Leucine-Rich Repeat-Containing G-Protein Coupled Receptor 6 Protects Cartilage from Diabetes-Driven Degeneration by Blocking Ferroptosis: A New Therapeutic Target for Osteoarthritis

Abstract

Background:

Osteoarthritis (OA), characterized by articular cartilage degeneration, is exacerbated by diabetes mellitus (DM), an independent risk factor whose molecular mechanisms remain incompletely understood. This study investigates novel regulators and pathways underlying DM-associated OA pathogenesis.

Methods:

We used bioinformatic analysis of transcriptomic data from OA and diabetic OA (DM-OA) cohorts to identify differentially expressed genes. We constructed functional enrichment and protein–protein interaction (PPI) networks. In vivo, we modeled diabetic OA in mice via high-fat diet/streptozotocin induction combined with destabilization of the medial meniscus surgery. In vitro, we exposed chondrocytes to high glucose to mimic diabetic conditions. We genetically modulated leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) through chondrocyte-specific knockout (KO) in LGR6-deficient mice and overexpression (OE) via intra-articular delivery of adeno-associated virus serotype 9. We validated key molecular changes using quantitative reverse transcription polymerase chain reaction, Western blotting, immunohistochemistry, and ferroptosis-associated assays (reactive oxygen species, glutathione, malondialdehyde [MDA], and mitochondrial morphology).

Results:

LGR6 expression was significantly downregulated in DM-OA cartilage. PPI analysis highlighted interactions between LGR6, collagen type II (COL2A1), and matrix metalloproteinase (MMP)13. LGR6 KO exacerbated OA severity, cartilage degradation, and inflammatory markers (MMP3, MMP13, and nitric oxide synthase-2) while reducing extracellular matrix (ECM) components (COL2A1 and SRY-box transcription factor 9). Conversely, LGR6 OE attenuated cartilage damage, suppressed catabolic factors, and restored ECM synthesis. Mechanistically, LGR6 deficiency intensified ferroptosis, evidenced by elevated lipid peroxidation (MDA), mitochondrial cristae disruption, and dysregulation of glutathione peroxidase 4/prostaglandin-endoperoxide synthase 2. LGR6 activation reversed these effects, restoring redox homeostasis and mitochondrial integrity.

Innovation:

This study identifies LGR6 as a pivotal inhibitor of chondrocyte ferroptosis in DM-OA, revealing a previously unexplored link between hyperglycemia, mitochondrial dysfunction, and iron-dependent cell death.

Conclusion:

LGR6 safeguards cartilage by suppressing ferroptosis and maintaining mitochondrial biogenesis in diabetic conditions. Targeting the LGR6 pathway offers a promising therapeutic strategy for DM-associated OA. Antioxid. Redox Signal. 44, 357–372.

Graphical Abstract

Keywords

LGR6 osteoarthritis diabetes ferroptosis chondrocytes

Innovation

This study demonstrates, for the first time, that leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) expression is significantly reduced in diabetic cartilage and chondrocytes under hyperglycemic conditions. Genetic ablation of LGR6 exacerbates osteoarthritis (OA) progression, whereas chondrocyte-specific LGR6 overexpression attenuates cartilage degeneration and mitochondrial dysfunction, establishing LGR6 as a pivotal modulator of diabetic OA (DM)-OA pathogenesis. The study connects hyperglycemia-induced metabolic stress with redox dysregulation in chondrocytes, highlighting LGR6 as a nexus for modulating mitochondrial biogenesis and oxidative damage in DM-OA. This dual regulatory role underscores the potential of LGR6 to counteract both metabolic and oxidative insults in diabetic joints.

