Effects and mechanisms of isoginkgetin on osteoclastogenesis

doi:10.12307/2026.552

Chinese Journal of Tissue Engineering Research ›› 2026, Vol. 30 ›› Issue (6): 1348-1358.doi: 10.12307/2026.552

Effects and mechanisms of isoginkgetin on osteoclastogenesis

Wen Guangwei1, 2, 3, Zhen Yinghao2, 4, Zheng Taikeng2, 4, Zhou Shuyi2, Mo Guoye2, Zhou Tengpeng2, 3, Li Haishan2, 4, Lai Yiyi2, 3

1Guangzhou Panyu District Hualong Hospital, Guangzhou 510405, Guangdong Province, China; 2Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 3Guangzhou Liwan District Orthopedic and Traumatology Hospital, Guangzhou 510405, Guangdong Province, China; 4Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China

Received:2024-11-14 Accepted:2025-01-25 Online:2026-02-28 Published:2025-07-12
Contact: Lai Yiyi, MS, Associate chief physician, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Guangzhou Liwan District Orthopedic and Traumatology Hospital, Guangzhou 510405, Guangdong Province, China Co-corresponding author: Li Haishan, PhD, Physician, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
About author:Wen Guangwei, Attending physician, Guangzhou Panyu District Hualong Hospital, Guangzhou 510405, Guangdong Province, China; Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Guangzhou Liwan District Orthopedic and Traumatology Hospital, Guangzhou 510405, Guangdong Province, China
Supported by:
Guangzhou Municipal Health and Wellness Science and Technology Project, No. 20241A010093 (to WGW); Liwan District Livelihood Science and Technology Project, No. 20230713 (to WGW); Guangdong Provincial Bureau of Traditional Chinese Medicine Scientific Research Project, No. 20241091 (to MGY); Guangzhou Municipal Health and Wellness Science and Technology Project, No. 20251A011106 (to ZTP)

Abstract

Abstract: BACKGROUND: During bone remodeling, bone formation and bone resorption are spatially and temporally coordinated, involving intricate interactions between osteoclasts and osteoblasts. Isoginkgetin, a flavonoid found in Ginkgo biloba, has a wide range of anticancer activity and anti-reactive oxygen species activity; however, the effect of isoginkgetin on osteoclast differentiation is unknown.
OBJECTIVE: To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.
METHODS: In vitro studies were performed on mouse bone marrow-derived macrophages, and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages. Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts. Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts. Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts. Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation, reactive oxygen species, and PI3K/AKT pathways. Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels. Flow cytometry technology was used to detect reactive oxygen species levels in cells.
RESULTS AND CONCLUSION: (1) Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia, and GSK3β, ESR1, MCL1 and CCNA2 were the key targets. (2) Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro, and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner. (3) Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins (NFATc1, c-Fos, CTSK, and MMP9). Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes (NFATc1, c-Fos, CTSK, and MMP9). (4) Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway. (5) The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production, and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

Key words: isoginkgetin, osteoporosis, bone marrow-derived macrophages, osteoclast, reactive oxygen species, PI3K, AKT, engineered tissue construction

CLC Number:

Wen Guangwei, Zhen Yinghao, Zheng Taikeng, Zhou Shuyi, Mo Guoye, Zhou Tengpeng, Li Haishan, Lai Yiyi. Effects and mechanisms of isoginkgetin on osteoclastogenesis[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(6): 1348-1358.

