Selenium Lessens Osteoarthritis by Protecting Articular Chondrocytes from Oxidative Damage through Nrf2 and NF-κB Pathways

Abstract

Osteoarthritis (OA) causes joint pain and disability due to the abnormal production of inflammatory cytokines and reactive oxygen species (ROS) in chondrocytes, leading to cell death and cartilage matrix destruction. Selenium (Se) intake can protect cells against oxidative damage. It is still unknown whether Se supplementation is beneficial for OA. This study investigated the effects of Se on sodium iodoacetate (MIA)-imitated OA progress in human chondrocyte cell line (SW1353 cells) and rats. The results showed that 0.3 μM of Se treatment could protect SW1353 cells from MIA-induced damage by the Nrf2 pathway by promoting the gene expression of glutathione-synthesis-related enzymes such as the glutamate-cysteine ligase catalytic subunit, the glutamate-cysteine ligase modifier subunit, and glutathione synthetase. In addition, glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase expressions are also elevated to eliminate excessive ROS production. Moreover, Se could downregulate NF-κB, leading to a decrease in cytokines, matrix proteases, and glycosaminoglycans. In the rats, MIA-induced cartilage loss was lessened after 2 weeks of Se supplementation by oral gavage; meanwhile, glutathione synthesis was increased, and the expressions of pro-inflammatory cytokines were decreased. These results suggest that Se intake is beneficial for OA due to its effects of decreasing cartilage loss by enhancing antioxidant capacity and reducing inflammation.

Keywords: anti-inflammation; antioxidant; chondrocyte; minerals; osteoarthritis; selenium.

Figure 1

Cell viability of SW1353 cells treated with MIA and/or Se. SW1353 cells were cultured for 24 h with or without 5 μM of MIA, accompanied by the presence or absence of 0.3–3 μM of Se. Subsequently, cell viability was assessed using trypan blue exclusion and represented as a percentage relative to the control. The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 2

Oxidative stress of SW1353 cells treated with MIA (M) and/or Se. SW1353 cells were cultured for 24 h with or without 5 μM of MIA, accompanied by the presence or absence of 0.3 μM of Se. Subsequently, levels of ROS were assessed using 10 μM of DCFH-DA staining, followed by analysis through flow cytometry. The untreated control is depicted by the gray-filled area with black lines, while the treated groups are represented by the blue lines. The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 3

Expression of antioxidants in SW1353 cells treated with MIA and/or Se. SW1353 cells were cultured for 24 h with or without 5 μM of MIA, accompanied by the presence or absence of 0.3 μM of Se. (A) The GSH levels were assayed by the enzymatic recycling method. (B) The mRNA expressions of GCLC, GCLM, GSS, and GR were evaluated by RT-qPCR and expressed as fold changes relative to the untreated control group. (C) The protein levels of GPx1, Cu/Zn-SOD, and Mn-SOD were determined using Western blotting, with β-actin used as a loading control. The right panel shows the relative density values of the representative Western blots compared to the untreated control (100%). The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 4

Effects of an inhibitor of GSH or SOD on the intracellular ROS levels and cell viability in SW1353 cells. SW1353 cells were subjected to treatment with or without 5 μM of MIA and in the presence or absence of 0.3 μM of Se, with the addition of 10 μM of BSO (a GSH inhibitor) or 0.2 μM of DETC (a SOD inhibitor) for 24 h. (A) Intracellular levels of ROS were assessed through 10 μM of DCFH-DA staining, followed by analysis using flow cytometry. The untreated control is depicted by the gray-filled area with black lines, while the treated groups are represented by the blue lines. (B) The cell viability was evaluated using trypan blue exclusion and expressed as a percentage of the control. The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group; ◆: p < 0.05 versus MIA+Se-treated group.

Figure 5

Extracellular matrix and MMP expressions of SW1353 cells treated with MIA and/or Se. SW1353 cells were cultured for 24 h with or without 5 μM of MIA, accompanied by the presence or absence of 0.3 μM of Se. (A) Glycosaminoglycan (GAG) contents were assessed by toluidine blue O staining, followed by a colorimetric assay. (B) The mRNA expressions of MMP-1, MMP-3, MMP-9, MMP-13, and ADAMTS-4 were evaluated by RT-qPCR and expressed as fold changes relative to the untreated control group. The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 6

Inflammatory cytokine expressions of SW1353 cells treated with MIA and/or Se. SW1353 cells were cultured for 24 h with or without 5 μM of MIA, accompanied by the presence or absence of 0.3 μM of Se. The mRNA levels of IL-1β, TNF-α, IL-6, and IL-17A were evaluated through RT-qPCR and expressed as fold changes relative to the untreated control group. The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 7

Transcription factor expressions of SW1353 cells treated with MIA and/or Se. SW1353 cells were cultured for 24 h with or without 5 μM of MIA, accompanied by the presence or absence of 0.3 μM of Se. The protein levels of Nrf2 and NF-κB were determined using Western blotting, with β-actin used as a loading control. The right panel shows the relative density values of the representative Western blots compared to the untreated control (100%). The results are presented as the mean ± standard deviation of experiments performed in triplicate. *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 8

Morphological and histological changes in articular cartilage in the MIA-induced OA rats with or without Se supplementation. The study design and timeline for the experiment involving MIA-induced OA in rats and Se administration are outlined in the Materials and Methods section. (A) Pictures capturing the articular surfaces of the femoral groove. (B) Photomicrographs depicting histomorphological alterations in joint cartilage stained with safranin O/fast green. Proteoglycan is represented by the red color. Scale bar: 100 μm. (C) Scoring of each joint based on the OARSI criteria. The results are presented as the mean ± standard deviation (n = 6). *: p < 0.05 versus untreated control group; #: p < 0.05 versus MIA-treated group.

Figure 9

Serum levels of GSH, SOD, MMP-13, and cytokines in the MIA-induced OA rats with or without Se supplementation. (A) GSH and SOD levels were assessed using enzymatic and colorimetric assay kits. (B) MMP-13, IL-1β, TNF-α, and IL-6 levels were determined through enzyme-linked immunosorbent assay. The results are presented as the mean ± standard deviation (n = 6). *: p < 0.05 compared to the untreated control group; #: p < 0.05 compared to the MIA-treated group.

Figure 10

Schematic diagram illustrating the effects of Se on MIA-treated chondrocytes. The findings of this study highlight that Se can protect against MIA-induced oxidative stress, elevated levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-17A, and TNF-α), and increased expression of MMPs (MMP-1, MMP-3, MMP-9, MMP-13, and ADAMTS-4). This protection is attributed to the activation of the Nrf2 pathway, which, in turn, upregulates the gene expression of antioxidants, including Cu/Zn-SOD, Mn-SOD, GPx1, GSH, GCLC, GCLM, GSS, and GR. This cascade leads to an augmentation of the anti-oxidative capacity, providing defense against MIA-induced oxidative stress. Furthermore, the addition of Se results in a decrease in NF-κB expression, counteracting the increase induced by MIA. This decrease contributes to a reduction in pro-inflammatory cytokines and MMP expression, ultimately mitigating matrix degradation. Red↑: enhanced by MIA; Red↓: decreased by MIA; Red=: did not change with MIA; Blue↑: enhanced by Se; Blue↓: decreased by Se.