Introduction

Osteoarthritis (OA) is the most common form of arthritis and one of the most common causes of disability worldwide. It is a chronic, progressive degenerative joint disease that affects the entire joint structure, including articular cartilage, subchondral bone, synovium, ligaments, and periarticular muscles (Courties, 2024; Lu et al., 2025; Schett et al., 2013). As one of the most common musculoskeletal disorders, OA poses a significant burden on individuals and global health care systems (Zhang et al., 2017). The global prevalence of OA is estimated to be ∼7% of the general population, with considerable regional and ethnic group variation (Wang et al., 2019). This disease is characterized by gradual progression, often leading to irreversible joint damage and chronic pain, with serious implications for the patient’s quality of life and independence (Centers for Disease and Prevention, 2010; Lai et al., 2022). OA features include cartilage degeneration, subchondral bone remodeling, osteophyte formation, and varying degrees of synovial inflammation. These changes result from a complex interplay between mechanical, metabolic, and biochemical factors that disrupt joint homeostasis, ultimately leading to joint dysfunction, pain, and impaired mobility (Englund, 2023). The disease is often associated with aging, but other risk factors, such as joint damage, obesity, type 2 diabetes mellitus (T2DM), and genetic predisposition, also contribute to its onset and progression (Laiguillon et al., 2015; Marshall et al., 2019).

Metabolic diseases, particularly T2DM, which is widely recognized as a chronic low-grade inflammatory status, serve as independent risk factors for the pathogenesis of OA (Schett et al., 2013). Although OA and diabetes share common risk factors such as obesity and aging, diabetic OA (DM-OA) exhibits distinct pathophysiological mechanisms and clinical characteristics. The association between diabetes and OA is increasingly recognized, with diabetes not only elevating the risk of OA but also accelerating its progression and exacerbating its severity. Unlike classical OA, which is primarily driven by mechanical stress, aging, and cumulative joint tissue damage, DM-OA is influenced by additional metabolic and systemic factors associated with diabetes (Schett et al., 2013). Particularly in T2DM, systemic inflammation, oxidative stress, altered cellular metabolism, and microvascular damage all contribute to the accelerated progression of OA in these patients (de Jonge et al., 2015; Shi et al., 2021). Hyperglycemia-driven inflammation, oxidative stress, mitochondrial dysfunction, and microvascular injury disrupt cartilage homeostasis, promoting extracellular matrix (ECM) degradation and synovitis, collectively termed DM-OA (Williams et al., 2016). Diabetic conditions elevate synovial proinflammatory cytokines (e.g., interleukin [IL]-1β and IL-6) and matrix metalloproteinases (MMPs), accelerating cartilage catabolism beyond the effects of mechanical loading alone. Consequently, diabetic individuals exhibit 1.5- to 2-fold higher OA risk and experience more rapid disease progression compared with nondiabetic individuals (Schett et al., 2013).

Various forms of chondrocyte death, including apoptosis and necroptosis, have been identified in OA cartilage, suggesting that chondrocyte death contributes to OA pathogenesis (Jeon et al., 2020; Park et al., 2020). Recently, ferroptosis, an iron-dependent programmed cell death that is entirely distinct from conventional forms of cell death, has been recognized (Stockwell et al., 2017). Unlike other regulated cell death forms, such as apoptosis and necrosis, ferroptosis is characterized by iron accumulation and lipid peroxidation (LPO), with morphological features including mitochondrial shrinkage and cristae alterations (Li et al., 2020a). Studies have extensively investigated ferroptosis in neurodegenerative disorders [such as Alzheimer’s disease (Derry et al., 2020), Parkinson’s diseases (Mahoney-Sanchez et al., 2021)], hemorrhagic stroke (Tuo et al., 2017), and acute kidney injury (Friedmann Angeli et al., 2014). In recent years, evidence has linked ferroptosis to OA, as iron accumulation and LPO in OA cartilage indicate a strong connection between the disease and ferroptosis (Sun et al., 2021; Zhang et al., 2023b). Iron accumulation and LPO in OA cartilage indicate a strong connection between ferroptosis and disease progression. Ferroptosis accelerates cartilage degradation by inducing chondrocyte dysfunction and death via LPO (Ru et al., 2023) and can influence inflammatory factor expression in synovial fibroblasts, thereby promoting ECM degradation (Hu et al., 2024). Inflammatory cytokines such as IL-1β, elevated in OA, can induce chondrocyte ferroptosis (Yao et al., 2021). Consequently, key ferroptosis regulators such as glutathione peroxidase 4 (GPX4; Miao et al., 2022), acyl-CoA synthetase long-chain family member 4 (ACSL4; He et al., 2023), and nuclear factor erythroid 2-related factor 2 (NRF2; Zhou et al., 2023) have emerged as potential therapeutic targets for OA.

Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6), homologous to LGR4 and LGR5, modulates inflammation resolution in cardiopulmonary diseases and ischemia-reperfusion injury (Elder et al., 2021; Leroy et al., 2023; Li et al., 2022; Zhang et al., 2023a; Zhao et al., 2024). As a Wnt signaling agonist, LGR6 binds R-spondin ligands to potentiate β-catenin-dependent transcription, which is crucial for skeletal development and osteogenesis (Bennett et al., 2005; Cui et al., 2011). Despite its established role in bone homeostasis, the function of LGR6 in chondrocyte survival and its potential involvement in DM-OA pathogenesis remain unexplored.

Given the accelerated cartilage degeneration in DM-OA and the emerging role of ferroptosis in OA progression, we hypothesized that LGR6 protects chondrocytes from diabetes-associated damage by modulating ferroptosis. Therefore, this study aimed to determine the expression pattern of LGR6 in diabetic cartilage, investigate its functional role in DM-OA progression using genetic approaches in vivo and in vitro, and elucidate the underlying mechanism with a focus on ferroptosis and mitochondrial dysfunction. Our findings identify LGR6 as a novel suppressor of ferroptosis in DM-OA, revealing a previously unrecognized link between hyperglycemia, LGR6 downregulation, and iron-dependent chondrocyte death.

Results

Here, we discovered that LGR6 was downregulated in destabilization of the medial meniscus (DMM) and DM-OA. Our results showed that LGR6 knockout (KO) aggravated, but cartilage-specific LGR6 overexpression (OE) ameliorated, the cartilage dysfunction and remodeling of diabetic mice. Mechanistically, in vivo and in vitro experiments revealed that LGR6 deficiency aggravated, whereas LGR6 OE alleviated, ferroptosis and mitochondrial biogenesis disorder in diabetic cartilages and chondrocytes under high glucose (HG) conditions. Taken together, these findings provide evidence that targeting the LGR6 in chondrocytes may provide a potential therapeutic option for diabetic cartilages. The findings of our studies herein are summarized in the graphical abstract.

Differential gene expression in OA of patients with DM

We searched and downloaded mRNA expression data of DM-OA from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) through the keyword “diabetic osteoarthritis.” We analyzed the transcriptomics results with 732 differential genes between the OA group and the DM-OA group (GSE198836). The volcano plot shows differentially expressed genes (DEGs) between OA and DM-OA, and DEGs with |log2FC| > 1 are labeled red or blue (Fig. 1A). Three hundred seventeen genes were upregulated in DM-OA, including receptor activator of nuclear factor kappa-B ligand, MMP13, and IL-6. Four hundred fifteen genes, including bone morphogenetic protein 2, collagen type II (COL2A1), insulin-like growth factor 1, and IL-4, were reduced in DM-OA. To further explore the biological functions of DEGs, the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation of hub genes was performed by Metascape, and the results are shown (Fig. 1B, C). Interestingly, enriched pathways are mainly immune-related cell chemotaxis, chemokine-mediated signaling pathways, neutrophil chemotaxis, lipid metabolism processes, complement activation, and amplification of cell signaling inflammatory responses, which are closely associated with OA. We presented the most statistically significant clusters according to the -log10(P) (Fig. 1D, E). To further explore the function of hub genes between OA and DM-OA, protein–protein interaction network analysis and functional enrichment analysis were constructed. Our results showed that there were some biological connections between these 21 hub genes (Fig. 1F). We applied gene set enrichment analysis (GSEA) analyses to bulk RNA-seq data of OA and DM-OA to find potential molecular mechanisms and biological processes in DM-OA. In the GSEA analysis, the NF-kappa B signaling pathway and collagen degradation pathway signatures were significantly enriched in the DM-OA dataset (Fig. 1G, H). On the contrary, compared with the OA group, the collagen synthesis signaling pathway was significantly inhibited in the DM-OA group (Fig. 1I). We identified a total of 16 genes closely related to the progression of OA, among which the expression in chondrocytes of genes encoding catabolic factors, including MMPs (MMP3 and MMP13) and nitric oxide synthase-2 (NOS2), was enriched in DM-OA (Fig. 1J). Interestingly, a G protein-coupled receptor, LGR6 (G protein-coupled receptor containing leucine-rich repeats 6), was significantly downregulated as well as COL2A1, suggesting a potential role of LGR6 in DM-OA in vivo.

