Mechanistic study on synovial macrophage-derived hepcidin inducing ferroptosis in chondrocytes

doi:10.3877/cma.j.issn.1674-134X.2026.03.009

Abstract

Abstract:

Objective

To explore the mechanism of synovial macrophage-derived hepcidin and iron exporter protein (FPN1) in chondrocyte ferroptosis of knee osteoarthritis (KOA).

Methods

Animal experiment: 24 SD rats of six to eight weeks old were selected and randomly divided using a random number tableinto the sham operation group (the sham group) and the modified-Hult surgery (M-Hult) group (the surgical group), 12 rats in each group. The sham group only incised the joint capsule, while the M-Hult group established a model of KOA. After the model was successful, enzyme linked immunosorbent assay (ELISA) was used to detect the changes in hepcidin expression in rat serum. Micro-CT was employed to observe the morphological and structural changes of the rat knee joints. Hematoxylin-eosin (HE) staining, toluidine blue staining and safranin O/fast green violet staining were used to observe the morphological changes of the knee joint cartilage tissue. Immunohistochemistry was used to detect the expression of hepcidin, matrix metalloproteinase (MMP3) and type Ⅱ collagen (Col2a1) in the knee joint synovium of rats. Immunofluorescence technique was used to detect the expression of glutathione peroxidase 4 (GPX4) and nuclear factor erythroid 2-related factor 2 (NRF2) in the knee joint cartilage. Cell experiment: RAW264.7 cells were treated with interleukin ( IL)-6 to investigate the optimal time and concentration for IL-6 to induce the secretion of hepcidin by macrophages; the supernatant obtained after IL-6 intervention on RAW264.7 cells was used to intervene chondrocytes, and the expression of FPN1 in chondrocytes was observed; the chondrocytes were divided into blank control group, model group (hepcidin treated), and positive control group (erastin treated). The changes in chondrocytes were directly observed, the effect of hepcidin in macrophages on FPN1 in chondrocytes was detected by western blot, the expression of redox-related factor reactive oxygen species (ROS) was detected by immunofluorescence technology, and the oxidative-reductive indicators were detected using glutathione/glutathione disulfide (GSH/GSSG), MDA, and SOD kits. Single factor variance analysis and t test were performed for data analysis.

Results

Compared with the sham group, the knee osteoarthritis model rats in the surgical group showed significant pathological changes in their knees: micro-CT revealed that the joint space in the model group narrowed, bone spurs formed, and the bone volume fraction (t=5.069, P<0.001), trabecular number (t=4.225, P<0.05), and thickness (t=5.879, P<0.001) significantly decreased, while the trabecular separation (t=2.691, P<0.01) increased, and the bone microstructure was severely damaged. Compared with the sham group, the osteoarthritis score in the surgical group was significantly elevated (t=7.778, P<0.001). Cartilage tissue staining revealed disorganized layers, sparse cell arrangement, and matrix fissure formation. Meanwhile, the expression of Col2a1 decreased (t=4.931, P<0.001), the expression of MMP3 upregulated (t=17.35, P<0.001), and a substantial loss of proteoglycans was observed. In the serum and synovial tissue of the model group rats, the expression of hepcidin significantly increased, while the expression of the iron export protein FPN1 in the cartilage tissue significantly down regulated (t=10.67, P<0.001). The expression levels of the ferroptosis core inhibitor GPX4 (t=15.93, P<0.001) and the antioxidant transcription factor NRF2 (t=14.26, P<0.001) in cartilage tissue were significantly decreased. Cell experiments demonstrated that 10 μg/L IL-6 could efficiently induce high expression of hepcidin in RAW264.7 cells, and its 10% conditioned medium could significantly inhibit the expression of FPN1 in chondrocytes. After hepcidin intervenes with chondrocytes, ROS accumulation occurs, the GSH/GSSG ratio decreases (F=395.1, P<0.001), the expression level of MDA increases (F=181.7, P<0.001), and the activity of SOD decreases (F=1160, P<0.001).

Conclusion

Hepcidin derived from synovial macrophages inhibits the function of FPN1 in chondrocytes, which triggers iron accumulation in chondrocytes, thereby activating the oxidative stress-lipid peroxidation pathway, and ultimately inducing ferroptosis of chondrocytes in KOA.

Key words: Knee Osteoarthritis, Synovial macrophages, Chondrocytes, Ferroptosis

Yutong Ji, Yuehui Zhou, Yangwenxiang Wei, Zhenqiu Chen, Haibin Wang, Chi Zhou. Mechanistic study on synovial macrophage-derived hepcidin inducing ferroptosis in chondrocytes[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2026, 20(03): 322-333.

