Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jun;25(12):5671-5680.
doi: 10.1111/jcmm.16581. Epub 2021 May 3.

The detrimental effect of iron on OA chondrocytes: Importance of pro-inflammatory cytokines induced iron influx and oxidative stress

Xingzhi Jing  1 , Ting Du  2 , Tao Li  1 , Xiaoxia Yang  1 , Guodong Wang  1 , Xiaoyang Liu  1 , Zhensong Jiang  1 , Xingang Cui  3
Affiliations

The detrimental effect of iron on OA chondrocytes: Importance of pro-inflammatory cytokines induced iron influx and oxidative stress

Xingzhi Jing et al. J Cell Mol Med. 2021 Jun.

Abstract

Iron overload is common in elderly people which is implicated in the disease progression of osteoarthritis (OA), however, how iron homeostasis is regulated during the onset and progression of OA and how it contributes to the pathological transition of articular chondrocytes remain unknown. In the present study, we developed an in vitro approach to investigate the roles of iron homeostasis and iron overload mediated oxidative stress in chondrocytes under an inflammatory environment. We found that pro-inflammatory cytokines could disrupt chondrocytes iron homeostasis via upregulating iron influx transporter TfR1 and downregulating iron efflux transporter FPN, thus leading to chondrocytes iron overload. Iron overload would promote the expression of chondrocytes catabolic markers, MMP3 and MMP13 expression. In addition, we found that oxidative stress and mitochondrial dysfunction played important roles in iron overload-induced cartilage degeneration, reducing iron concentration using iron chelator or antioxidant drugs could inhibit iron overload-induced OA-related catabolic markers and mitochondrial dysfunction. Our results suggest that pro-inflammatory cytokines could disrupt chondrocytes iron homeostasis and promote iron influx, iron overload-induced oxidative stress and mitochondrial dysfunction play important roles in iron overload-induced cartilage degeneration.

Keywords: iron overload; mitochondrial dysfunction; osteoarthritis; oxidative stress; pro-inflammatory cytokines.