FIG. 1.

LGR6 was downregulated in DM-OA. (A) The volcano plot of differentially expressed genes (DEGs) in DM-OA and OA. DEGs with |log2FoldChange| > 1 are labeled red or blue. (B) GO term enrichment analysis of downregulated genes in DM-OA. (C) KEGG enrichment analysis of downregulated genes in DM-OA. (D) Gene enrichment clusters and nodes colored by cluster ID. (E) Gene enrichment clusters and nodes colored by cluster ID. (F) Visualization of PPI network of hub genes. (G–I) GSEA of differentially expressed genes in human DM-OA for the NF-kappa B signaling pathway (NES = 2.33, p < 0.001, FDR < 0.001) (G), the collagen degradation pathway (NES = 2.7, p < 0.001, FDR < 0.001) (H), and collagen-containing extracellular matrix (NES = 2.83, p < 0.001, FDR < 0.001) (I). False discovery rate (q-value) and normalized enrichment score (NES) calculated by GSEA. (J) Heatmap of differentially expressed genes (DEGs) in DM-OA.DM, diabetes mellitus; OA, osteoarthritis.

DM promoted cartilage OA and LGR6 expression in DMM mice

To confirm that DM promotes the progression of OA in DMM mice, we established a mouse model of DM +DMM in mice using HG in combination with DMM. OARSI scoring based on SO staining showed that the cartilage damage was aggravated in the joints of the DM +DMM treatment (Fig. 2A, B). DM decreased the expression of ECM molecules, including COL2A1 and SRY-box transcription factor 9 (SOX9), in DM+DMM mouse chondrocytes (Fig. 2C–E). The expression of MMP3, MMP13, and NOS2 was increased in mice with DM (Fig. 2C–E). We isolated adult mouse chondrocytes subjected to sham or DMM conditions to investigate LGR6 expression in chondrocytes. As expected, LGR6 protein levels were markedly decreased in chondrocytes from DMM mice (Fig. 2F). Consistent with the transcriptional data, LGR6 protein levels in the mouse cartilages gradually decreased with the progression of diabetes (Fig. 2G). Consistent with our findings in DMM mice, LGR6 expression was markedly reduced in HG-treated chondrocytes compared with LG-treated chondrocytes, as shown by the immunoblot assays (Fig. 2F). Immunofluorescent staining also showed DM increased the expression of MMP3 but reduced LGR6 in mice (Fig. 2H). These results suggest that the cartilage LGR6 expression is markedly decreased in the diabetic mice. Proinflammatory cytokines in chondrocytes trigger expression of various catabolic factors. HG increased IL-1β-induced MMP3 as well as inhibited LGR6 in primary culture of articular chondrocytes (Fig. 2I). Together, these data suggested that LGR6 expression was reduced in DMM and diabetic mice.

FIG. 2.

