by reducing receptor activator of nuclear factor kappa B ligand (RANKL)-induced autophagy. GB inhibits the phosphorylation of RANKL downstream signaling pathways, such as Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). Anisomycin (ANI), a JNK activator, reversed GB's inhibitory effects on RANKL-induced autophagy and osteoclastogenesis. Inhibiting autophagy using 3-Methyladenine (3-MA) or small interfering RNA significantly suppressed osteoclast differentiation both in vitro and in vivo. Our findings suggested that GB inhibits osteoclast formation by decreasing JNK phosphorylation and autophagy under RANKL stimulation. Interestingly, GB also directly promotes autophagy in OCPs. Thus, GB markedly reduces osteoclast differentiation and prevents bone loss, with its anti-osteoclastogenesis effect being enhanced by 3-MA. Accordingly, inhibiting GB-induced direct autophagy could further increase its therapeutic effect on bone disease resulting from excessive osteoclast activity.
破骨细胞活性过高会引发包括骨质疏松症在内的骨骼疾病。此外，自噬在破骨细胞的分化和功能中起初始作用。银杏内酯B（GB）是银杏中的关键化合物，可增加骨量并在体外抑制成熟破骨细胞的形成。本研究探讨GB通过自噬调节破骨细胞形成的作用。利用体外培养的小鼠骨髓来源巨噬细胞，我们探究了GB对自噬和破骨细胞形成的影响。我们还评估了GB联合自噬抑制剂在去卵巢（OVX）小鼠模型中预防骨质流失的效果。采用抗酒石酸酸性磷酸酶染色观察破骨细胞的形成。利用蛋白质免疫印迹法和免疫荧光法观察自噬相关蛋白和细胞内微管相关蛋白1轻链3β斑点。使用显微计算机断层扫描和苏木精-伊红染色评估GB对OVX小鼠的影响。GB直接促进破骨细胞前体（OCPs）的自噬，但通过减少核因子κB受体活化因子配体（RANKL）诱导的自噬来抑制破骨细胞分化。GB抑制RANKL下游信号通路的磷酸化，如c-Jun氨基末端激酶（JNK）、p38和细胞外信号调节激酶（ERK）。JNK激活剂茴香霉素（ANI）可逆转GB对RANKL诱导的自噬和破骨细胞生成的抑制作用。使用3-甲基腺嘌呤（3-MA）或小干扰RNA抑制自噬，在体外和体内均显著抑制破骨细胞分化。我们的研究结果表明，GB在RANKL刺激下通过降低JNK磷酸化和自噬来抑制破骨细胞形成。有趣的是，GB还直接促进OCPs的自噬。因此，GB显著减少破骨细胞分化并预防骨质流失，其抗破骨细胞生成作用可被3-MA增强。因此，抑制GB诱导的直接自噬可能会进一步提高其对破骨细胞活性过高所致骨疾病的治疗效果。
