α-Ketoglutarate alleviates osteoarthritis by inhibiting ferroptosis via the ETV4/SLC7A11/GPX4 signaling pathway

Abstract

Osteoarthritis (OA) is the most common degenerative joint disorder that causes disability in aged individuals, caused by functional and structural alterations of the knee joint. To investigate whether metabolic drivers might be harnessed to promote cartilage repair, a liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics approach was carried out to screen serum biomarkers in osteoarthritic rats. Based on the correlation analyses, α-ketoglutarate (α-KG) has been demonstrated to have antioxidant and anti-inflammatory properties in various diseases. These properties make α-KG a prime candidate for further investigation of OA. Experimental results indicate that α-KG significantly inhibited H₂O₂-induced cartilage cell matrix degradation and apoptosis, reduced levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) and glutathione (GSH)/glutathione disulfide (GSSG) levels, and upregulated the expression of ETV4, SLC7A11 and GPX4. Further mechanistic studies observed that α-KG, like Ferrostatin-1 (Fer-1), effectively alleviated Erastin-induced apoptosis and ECM degradation. α-KG and Fer-1 upregulated ETV4, SLC7A11, and GPX4 at the mRNA and protein levels, decreased ferrous ion (Fe²⁺) accumulation, and preserved mitochondrial membrane potential (MMP) in ATDC5 cells. In vivo, α-KG treatment inhibited ferroptosis in OA rats by activating the ETV4/SLC7A11/GPX4 pathway. Thus, these findings indicate that α-KG inhibits ferroptosis via the ETV4/SLC7A11/GPX4 signaling pathway, thereby alleviating OA. These observations suggest that α-KG exhibits potential therapeutic properties for the treatment and prevention of OA, thereby having potential clinical applications in the future.

Keywords: ETV4/SLC7A11/GPX4 signaling pathway; Ferroptosis; Osteoarthritis; α-ketoglutarate.

Fig. 1

Serum metabolic profiling by LC‒MS in OA rats. A PCA score plot of QC analysis in the Blank and Model groups in positive ion mode. B PCA score plot of QC analysis in the Blank and Model groups in negative ion mode. C Plots of PLS-DA scores in the Blank and Model groups in positive ion mode. D Plots of PLS-DA scores in the Blank and Model groups in negative ion mode. E, F Volcano plots of metabolites in positive and negative ion modes in the Blank and Model groups. Metabolites significantly expressed are positioned above the dashed line, whereas those not significantly expressed are located below it. G Heatmap analysis of differential metabolites: blue bars denote decreased levels, and red bars signify increased levels. H, I Levels of α-KG in rat serum and knee cartilage. The statistical significance of the differences between the two groups was assessed using Student's t-test. Blank vs Model, ###p < 0.001 (n = 6)

Fig. 2

α-KG alleviated oxidative stress and ferroptosis in H₂O₂-induced ATDC5 cells. A Assessing the changes in cell proliferation 24 h after α-KG treatment. B Measuring the effects of different α-KG concentrations on H₂O₂-induced ATDC5 cells. C, D Flow cytometry was used to assess the apoptosis rate in each group. E Fluorescence images of ROS in ATDC5 cells, where the green fluorescence intensity of DCFH-DA represents the ROS level. F ROS levels were analyzed using flow cytometry. G–I Levels of MDA, SOD, and GSH/GSSG in ATDC5 cells. J, K Alcian blue quantification and staining. L qRT-PCR analysis of ETV4, SLC7A11, and GPX4. M, N Western blot analysis and quantification of ETV4, SLC7A11, and GPX4. The statistical significance of the differences among groups was assessed using one-way ANOVA. Control vs H₂O₂, ###p < 0.001; H₂O₂ vs α-KG, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3)

Fig. 3

α-KG inhibited ferroptosis in Erastin-induced ATDC5 cells. A, B Apoptosis rate of each group. C, D Alcian blue staining and quantification. E The mitochondrial membrane potential was examined utilizing JC-1. Red, JC-1 aggregates; green, JC-1 monomers. F Red fluorescence intensity divided by green fluorescence intensity was used to determine the relative MMP. G Fe²⁺ concentration measurement. H qRT‒PCR analysis of ETV4, SLC7A11, and GPX4. I-J. Western blot analysis and quantification of ETV4, SLC7A11, and GPX4. The statistical significance of the differences among groups was assessed using one-way ANOVA. Control vs Erastin, ###p < 0.001; Erastin vs α-KG/Fer-1, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3)

Fig. 4

α-KG improved the function of joints in OA. A Time plots of the animal experiments (ID: IROIS7887a). B Changes in weight-bearing time. C, D The gait analysis demonstrated the weight-bearing capacity and contact area of the right hind paw. The statistical significance of within-group differences was assessed using Student's t-test. Rear Left Paw vs Rear Right Paw, *p < 0.05, **p < 0.01 (n = 5). The statistical significance of differences among groups was assessed using one-way ANOVA. Blank vs Model, ##p < 0.01, ###p < 0.001. Model vs α-KG/PC, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5)

Fig. 5

Protection of OA rats by α-KG. A Micro-CT three-dimensional reconstructed images and two-dimensional images of knee joints from the different groups. B–D BS/TV, Tb.N, and Tb.Sp were analyzed by CT-AN software. E, F Safranin O-fast green staining and OARSI scores. G, I IHC staining of COL2A1 and quantitative analysis of the COL2A1-positive area. H, J IHC staining of MMP9 and quantitative analysis of the MMP9-positive area. K–M Detection of IL-6, IL-1β, and TNF-α levels in the joint fluid by ELISA. The statistical significance of the differences among groups was assessed using one-way ANOVA. Blank vs Model, #p < 0.05, ##p < 0.01, ###p < 0.001; Model vs α-KG/PC, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5)

Fig. 6

α-KG inhibited ferroptosis by activating the ETV4/SLC7A11/GPX4 pathway. A The concentrations of MDA and Fe²⁺, and the activity of SOD in cartilage tissues, were measured. B qRT-PCR analysis of ETV4, SLC7A11, and GPX4 in cartilage tissues. C Western blot analysis and quantification of ETV4, SLC7A11, and GPX4 in cartilage tissues. D A schematic diagram illustrating the mechanism was generated via FigDraw (ID: PISSYcd7bc). The statistical significance of the differences among groups was assessed using one-way ANOVA. Blank vs Model, ###p < 0.001; Model vs α-KG, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5)