DM increased inflammatory and OA of DMM. (A) Representative images of Safranin O/Fast Green staining of the medial knee joint section of male mice (n = 5) subjected to sham operation, DMM, or DM+DMM surgery. Scale bar, 100 μm. (B) Scoring of OARSI grade to evaluate the extent of cartilage destruction of male mice in (A) (n = 5). (C) The relative mRNA levels of several representative genes in mice subjected to sham operation, DMM, or DM+DMM surgery (n = 5). Significant p-values between groups are presented as **p < 0.01, ***p < 0.001, and ****p < 0.0001 by Student’s t-test. (D) Western blotting of several representative protein levels in mice subjected to sham operation, DMM, or DM+DMM surgery. (E) Qualitative analysis of Western blotting in (D). (F) Western blotting of LGR6 in mice subjected to sham operation, DMM surgery, or DM+DMM surgery. (G) Western blotting of LGR6 protein levels in mice subjected to sham operation or DM surgery. (H) Representative immunofluorescent staining images and qualitative analysis of MMP3 and LGR6 in cartilage sections of mice subjected to DM or DM+DMM surgery. Scale bar: 100 μm. (I) Representative immunofluorescent staining images of levels of MMP3 and LGR6 in chondrocytes treated with IL-1β (5 ng/mL) and IL-1β+HG (25 mM) for 24 h. Scale bar: 10 μm. DM, diabetes mellitus; DMM, medial meniscus amputation; HG, high glucose; OA, osteoarthritis.

LGR6 KO aggravated inflammatory and OA of DMM

To directly assess the functional role of LGR6, we subjected LGR6-KO mice to DMM surgery. LGR6 deficiency markedly exacerbated OA progression, as demonstrated by significantly higher OARSI scores and more severe cartilage damage compared with wild-type (WT) controls (Fig. 3A, B). The aggravated phenotype in LGR6-KO mice was mechanistically linked to a further reduction in anabolic markers (COL2A1 and SOX9) and a synergistic increase in catabolic markers (MMP3, MMP13, and NOS2; Fig. 3C–E).

FIG. 3.

LGR6 knockout aggravated inflammatory and osteoarthritis (OA) of DMM. (A) Representative images of Safranin O/Fast Green staining of mice cartilages in sham, DMM, sham +KO, and DMM +KO. Scale bar: 500 μm in black, 100 μm in red. (B) Qualitative analysis of OA severity by OARSI scores for mice in (A); n = 5 per group. Significant p-values among the treatment groups are presented as ***p < 0.001 and ****p < 0.0001 by two-way ANOVA with post hoc Tukey test. (C) The relative mRNA levels of several representative genes in sham, DMM, sham +KO, and DMM +KO (n = 5). Significant p-values between groups are presented as **p < 0.01, ***p < 0.001, and ****p < 0.0001 by Student’s t-test. (D, E) Western blotting of several representative protein levels in LGR6^+/+ and LGR6^−/−mice subjected to sham operation or DMM surgery (D) and quantitative analysis (E). The values represent the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, n = 5. (F) Quantitative analysis of LGR6 in chondrocytes (Adv-shLGR6). (G) Representative immunofluorescent staining images of NOS2 and COL2A1 in primary chondrocytes of Adv-Null and Adv-shLGR6 treated with IL-1β or not; scale bar: 10 μm. (H–I) Western blot (H) and quantitative analysis (I) of MMP3, MMP13, NOS2, COL2A1, SOX9 and LGR6 protein levels in Adv-null and Adv-shLGR6 chondrocytes treated with IL-1β or PBS.

We replicated these findings in vitro by knocking down LGR6 in primary mouse chondrocytes using an adenovirus encoding shRNA (Adv-shLGR6). LGR6 knockdown significantly potentiated the IL-1β-induced inflammatory response, leading to a greater upregulation of NOS2 and a more severe suppression of COL2A1 (Fig. 3F, G). Western blotting analysis confirmed that LGR6 knockdown enhanced the expression of MMP3, MMP13, and NOS2 while suppressing COL2A1 and SOX9 protein levels upon IL-1β stimulation (Fig. 3H, I). These results demonstrate that LGR6 deficiency disrupts cartilage homeostasis and accelerates OA progression.

