Active vitamin D analog eldecalcitol is clinically applied in treatment of postmenopausal osteoporosis. This study aims to determine the role of eldecalcitol in the protection of osteocytes from senescence and the associated ferroptosis. The MLO-Y4 osteocytes were exposed to D-gal inducing senescence. The ovariectomized (OVX) mice treated with D-gal using as an aging inducer were intraperitoneally injected with eldecalcitol. The multiplexed confocal imaging, fluorescence hybridization and transmission electron microscopy were applied in assessing osteocytic properties. Immunochemical staining and immunoblotting were carried out to detect abundance and expression of molecules. The ablation of vitamin D receptor led to a reduction in amounts of osteocytes, a loss of dendrites, an increase in mRNA expression of SASP factors and in protein expression of senescent factors, as well as changes in mRNA expression of ferroptosis-related genes (PTGS2 & RGS4). Eldecalcitol reversed senescent phenotypes of MLO-Y4 cells shown by improving cell morphology and density, decreasing β-gal-positive cell accumulation, and down-regulating protein expression (P16, P21 & P53). Eldecalcitol reduced intracellular ROS and MDA productions, elevated JC-1 aggregates, and up-regulated expression of Nrf2 and GPX4. Eldecalcitol exhibited osteopreserve effects in D-gal-induced aging OVX mice. The confocal imaging displayed its improvement on osteocytic network organization. Eldecalcitol decreased the numbers of senescent osteocytes at tibial diaphysis by SADS assay and attenuated mRNA expression of SASP factors as well as down-regulated protein expression of senescence-related factors and restored levels of ferroptotic biomarkers in osteocytes-enriched bone fraction. It reduced 4-HNE staining area, stimulated Nrf2-positive staining, and promoted nuclear translocation of Nrf2 in osteocytes of mice as well as inhibited and promoted protein expression of 4-HNE and Nrf2, respectively, in osteocytes-enriched bone fraction. The present study revealed the ameliorative effects of eldecalcitol on senescence and the associated ferroptosis of osteocytes, contributing to its preservation against osteoporosis of D-gal-induced senescent ovariectomized mice.

中文翻译：

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艾德骨化醇保护骨细胞免受 D-gal 诱导的衰老 MLO-Y4 细胞和卵巢切除小鼠的铁死亡

活性维生素D类似物艾德骨化醇临床应用于治疗绝经后骨质疏松症。本研究旨在确定艾德骨化醇在保护骨细胞免于衰老和相关铁死亡中的作用。 MLO-Y4 骨细胞暴露于 D-gal 诱导衰老。用D-gal作为衰老诱导剂处理的去卵巢（OVX）小鼠腹腔注射艾德骨化醇。应用多重共焦成像、荧光杂交和透射电子显微镜来评估骨细胞特性。进行免疫化学染色和免疫印迹以检测分子的丰度和表达。维生素D受体的消除导致骨细胞数量减少、树突损失、SASP因子mRNA表达和衰老因子蛋白表达增加，以及铁死亡相关基因（PTGS2）mRNA表达的变化。 ＆RGS4）。 Eldecalcitol 通过改善细胞形态和密度、减少 β-gal 阳性细胞积累以及下调蛋白表达（P16、P21 和 P53）来逆转 MLO-Y4 细胞的衰老表型。 Eldecalcitol 减少细胞内 ROS 和 MDA 的产生，升高 JC-1 聚集体，并上调 Nrf2 和 GPX4 的表达。艾德骨化醇在 D-gal 诱导的老化 OVX 小鼠中表现出骨保护作用。共聚焦成像显示其对骨细胞网络组织的改善。通过 SADS 测定，艾德骨化醇减少了胫骨骨干处衰老骨细胞的数量，减弱了 SASP 因子的 mRNA 表达，下调了衰老相关因子的蛋白表达，并恢复了富含骨细胞的骨成分中铁死亡生物标志物的水平。它减少了 4-HNE 染色面积，刺激了 Nrf2 阳性染色，促进了小鼠骨细胞中 Nrf2 的核转位，并分别抑制和促进了富含骨细胞的骨成分中 4-HNE 和 Nrf2 的蛋白表达。本研究揭示了艾地骨化醇对骨细胞衰老和相关铁死亡的改善作用，有助于其预防 D-gal 诱导的衰老卵巢切除小鼠的骨质疏松症。