Second Affiliated Hospital of Soochow University, Orthopedic Department, China; Osteoporosis Research Institute of Soochow University, China.
Shandong Provincial Qianfoshan Hospital, The First Affiliated Hospital of Shandong First Medical University, Orthopedic Department, China.
Bone. 2022 Nov;164:116511. doi: 10.1016/j.bone.2022.116511. Epub 2022 Aug 4.
Numerous studies have demonstrated that iron overload is a risk factor of osteoporosis. However, there has been no systematic and in-depth studies on the effect of iron overload on osteocytes and its role in iron overload-induced bone loss. Therefore, to address this problem, we carried out in vitro and in vivo studies using MLO-Y4 osteocyte-like cells and Hepcidin mice as iron overload models.
(1) MLO-Y4 cells were treated with ferric ammonium citrate (FAC). Intracellular reactive oxygen species (ROS) levels and apoptosis of MLO-Y4 cells were determined by flow cytometry. Western blotting was performed to evaluate the effect of FAC on the expression of sclerostin and RANKL/OPG. (2) The conditioned medium of MLO-Y4 cells after treatment with FAC was collected and used to treat pre-osteoblasts and monocytes. Alkaline phosphatase (ALP) staining and alizarin red (AR) staining were used to evaluate osteogenic differentiation capacity, and tartrate-resistant acid phosphatase (TRAP) staining was performed to demonstrate osteoclast differentiation capacity. (3) In vivo studies included a wild type mouse, Hepcidin mice, Hepcidin mice + deferoxamine (DFO), and Hepcidin mice + N-actyl-l-cysteine (NAC) group. Micro-CT was performed to evaluate the bone mineral density (BMD), bone volume, and bone micro-architecture of the mice, and three bending tests were used to assess bone strength. Histological analysis was used to detect alterations in bone turnover. TUNEL staining and scanning electron microscopy (SEM) were performed to evaluate the apoptosis and morphology of osteocytes. Immunohistochemical staining and Western blotting were used to determine alterations in sclerostin and RANKL/OPG expression levels in mice.
(1) FAC increased intracellular ROS and apoptosis in MLO-Y4 cells, while FAC enhanced the expression of sclerostin and RANKL/OPG in MLO-Y4 cells. (2) Conditioned medium of MLO-Y4 cells inhibited the osteogenic capacity of osteoblasts while stimulating osteoclast differentiation. (3) By increasing oxidative stress, iron overload promotes the apoptosis of osteocytes and undermines the morphology of osteocytes in Hepcidin mice, further increasing the expression levels of sclerostin and RANKL/OPG in osteocytes, which is considered to be the causative factor for reduced bone formation and enhanced bone resorption. DFO administration reduced iron levels, and NAC treatment decreased oxidative stress in Hepcidin mice. Therefore, DFO or NAC treatment rescued the decrease in BMD, bone volume, and bone strength and attenuated the deterioration of bone architecture in Hepcidin mice by attenuating the effect of iron overload on osteocytes.
Osteocyte apoptosis due to increased ROS and resultant sclerostin and RANKL/OPG expression alteration was the main reason for bone loss in Hepcidin mice. Osteocytes are the main targets for the prevention and treatment of iron overload-induced osteoporosis.
大量研究表明，铁过载是骨质疏松症的一个危险因素。然而，目前还没有关于铁过载对骨细胞的影响及其在铁过载诱导的骨丢失中的作用的系统和深入的研究。因此，为了解决这个问题，我们使用 MLO-Y4 成骨细胞样细胞和 Hepcidin 小鼠作为铁过载模型进行了体外和体内研究。
（1）用柠檬酸铁铵（FAC）处理 MLO-Y4 细胞。通过流式细胞术测定 MLO-Y4 细胞内活性氧（ROS）水平和细胞凋亡。通过 Western blot 评估 FAC 对骨硬化蛋白和 RANKL/OPG 表达的影响。（2）收集用 FAC 处理后的 MLO-Y4 细胞的条件培养基，并用于处理成骨前体细胞和单核细胞。碱性磷酸酶（ALP）染色和茜素红（AR）染色用于评估成骨分化能力，抗酒石酸酸性磷酸酶（TRAP）染色用于证明破骨细胞分化能力。（3）体内研究包括野生型小鼠、Hepcidin 小鼠、Hepcidin 小鼠+去铁胺（DFO）和 Hepcidin 小鼠+N-乙酰-L-半胱氨酸（NAC）组。通过 micro-CT 评估小鼠的骨矿物质密度（BMD）、骨量和骨微结构，并进行三次弯曲试验评估骨强度。组织学分析用于检测骨转换的变化。TUNEL 染色和扫描电子显微镜（SEM）用于评估成骨细胞的凋亡和形态。免疫组织化学染色和 Western blot 用于确定小鼠中骨硬化蛋白和 RANKL/OPG 表达水平的变化。
（1）FAC 增加了 MLO-Y4 细胞内的 ROS 和细胞凋亡，同时 FAC 增强了 MLO-Y4 细胞中骨硬化蛋白和 RANKL/OPG 的表达。（2）MLO-Y4 细胞的条件培养基抑制成骨细胞的成骨能力，同时刺激破骨细胞分化。（3）通过增加氧化应激，铁过载促进了成骨细胞的凋亡，并破坏了 Hepcidin 小鼠中成骨细胞的形态，进一步增加了成骨细胞中骨硬化蛋白和 RANKL/OPG 的表达水平，这被认为是导致骨形成减少和骨吸收增强的原因。DFO 给药降低了铁水平，NAC 处理降低了 Hepcidin 小鼠中的氧化应激。因此，DFO 或 NAC 治疗通过减轻铁过载对成骨细胞的影响，挽救了 Hepcidin 小鼠中 BMD、骨量和骨强度的降低，并减轻了骨结构的恶化。
由于 ROS 增加和由此导致的骨硬化蛋白和 RANKL/OPG 表达改变引起的成骨细胞凋亡是 Hepcidin 小鼠骨丢失的主要原因。成骨细胞是预防和治疗铁过载诱导的骨质疏松症的主要靶点。