Figures/Tables 7

2.1 异银杏素作用骨质疏松症靶点筛选和蛋白互作网络构建通过检索Swiss Target Prediction数据库共获得骨质疏松症潜在靶点7 023个；通过Genecards、OMIM、TTD和DisGeNET数据库检索去重后得到121个破骨细胞分化相关靶点，韦恩图取交集后得到75个异银杏素干预骨质疏松症的潜在靶点(图1A)。将75个潜在的交叉靶点导入STRING数据库，获得蛋白互作网络图(图1B，C)；使用Cytoscape软件进行可视化，获得GSK3β、ESR1、MCL1及CCNA2等关键靶点(图1D)。 2.2 GO/KEGG富集分析功能富集分析结果发现异银杏素作用骨质疏松症在生物学过程主要涉及药物反应、肽基丝氨酸磷酸化和对缺氧的反应等，细胞组分主要涉及丝氨酸/苏氨酸蛋白激酶复合物和蛋白激酶复合物等，分子功能主要涉及蛋白丝氨酸/苏氨酸激酶活性和转录激活子结合等(图2A)。异银杏素作用骨质疏松症关联的通路主要是PI3K/AKT信号通路等(图2B)，这些结果表明异银杏素可能通过激活PI3K/AKT通路和细胞对药物、缺氧的反应影响骨质疏松症。 2.3 异银杏素抑制BMDM向破骨细胞分化图3A展示了异银杏素的CAS号和结构式。CCK-8结果显示异银杏素在10 μmol/L以下对BMDM增殖无明显细胞毒性(图3B)。用不同浓度的异银杏素干预BMDM并诱导其向破骨细胞分化，破骨细胞特异性抗酒石酸酸性磷酸酶染色结果显示在没有异银杏素影响时，BMDM向破骨细胞分化显著，随着异银杏素浓度增加，BMDM向破骨细胞分化受到明显抑制，当异银杏素浓度为8 μmol/L时抑制效果最显著(图3C，D)。综上可见，异银杏素对BMDM增殖无明显细胞毒性影响，并且呈浓度依赖性抑制BMDM向破骨细胞分化。 2.4 异银杏素抑制细胞骨架F-actin环形成破骨细胞分化过程中伴随着细胞核的聚集和细胞形态的巨大化，F-actin环帮助重组细胞骨架，为破骨细胞的融合分化提供结构支持[5，11]。使用罗丹明鬼笔环肽染液探讨异银杏素对F-actin环的影响，荧光染色结果显示与4 μmol/L异银杏素相比，8 μmol/L异银杏素更加明显抑制F-actin环面积的扩大和环内细胞核的聚集(图4A，B)，从而抑制BMDM向破骨细胞分化。 2.5 异银杏素与破骨细胞分化靶点的分子动力学模拟众所周知，NFATc1是破骨细胞分化过程中的关键转录因子，NFATc1通过结合自身启动子来自我扩增，引起下游基因(如c-fos、CTSK和MMP9)表达上升[9]。通过分子动力学模拟探讨异银杏素对破骨细胞分化标志蛋白的直接结合情况，结果显示异银杏素-NFATc1和异银杏素-c-Fos复合物在整个分子动力学模拟过程中处于较稳定状态，而异银杏素-CTSK和异银杏素-MMP9复合物的结合稳定性较差(图5A-H)。分子动力学模拟结果表明异银杏素与NFATc1、c-fos、CTSK和MMP9均能直接结合，可能通过调控破骨细胞分化相关蛋白和基因来抑制破骨细胞分化。 2.6 异银杏素抑制破骨细胞标志蛋白和基因的表达 NFATc1的表达是破骨细胞发生的关键，而c-Fos能与NFATc1相互作用，引起破骨细胞基因的转录与表达，促进破骨细胞分化。CTSK和MMP9参与骨基质的降解，其表达在破骨细胞分化中明显升高[20]。通过Western blot检测异银杏素对破骨细胞分化相关蛋白(NFATc1、c-Fos、CTSK和MMP9)表达的影响，结果显示异银杏素在破骨细胞分化全程(第1-5天)抑制c-fos的表达(图6A，B)，在破骨细胞分化的中后期(第3-5天)抑制NFATc1、CTSK和MMP9的表达(图6C-E)。PCR检测结果以仅RANKL诱导为阳性对照，异银杏素呈浓度依赖性抑制c-fos、CTSK、MMP9和NFATc1的mRNA表达(图6F-I)。以上结果表明异银杏素通过降低破骨细胞分化蛋白和基因的表达抑制破骨细胞分化成熟。 2.7 异银杏素抑制PI3K/AKT通路激活进而抑制破骨细胞分化当RANKL与受体RANK结合时，会激活下游的PI3K/AKT信号通路，促进破骨细胞前体细胞的分化和成熟，而下游GSK3β的活性变化与破骨细胞分化相关蛋白MMP9、CTSK和NFATc1的表达水平有关[12]。结合2.1和2.2的网络药理学结果，采用Western blot检测异银杏素对PI3K/AKT通路及下游GSK3β的激活情况，结果显示异银杏素在RANKL激活破骨细胞早期分化的15-60 min内显著抑制了PI3K、AKT和GSK3β的磷酸化水平(图7A-D)。综上可见，PI3K/AKT通路确实是影响异银杏素抑制破骨细胞分化的主要信号通路。 2.8 异银杏素抑制RANKL诱导的活性氧产生在RANKL诱导破骨细胞分化中，细胞利用活性氧作为第二信使传递信号促进破骨细胞分化[21]，而线粒体是细胞产生活性氧的主要场所。通过使用细胞活性氧H2DCFDA探针和线粒体MitoSOX Red探针检测细胞活性氧和线粒体活性氧的产生情况[4]。荧光结果"

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Effects and mechanisms of isoginkgetin on osteoclastogenesis

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