LGR6 OE alleviates inflammatory and OA of DMM

We next specifically overexpressed LGR6 in chondrocytes by delivering adeno-associated virus serotype 9 (AAV9)-LGR6 into WT mice through tail vein injection 4 weeks postdiabetes, with AAV9-null as a control. Comparative histological assessments using Safranin O/Fast Green staining showed substantial ECM component loss in the OA group, which was mitigated in LGR6 OE (Fig. 4A, B). This protective effect was confirmed at the molecular level, as LGR6 OE suppressed the mRNA and protein levels of MMP3, MMP13, and NOS2, while rescuing the expression of COL2A1 and SOX9 (Fig. 4C–E).

FIG. 4.

LGR6 overexpression alleviates inflammatory and osteoarthritis (OA) of DMM. (A) Representative images of Safranin O/Fast Green staining of mice cartilages in sham, DMM, and DMM +OE. Scale bar: 500 μm in black, 100 μm in red. (B) Qualitative analysis of OA severity by OARSI scores for mice in (A); n = 5 per group. Significant p-values among the treatment groups are presented as ***p < 0.001 and ****p < 0.0001 by two-way ANOVA with post hoc Tukey test. (C) Western blotting of several representative protein levels in LGR6 OE mice subjected to sham operation or DMM surgery. (D) The relative mRNA levels of several representative genes in sham, DMM, and DMM +OE (n = 5). Significant p-values between groups are presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by Student’s t-test. (E) Immunofluorescent staining for COL2A1 and MMP13 expressions in cartilage samples. (F) Representative immunofluorescent staining images of MMP13 and COL2A1 in control, DMM, and DMM with Adv-shLGR6 chondrocytes; scale bar: 10 μm. (G) The relative mRNA levels of several representative genes in control, DMM, and DMM with Adv-shLGR6 chondrocytes (n = 5). Significant p-values between groups are presented as **p < 0.01, ***p < 0.001, and ****p < 0.0001 by Student’s t-test, n = 5.

Similarly, ectopic expression of LGR6 (Ad-LGR6) in mouse chondrocytes in vitro effectively counteracted the IL-1β-induced reduction of COL2A1 and upregulation of MMP13 (Fig. 4F). Quantitative polymerase chain reaction further verified that LGR6 OE reversed the IL-1β-driven dysregulation of key catabolic and anabolic genes (Fig. 4G). These data unequivocally show that LGR6 OE alleviates inflammatory and osteoarthritic changes.

