Chen Shuangliu, Xiao Jiřimutu, Zhou Shijie, Wumiti Taxi, Zhao Zitong, Zhao Ruihua, Pan Yalan, Wang Qing, Ma Yong, Wu Lan, Guo Yang
School of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Laboratory of New Techniques of Restoration & Reconstruction, Institute of Traumatology & Orthopedics, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Int J Mol Sci. 2025 Feb 26;26(5):2027. doi: 10.3390/ijms26052027.
Dysregulated iron metabolism-induced ferroptosis is considered a key pathological mechanism in the development of osteoporosis (OP). G protein-coupled receptor 30 (GPR30, also known as Gper1) is an estrogen-binding receptor that has shown therapeutic benefits in patients with certain degenerative diseases. Moreover, several studies have demonstrated the anti-ferroptotic effects of estrogen receptor activation. However, its role in the prevention and treatment of OP remains unclear, and there are currently no reports on the anti-ferroptotic function of GPR30 in OP. Therefore, this study aimed to investigate the ferroptosis-related effects and mechanisms of GPR30 in the context of OP. In vivo and in vitro experiments were conducted using wild-type () female mice and GPR30-knockout () female mice. The microarchitecture of the distal femur was assessed using micro-computed tomography (micro-CT), and histomorphological changes were analyzed via hematoxylin and eosin (H&E) staining. Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured to establish an iron overload model using ferric ammonium citrate (FAC). Interventions included GPR30 overexpression via transfection and BMP-6 inhibition using LDN-214117. Cell viability was evaluated with the CCK-8 assay, while osteogenic differentiation and mineralization levels were assessed using ALP and Alizarin Red S (ARS) staining. Iron accumulation was detected via Prussian blue staining, oxidative stress levels were evaluated using ROS staining, and mitochondrial membrane potential changes were analyzed using JC-1 staining. Transmission electron microscopy (TEM) was employed to observe mitochondrial ultrastructural changes. Additionally, key gene and protein expression levels were measured using immunofluorescence and Western blotting. The micro-CT analysis revealed significant bone microarchitecture deterioration and bone loss in the GPR30-KO mouse model. At the cellular level, GPR30 overexpression markedly reduced iron accumulation and oxidative stress in BMSCs, restored the mitochondrial membrane potential, and improved the mitochondrial ultrastructure. Furthermore, GPR30 enhanced osteogenic differentiation in BMSCs by promoting the activation of the BMP-6/HEP/FPN signaling pathway, leading to increased expression of osteogenic markers. The protective effects of GPR30 were reversed by the BMP-6 inhibitor LDN-214117, indicating that BMP-6 is a critical mediator in GPR30-regulated iron metabolism and ferroptosis inhibition. GPR30 inhibits ferroptosis in BMSCs and enhances osteogenic differentiation by activating the BMP-6/HEP/FPN signaling pathway. This provides new insights and potential therapeutic targets for the treatment of osteoporosis OP.
铁代谢失调诱导的铁死亡被认为是骨质疏松症（OP）发展的关键病理机制。G蛋白偶联受体30（GPR30，也称为Gper1）是一种雌激素结合受体，已显示对某些退行性疾病患者具有治疗益处。此外，多项研究已证明雌激素受体激活具有抗铁死亡作用。然而，其在OP预防和治疗中的作用仍不清楚，目前尚无关于GPR30在OP中的抗铁死亡功能的报道。因此，本研究旨在探讨GPR30在OP背景下与铁死亡相关的作用及机制。使用野生型（WT）雌性小鼠和GPR30基因敲除（KO）雌性小鼠进行体内和体外实验。使用显微计算机断层扫描（micro-CT）评估股骨远端的微观结构，并通过苏木精和伊红（H&E）染色分析组织形态学变化。分离并培养骨髓间充质干细胞（BMSCs），使用柠檬酸铁铵（FAC）建立铁过载模型。干预措施包括通过转染进行GPR30过表达和使用LDN-214117抑制BMP-6。用CCK-8法评估细胞活力，同时使用碱性磷酸酶（ALP）和茜素红S（ARS）染色评估成骨分化和矿化水平。通过普鲁士蓝染色检测铁积累，使用活性氧（ROS）染色评估氧化应激水平，使用JC-1染色分析线粒体膜电位变化。采用透射电子显微镜（TEM）观察线粒体超微结构变化。此外，使用免疫荧光和蛋白质免疫印迹法测量关键基因和蛋白质表达水平。Micro-CT分析显示GPR30基因敲除小鼠模型中骨微观结构明显恶化和骨质流失。在细胞水平上，GPR30过表达显著减少了BMSCs中的铁积累和氧化应激，恢复了线粒体膜电位，并改善了线粒体超微结构。此外，GPR30通过促进BMP-6/HEP/FPN信号通路的激活增强了BMSCs中的成骨分化，导致成骨标志物表达增加。GPR30的保护作用被BMP-6抑制剂LDN-214117逆转，表明BMP-6是GPR30调节铁代谢和抑制铁死亡的关键介质。GPR30通过激活BMP-6/HEP/FPN信号通路抑制BMSCs中的铁死亡并增强成骨分化。这为骨质疏松症OP的治疗提供了新的见解和潜在的治疗靶点。