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URL: https://zhgjwkzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-134X.2026.03.009

https://zhgjwkzz.cma-cmc.com.cn/EN/Y2026/V20/I03/322

Figures/Tables 5

Figure 1 Joint structure and histopathological changes in the modified-Hult surgery-induced KOA (knee osteoarthritis) model. A shows the 3D reconstructed images and cross-sectional images of the knee joints in the sham group and the surgical group (scale=5 mm); B is quantitative analysis on BV/TV (%) (bone volume/tissue volume) of subchondral bone in the two groups; C is quantitative analysis on Tb.N (the number of trabeculae) of subchondral bone in the two groups; D is quantitative analysis on Tb.Sp (separation degree of trabeculae)of subchondral bone in the two groups; E is quantitative analysis on Tb.Th (the thickness of trabeculae) of subchondral bone in the two groups; F is HE staining of the knee joint cartilage tissue in the two groups (scale=100 μm); G is TB staining of the knee joint cartilage tissue in the two groups (scale=100 μm); H is SO&FG staining of the knee joint cartilage tissue in the two groups (scale=100 μm); I is the relative content of GAGs analysis in the two groups; J is the histological score of OARSI; K and L are the results of immunohistochemical staining (scale=100 μm) and quantitative analysis of Col2a1 in the knee joint tissues of the two groups; M and N are results of immunohistochemical staining (scale=100 μm) and quantitative analysis of MMP3 in the knee joint tissues of the two groups Note: *-P＜0.05; **-P＜0.01; ***-P＜0.001; Col2a1-type Ⅱcollagen alpha one chain; MMP-matrix metalloproteinase; HE-hematoxylin-eosin staining; TB-toluidine blue staining; SO&FG-safranin O-gentian violet staining; BV/TV-bone volume / tissue volume; Tb.N-trabecular bone number; Tb.Sp-trabecular separation degree; Tb.Th-trabecular thickness; GAGs-glycosaminoglycans; OARSI-Osteoarthritis Research Society International score

Figure 2 Immunofluorescence staining results of GPX4 and NRF2 in the knee joint tissues of the sham group and the surgical group. A shows the immunofluorescence staining of glutathione peroxidase 4 (GPX4) in the Sham group and the M-Hult group (green fluorescence represents GPX4, blue is DAPI nuclear staining, Merge is the fluorescence superimposition image) (scale=100 μm); B shows the immunofluorescence staining of NRF2 in the two groups (green fluorescence represents NRF2, blue is DAPI nuclear staining, Merge is the fluorescence superimposition image) (scale=100 μm); C is the quantitative analysis on the relative intensity of fluorescence staining for GPX4; D is the quantitative analysis on the relative intensity of fluorescence staining for NRF2 Note: ***-P＜0.001; GPX4-glutathione peroxidase 4; NRF2-nuclear factor E2-related factor 2; DAPI-4’, 6-diaminobiphenylindole (nuclear stain)

Figure 3 Expression characteristics of iron metabolism-related molecules in the knee osteoarthritis model under different treatment conditions. A shows immunohistochemical staining of hepcidin and FPN1 (ferroportin 1) in the knee joint tissues of the sham group and the surgical group (arrows indicate positive expression areas, scale bar=100 μm); B shows the quantitative analysis of relative serum free hepcidin levels in the two groups of rats; Figures C and D show the quantitative analysis results of immunohistochemical positive expression in the two groups (the number of FPN1-and hepcidin-positive cells per unit area (mm²); E shows the western blot results of hepcidin under different concentrations of IL-6 (0 to 100 μg/L); F shows the western blot results of FPN1 under different concentrations of conditioned medium (0% to 30%); G shows the relative protein expression levels of hepcidin under different concentrations of IL-6 (0 to 100 μg/L); H shows the relative protein expression levels of FPN1 under different concentrations of conditioned medium (0% to 30%) Note: ***-P<0.001; hepcidin-iron regulatory protein; FPN1-membrane iron efflux protein; IL-6-interleukin 6; CM-conditioned medium (the supernatant of RAW264.7 cells treated with IL-6 of 10 μg/L, mixed with DMEM/F12 complete medium at different concentrations); β-actin-β-actin (reference protein)

Figure 4 Detection results of ROS (reactive oxygen species) and oxidative stress-related indicators in cells of different treatment groups. A shows the results of ROS immunofluorescence staining for the control group, the ferrostatin treatment group (Hep), and the erastin (ferroptosis inducer) treatment group, green fluorescence presents ROS, blue fluorescence presents cell nucleus, Merge is the fluorescence superimposition image, scale=200 μm); B is the quantitative analysis of ROS fluorescence staining; C is the quantitative analysis of GSH/GSSG ratio in each groups; D is the quantitative analysis of the malondialdehyde level (μmol/mg) in each group; E is the quantitative analysis of the catalytic activity of superoxide dismutase in each group Note: *-P<0.05, **-P<0.01, ***-P<0.001, ns-no statistically significant difference; NC-control group without treatment; hepcidin group-model group; erastin-positive control group; ROS-reactive oxygen species; DAPI-4’, 6-diamidino-2-phenylindole (nucleus dye); GSH/GSSG-reduced glutathione/oxidized glutathione; MDA-malondialdehyde; SOD-superoxide dismutase

Figure 5 Key design elements and molecular regulatory pathways of in vivo animal experiments and in vitro cell experiments in this study. The left side shows the in vivo animal experiment system, the middle part presents the in vitro cell experiment system, and the right side illustrates the mechanism transduction process of core molecules Note: IL-6-interleukin-6; hepcidin-hepcidin; FPN1-ferroportin 1; Fe²⁺-ferrous ion; HE-hematoxylin-eosin staining; TB-toluidine blue staining; SO/FG-safranin O-fast green staining; ROS-reactive oxygen species; MDA-malondialdehyde; SOD-superoxide dismutase; GSH/GSSG-reduced glutathione/oxidized glutathione

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