Wei Fanhao, Ruan Binjia, Dong Jian, Yang Bin, Zhang Guofu, Kelvin Yeung Wai Kwok, Wang Hongwei, Cao Wangsen, Wang Yongxiang
Department of Graduate School, Dalian Medical University, No.9 of West Section of Lushun South Road, Dalian 116044, China; The Yangzhou School of Clinical Medicine of Dalian Medical University, 98 West Nantong Road, Yangzhou 225001, China.
Northern Jiangsu People's Hospital, Clinical Teaching Hospital of Medical School, Nanjing University, 98 West Nantong Road, Yangzhou 225001, China.
J Adv Res. 2024 Dec 6. doi: 10.1016/j.jare.2024.11.036.
Diabetic osteoporosis (DOP) is an insidious complication of diabetes with limited therapeutic options. DOP is pathologically associated with various types of regulated cell death, but the precise role of ferroptosis in the process remains poorly understood. Asperosaponin VI (AVI), known for its clinical efficacy in treating bone fractures and osteoporosis, may exert its osteoprotective effects through mechanisms involving ferroptosis, however this has not been established.
This study aimed to investigate the role of AVI in modulating ferroptosis in a mouse model of DOP and to explore the underlying mechanisms.
We assessed OP alterations in femurs of DOP-conditioned mice and primary bone cells. We generated a strain of osteoblast-specific Gpx4-deficient mice. A combination of micro-CT, immunohistochemistry, immunofluorescence, methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), western blotting (WB), and AVI pull-down assays were employed to elucidate the mechanism and therapeutic target of AVI in DOP.
Our findings revealed that femurs from DOP-conditioned mice exhibited significant ferroptosis and suppression of the core anti-ferroptosis factor GPX4, mainly due to hypermethylation of the Gpx4 promoter mediated by DNA methyltransferases DNMT1and DNMT3a. Notably, treatment with AVI effectively reversed the hypermethylation, restored GPX4 expression, and reduced ferroptotic pathologies associated with DOP by inhibiting DNMT1/3a. In primarily-cultured osteoblasts and osteoclasts, AVI alleviated GPX4 suppression and reduced ferroptosis in DOP-conditioned osteoblasts through a mechanism dependent on DNMT inhibition and GPX4 restoration. Importantly, the anti-ferroptotic and osteoprotective effects of AVI were abolished in osteoblastic Gpx4 haplo-deficient mice (Gpx4) or when GPX4 was pharmacologically inactivated with RSL3.
Our study identifies a pivotal epigenetic ferroptotic pathway that contributes significantly to DOP and uncovers a crucial pharmacological property of AVI that is potentially effective in treating patients with DOP and related osteoporotic disorders.
糖尿病性骨质疏松症（DOP）是糖尿病的一种隐匿性并发症，治疗选择有限。DOP在病理上与多种类型的程序性细胞死亡相关，但铁死亡在这一过程中的具体作用仍知之甚少。穿龙薯蓣皂苷VI（AVI）以其在治疗骨折和骨质疏松症方面的临床疗效而闻名，可能通过涉及铁死亡的机制发挥其骨保护作用，然而这一点尚未得到证实。
本研究旨在探讨AVI在DOP小鼠模型中调节铁死亡的作用，并探索其潜在机制。
我们评估了DOP条件小鼠和原代骨细胞股骨中的骨质疏松改变。我们构建了一种成骨细胞特异性Gpx4基因缺陷小鼠品系。采用显微CT、免疫组织化学、免疫荧光、甲基化特异性PCR（MSP）、亚硫酸氢盐测序PCR（BSP）、蛋白质免疫印迹（WB）和AVI下拉试验等方法，阐明AVI在DOP中的作用机制和治疗靶点。
我们的研究结果显示，DOP条件小鼠的股骨表现出明显的铁死亡以及核心抗铁死亡因子GPX4的抑制，这主要是由于DNA甲基转移酶DNMT1和DNMT3a介导的Gpx4启动子高甲基化所致。值得注意的是，AVI治疗通过抑制DNMT1/3a有效逆转了高甲基化，恢复了GPX4表达，并减少了与DOP相关的铁死亡病理变化。在原代培养的成骨细胞和破骨细胞中，AVI通过一种依赖于DNMT抑制和GPX4恢复的机制减轻了GPX4抑制，并减少了DOP条件下成骨细胞中的铁死亡。重要的是，在成骨细胞特异性Gpx4单倍体缺陷小鼠（Gpx4）中，或者当GPX4被RSL3药理失活时，AVI的抗铁死亡和骨保护作用被消除。
我们的研究确定了一条对DOP有显著贡献的关键表观遗传铁死亡途径，并揭示了AVI的一种关键药理特性，其可能对治疗DOP患者及相关骨质疏松症有效。
