Iron overload is closely associated with osteoporosis, the potential cellular mechanism involved in decreased osteoblast differentiation and increased osteoclast formation. However, the effect of iron overload on the biological behavior in osteocytes has not been reported. This study aims to investigate the changes of osteocytic activity, apoptosis, and its regulation on osteoclastogenesis in response to iron overload. MLO-Y4 osteocyte-like cells and primary osteocytes from mice were processed with ferric ammonium citrate (FAC) and deferoxamine (DFO), the conditioned medium (CM) was harvested and co-cultured with Raw264.7 cells and bone marrow-derived macrophages (BMDMs) to induce them to differentiate into osteoclasts. Osteocyte apoptosis, osteoclast differentiation, osteocytic gene expression and protein secretion of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) was examined. Excessive iron has a toxic effect on MLO-Y4 osteocyte-like cells. Increased cell apoptosis in MLO-Y4 cells and primary osteocytes was induced by iron overload. The osteoclastic formation, differentiation-related gene expression, and osteoclastic bone‐resorption capability were significantly increased after treated with the CM from iron overload‐exposed osteocytes. Excessive iron exposure significantly promoted the gene expression and protein secretion of the RANKL in MLO‐Y4 cells. Addition of RANKL-blocking antibody completely abolished the increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron. Moreover, the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone) was used to inhibit osteocyte apoptosis. The results showed osteocyte apoptosis induced by iron overload was reduced by QVD and accompanied by the decrease of soluble RANKL (sRANKL) in supernatant. The increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron was significantly decreased by QVD. These results indicated that iron overload-induced osteocyte apoptosis is required to regulate osteoclast differentiation by increasing osteocytic RANKL production. This study, for the first time, reveals the indirect effect of iron overload on osteoclast differentiation through regulating osteocytes.

铁过载与骨质疏松症密切相关，骨质疏松症的潜在细胞机制与成骨细胞分化减少和破骨细胞形成增加有关。然而，铁过载对骨细胞生物学行为的影响尚未见报道。这项研究旨在调查骨质疏松活性，细胞凋亡及其对破骨细胞生成的调节，以应对铁超负荷。用柠檬酸铁铵（FAC）和去铁胺（DFO）处理小鼠的MLO-Y4骨细胞样细胞和原代骨细胞，收获条件培养基（CM）并与Raw264.7细胞和骨髓巨噬细胞共培养（BMDM）诱导它们分化为破骨细胞。骨细胞凋亡，破骨细胞分化，检查核因子κB配体（RANKL）和骨保护素（OPG）受体激活剂的骨细胞基因表达和蛋白分泌。过量的铁对MLO-Y4骨细胞样细胞具有毒性作用。铁过载导致MLO-Y4细胞和原代骨细胞的细胞凋亡增加。用铁超负荷暴露的骨细胞进行CM处理后，破骨细胞形成，分化相关基因表达和破骨细胞骨吸收能力显着增加。铁的过量暴露显着促进了MLO-Y4细胞中RANKL的基因表达和蛋白质分泌。RANKL阻断抗体的加入完全消除了CM暴露于过量铁中的骨细胞引起的破骨细胞形成和骨吸收能力的增加。此外，使用泛半胱天冬酶凋亡抑制剂QVD（喹啉基-戊基-O-甲基天冬氨酰-[-2,6-二氟苯氧基]-甲基酮）抑制骨细胞凋亡。结果表明，QVD降低了铁超负荷诱导的骨细胞凋亡，并伴随上清液中可溶性RANKL（sRANKL）的降低。QVD显着降低了由CM暴露于过量铁引起的破骨细胞引起的破骨细胞形成和骨吸收能力的增加。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。使用6-二氟苯氧基]-甲基酮抑制骨细胞凋亡。结果表明，QVD降低了铁超负荷诱导的骨细胞凋亡，并伴随上清液中可溶性RANKL（sRANKL）的降低。QVD显着降低了由CM暴露于过量铁引起的破骨细胞引起的破骨细胞形成和骨吸收能力的增加。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。使用6-二氟苯氧基]-甲基酮抑制骨细胞凋亡。结果表明，QVD降低了铁超负荷诱导的骨细胞凋亡，并伴随上清液中可溶性RANKL（sRANKL）的降低。QVD显着降低了由CM暴露于过量铁引起的破骨细胞引起的破骨细胞形成和骨吸收能力的增加。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。结果表明，QVD降低了铁超负荷诱导的骨细胞凋亡，并伴随上清液中可溶性RANKL（sRANKL）的降低。QVD显着降低了由CM暴露于过量铁引起的破骨细胞引起的破骨细胞形成和骨吸收能力的增加。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。结果表明，QVD降低了铁超负荷诱导的骨细胞凋亡，并伴随上清液中可溶性RANKL（sRANKL）的降低。QVD显着降低了由CM暴露于过量铁引起的破骨细胞引起的破骨细胞形成和骨吸收能力的增加。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。这些结果表明需要铁超负荷诱导的骨细胞凋亡以通过增加骨细胞RANKL的产生来调节破骨细胞的分化。这项研究首次揭示了铁超负荷通过调节骨细胞对破骨细胞分化的间接作用。