PubMed Disclaimer

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Pro‐inflammatory cytokines IL‐1β and TNF‐α disrupted iron homeostasis in chondrocytes via upregulating iron influx protein, downregulating iron efflux protein. A‐D, Chondrocytes were treated with increasing concentrations of IL‐1β or TNF‐α and iron regulators, IRP1/2, TfR1 and FPN proteins expression were determined by western blot analysis. The band density of IRP1/2, TfR1 and FPN were quantified and normalized to control. E, Chondrocytes were treated with 10 ng/mL IL‐1β for 24 h and RT‐PCR was conducted to evaluated TfR1 and FPN gene expression. F, Chondrocytes were treated with 10 ng/mL IL‐1β with or without 100 μmol/L FAC for different time intervals and iron regulators, IRP1/2, TfR1 and FPN protein expression were determined by western blot analysis. G‐J, The band density of IRP1/2, TfR1 and FPN were quantified and normalized to control. Data are presented as mean ± SD from three different experiments. *P < .05; **P < .01; ***P < .001 vs Ctrl; # P < .05; ## P < .01; ### P < .001vs FAC (100 μmol/L, 0 min)
FIGURE 2
FIGURE 2
Elevated iron influx induced by IL‐1β promoted chondrocytes matrix‐degrading proteins (MMP3, MMP13) expression. A‐B, Chondrocytes were treated with 100 μmol/L FAC in the presence or absence of 10 ng/mL IL‐1β. The quenching of green fluorescence was observed by confocal microscopy at 15 min after treatment. Scale Bars = 200 μmol/L. *P < .05 vs Ctrl group; ## P < .01 vs FAC treatment group. C‐E, Chondrocytes were treated with 10 ng/mL IL‐1β with or without 100 μmol/L FAC for different time intervals and MMP3, MMP13 proteins expression were determined by western blot analysis. The band density of MMP3, MMP13 were quantified and normalized to control. F‐H, Chondrocytes were treated with 10 ng/mL IL‐1β with or without 100 μmol/L FAC for 24 h and RT‐PCR analysis was conducted to examine ADAMTS5, MMP3 and MMP13 gene expression. Data are presented as mean ± SD from three different experiments; *P < .05; ***P < .001
FIGURE 3
FIGURE 3
Iron overload promoted ROS production and impaired mitochondrial dynamics in chondrocytes. A, Chondrocytes were treated with various concentrations of FAC and flow cytometric analysis was conducted to quantify ROS production. MFI (mean fluorescence intensity). B, Representative fluorescence photographs of mitochondria after 100 μmol/L FAC treatment for 24 h. The morphology of the mitochondria is stained by mito‐tracker green. Scale bar = 100 μmol/L. C, Chondrocytes were treated with various concentrations of FAC for 24 h and mitochondrial fission proteins, DRP1, MFF and FIS1 were determined by western blot. D, The band density of DRP1, MFF and FIS1 were quantified and normalized to control. Data are presented as mean ± SD from three different experiments. **P < .01; ***P < .001
FIGURE 4
FIGURE 4
Iron chelator DFO or antioxidant NAC inhibited ROS production and protected against iron overload induced collapse of mitochondrial membrane potential. A, Representative fluorescence photographs of intracellular ROS in chondrocytes. Scale bars = 400 μmol/L. B‐D, Chondrocytes were treated with 100 μmol/L FAC with 100 μmol/L NAC or DFO for 24 h, JC‐1dye immunofluorescence and flow cytometric analysis were conducted to detect the mitochondrial membrane potential. Scale bars = 200 μmol/L. Data are presented as mean ± SD from three different experiments. ***P < .001 vs Ctrl; # P < .05; ## P < .01 vs FAC treatment
FIGURE 5
FIGURE 5
Iron chelator DFO or antioxidant NAC inhibited iron overload induced apoptosis, mitochondrial dysfunction and matrix metalloproteinases (MMPs) upregulation. A‐B, Chondrocytes were treated with 100 μmol/L FAC with or without 100 μmol/L NAC for 24 h and mitochondrial fission protein (DRP1, MFF, FIS1) were detected using western blot analysis. The band density were quantified and normalized to control. C‐D, Chondrocytes were treated with 100 μmol/L FAC with 100 μmol/L NAC or DFO for 24 h, Annexin V‐FITC/PI flow cytometric analysis was conducted to detect the apoptosis rate. Data are presented as mean ± SD. *P < .05 vs Ctrl group; # P < .05 vs FAC treatment group. E‐F, Chondrocytes were treated with 100 μmol/L FAC with or without 100 μmol/L NAC for 24 h and matrix metalloproteinases (MMP3, MMP13) were detected using western blot analysis. The band density were quantified and normalized to control. Data are presented as mean ± SD from three different experiments. *P < .05; ***P < .001
See this image and copyright information in PMC

References

    1. Glyn‐Jones S, Palmer AJ, Agricola R, et al. Osteoarthritis. Lancet. 2015;386(9991):376‐387. - PubMed
    1. Gozzelino R, Arosio P. Iron homeostasis in health and disease. Int J Mol Sci. 2016;17(1):130. - PMC - PubMed
    1. Kennish L, Attur M, Oh C, et al. Age‐dependent ferritin elevations and HFE C282Y mutation as risk factors for symptomatic knee osteoarthritis in males: a longitudinal cohort study. BMC Musculoskelet Disord. 2014;15:8. - PMC - PubMed
    1. Nieuwenhuizen L, Schutgens RE, van Asbeck BS, et al. Identification and expression of iron regulators in human synovium: evidence for upregulation in haemophilic arthropathy compared to rheumatoid arthritis, osteoarthritis, and healthy controls. Haemophilia. 2013;19(4):e218‐e227. - PubMed
    1. Roosendaal G, Tekoppele JM, Vianen ME, van den Berg HM, Lafeber FP, Bijlsma JW. Articular cartilage is more susceptible to blood induced damage at young than at old age. J Rheumatol. 2000;27(7):1740‐1744. - PubMed

Publication types

MeSH terms