Iron overload involved in the enhancement of unloading-induced bone loss by hypomagnetic field

Jiancheng Yang, Xiaofeng Meng, Dandan Donga,d, Yanru Xue, Xin Chen, Shenghang Wang, Ying Shen, Gejing Zhang, Peng Shang

[ABSTRACT]
During deep-space exploration missions, astronauts will be exposed to abnormal space environments including microgravity and hypomagnetic field (HyMF) that is 10,000 times weaker than geomagnetic field (GMF). It is well known that microgravity in space can induce bone loss; however, it is ill-defined whether HyMF involved in this process. Herein, we aimed to investigate the combined effects of HyMF and microgravity on bone loss. A mouse model of hindlimb suspension (HLU) was adopted to simulate microgravity-induced bone loss, that was exposed to a hypomagnetic field of<300 nanotesla (nT) generated by a geomagnetic field-shielding chamber. Besides, a recent study showed that HLU induced bone loss was orchestrated by iron overload. Therefore, the  changes of iron content in unloading-induced bone loss under HyMF condition were detected simultaneously. The results showed HyMF exacerbated the loss of bone mineral content (BMC), induced more detrimental effects on microstructure of cancellous bone but not cortical bone and yielded greater negative effects on biomechanical characteristics in mice femur under unloading status. Concomitantly, there was more iron accumulation in serum, liver, spleen and bone in the combined treatment group than in the separate unloading group or HyMF exposure group. These results showed that HyMF promoted additional bone loss in mice femur during mechanical unloading, and the potential mechanism may be involved in inducing iron overload of mice.

[Biochemical assay]
The blood samples were centrifuged (3000 rpm; 15 min; 4 °C), serum was fractionated and stored in a ?80 °C freezer. Serum ferritin, hepcidin, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase 5b (TRAP5b) were measured by using a mouse enzyme linked immunosorbent assay (ELISA) kit (Shanghai Jianglai Biotech, Shanghai, China [1]). All steps followed the manufacturer's instructions.

The effects of HyMF and HyMF+HLU on femoral BMD and BMC. DXA analysis of whole femoral bone mineral density (BMD, A) and bone mineral content (BMC, B). Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/group). Data shown as Mean ± SD. P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

The Effects of HyMF and HyMF+HLU on the mechanical properties of femur in mice. Ultimate bending moment (A), elastic modulus, (B) Stiffness (C) and Ultimate stress (D) were calculated by using the raw data 3-point bending. Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/group). Data shown as Mean ± SD. P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

Effects of HyMF and HyMF+HLU on trabecular and cortical microstructure parameters of femurs in mice. Trabecular and cortical microstructure parameters obtained via microcomputed tomography scans. (A) Three-dimensional trabecular architecture of an ROI located in 0.5mm proximal to the distal growth plate. (B) Measured parameters at the distal femur, including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), connectivity density (Conn.D), structure model index (SMI). (C) Three-dimensional cortical architecture of an ROI located in 5mm proximal to the distal growth plate. (D) Measured parameters at the midshaft femur, including cortical area (Ct.Ar), total area (Tt.Ar), marrow area (Ma.Ar), cortical thickness (Ct.Th), cortical porosity (Ct.Po), polar moment of inertia (PMOI). Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/group). Data shown as Mean ± SD. P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

Effects of HyMF and HyMF+HLU on bone formation and bone resorption of femurs in mice. A) Hematoxylin and Eosin (H&E) staining of trabecular bone on the distal of femur and osteoblasts were marked in black arrow (Bar=20 μm); B) The number of osteoblasts were evaluated by osteoblast number per bone surface (N.Ob/BS); C) Serum levels of the bone formation markers alkaline phosphatase (ALP); TRAP staining of trabecular bone on the distal of femur and osteoclasts were identified on the bone surfaces by red TRAP staining (D, Bar=50 μm; E, Bar=10 μm); F) The number of osteoclasts were evaluated by osteoclast number per bone surface (N.Oc/BS); G) The osteoclast surface per bone surface (Oc.S/BS); H) The eroded surface per bone surface (ES/BS); I) Serum levels of tartrate resistant acid phosphatase (TRAP5b) were evaluated as markers of bone resorption. Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/group). Data shown as Mean ± SD. P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

Effects of HyMF and HyMF+HLU on iron, ferritin and hepcidin levels in serum of mice. The level of serum iron (A), ferritin (B) and hepcidin (B) were calculated by using mouse ELISA kits. Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/ group). Data shown as Mean ± SD. P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

Effects of HyMF and HyMF+HLU on iron distribution in tissues of mice. Histological analysis of iron deposits in liver (A, Bar=20 μm), spleen (C, Bar=50 μm) and duodenum (E, Bar=20 μm) sections by Perls' iron staining. Atomic absorption spectrometry detected the total iron content in liver (B), spleen (D) and duodenum (F). Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/group). Data shown as Mean ± SD. ?P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

Effects of HyMF and HyMF+HLU on iron content in femurs of mice. The total iron content in diaphysis (A) and bone marrow (B) of femur was detected by AAS. DAB-enhanced Perls' iron staining in cancellous bone (C, Bar=20 μm) and cortical bone (D, Bar=50 μm) of femur. Ctrl: Mice were kept in a wooden experimental box with the normal GMF for 28 days; HLU: Mice were suspended and raised in a wooden box; HyMF: Mice were raised normally in a GMF-shielded room; HyMF+HLU: HLU mice were raised in a GMF-shielded room (n=6–8/group). Data shown as Mean ± SD. ?P < 0.05 vs. Ctrl, #P < 0.05 vs. HyMF.

[References]
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[3] W. Mo, Y. Liu, R. He, Hypomagnetic field, an ignorable environmental factor in space? Sci. China Life Sci. 57 (7) (2014) 726–728.
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[備 注] [1]“江萊生物”10年以來(lái)一直致力于免疫學(xué)技術(shù)的積累與沉淀，目前江萊生物實(shí)驗(yàn)室占地約3000平米，技術(shù)研發(fā)團(tuán)隊(duì)50余人，配備了全球最先進(jìn)的分析和檢測(cè)設(shè)備，我們通過(guò)自主創(chuàng)新以及與國(guó)內(nèi)外的科研單位合作研發(fā)，不斷突破新技術(shù)，目前我們研發(fā)了上千種指標(biāo)，質(zhì)量成熟，并長(zhǎng)期出口到歐美市場(chǎng)，贏得了眾多科研機(jī)構(gòu)的認(rèn)可 ...

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