LGR6 OE ameliorates ferroptosis in diabetic cartilage

To identify how LGR6 ameliorates DM-OA, we performed comprehensive transcriptomics analysis on cartilage tissues of DMM mice with AAV9-LGR6 or AAV9-null treatment. A total of 393 genes were significantly dysregulated, consisting of 125 upregulated genes and 168 downregulated genes (Fig. 5A). KEGG analysis among DEGs showed that the KEGG ferroptosis signal pathway was significantly enriched (Fig. 5B). While other pathways, including chemokine-mediated signaling and lipid metabolism, were also enriched, ferroptosis was prioritized for investigation based on converging biological evidence. Our prior phenotypic data revealed hallmark features of ferroptosis in diabetic cartilage, including reactive oxygen species (ROS) accumulation, elevated LPO, mitochondrial cristae disruption, and downregulation of GPX4. Further supporting this, key ferroptosis-related genes (e.g., GPX4, prostaglandin-endoperoxide synthase 2 [PTGS2], and ACSL4) exhibited coordinated expression changes in DM-OA cartilage, aligning with these pathological alterations. Critically, OE of LGR6 specifically ameliorated ferroptosis markers both in vivo and in vitro, establishing a direct mechanistic link to this cell death pathway. Therefore, although multiple pathways likely contribute to DM-OA pathogenesis, we selected ferroptosis as the focal pathway due to its established role in hyperglycemia-induced oxidative damage and the compelling experimental validation within our study. According to the KEGG database, LGR6 OE significantly downregulated the expression of genes associated with ferroptosis signaling (Fig. 5C). We then assessed ferroptosis in the cartilages to investigate the role of LGR6 OE in DMM. The increase of MMP13 was reversed by LGR6 OE (Fig. 5D). Iron-dependent accumulation of toxic lipid-based ROS is a critical pathogenic factor during ferroptosis. We detected ROS in mouse cartilage tissue by dihydroethidium staining. Compared with sham mice, higher ROS level in articular cartilage was observed in DMM mice (Fig. 5E). The glutathione (GSH)/GPX4-based ROS scavenging mechanism plays indispensable roles in preventing LPO during ferroptosis. Compared with those in sham mice, the GSH levels in the synovium and articular cartilage of DMM mice were significantly lower (Fig. 5F). LPO, a hallmark of ferroptosis, was elevated in diabetic cartilages, as shown by the malondialdehyde (MDA) and lipid hydroperoxide (LPO) levels (Fig. 5G). We observed abnormal mitochondria with significant mitochondrial cristae damage, manifested by loss and swelling of cristae, in the cartilage tissues of DMM mice, a morphological feature of ferroptosis (Fig. 5H). In addition, we observed downregulated GPX4 expression and upregulated PTGS2 expression in the cartilage tissues of DMM mice (Fig. 5I). However, LGR6 OE mitigated ferroptosis in diabetic cartilage. Compared with LG treatment, HG treatment resulted in significant chondrocyte cell death as shown by decreases in cell viability observed via the CCK8 assay, which was ameliorated by Ad-LGR6 treatment (Fig. 5J). LGR6 OE in vitro reduced abnormal mitochondria ratios, ROS and MDA levels, and increased GSH level in chondrocyte cells treated with HG (Fig. 5K–M). Together, these data suggest LGR6 OE ameliorates HG-induced ferroptosis in chondrocytes.

FIG. 5.

LGR6 overexpression ameliorates ferroptosis in the diabetic chondrocytes. (A) Principal component analysis (PCA) and volcano plot of differentially expressed genes (DEGs) in chondrocytes treated with AAV9-null and AAV9-lgr6. DEGs with |log2FoldChange| > 0.5 are labeled red or green. (B) KEGG enrichment analysis of downregulated genes in AAV9-lgr6 chondrocytes. (C) Heatmap of DEGs involved in ferroptosis in AAV9-null and AAV9-lgr6 chondrocytes. (D) Serum levels of cardiac troponin I (n = 5) in AAV9-lgr6 chondrocytes treated with DMM. (E) Representative immunofluorescent staining images and corresponding qualitative analysis of reactive oxygen species (ROS) levels in chondrocytes tissues (n = 5). (F, G) The levels of GSH, MDA, and LPO in the synovium and articular cartilage of mice at 30 weeks of age (n = 5). (H) Representative electron microscopy images showing damaged mitochondria with loss of cristae in chondrocytes and qualitative analysis of the damaged mitochondria in chondrocytes (n = 5). (I) Western blotting and quantitative analysis of GPX4 and PTGS2 expression in AAV9-null and AAV9-lgr6 treated with DMM (n = 5). (J) The viability of chondrocyte cells from the indicated groups was detected via a CCK-8 assay (n = 5). (K, L) The levels of MDA and GSH in chondrocyte cells from the indicated groups (n = 5). (M) Representative electron microscopy images showing damaged mitochondria with loss of cristae in chondrocytes and qualitative analysis of damaged mitochondria in chondrocyte cells (n = 5). Significant p-values between groups are presented as **p < 0.01, ***p < 0.001, and ****p < 0.0001 by Student’s t-test, n = 5. AAV9, adeno-associated virus serotype 9; DMM, destabilization of the medial meniscus; GPX4, glutathione peroxidase 4; GSH, glutathione; HG, high glucose; LG, low glucose; LPO, lipid hydroperoxide; MDA, malondialdehyde; PTGS2, prostaglandin-endoperoxide synthase 2; ROS, reactive oxygen species.

LGR6 deficiency aggravates ferroptosis of chondrocytes

Next, we detected the ferroptosis in chondrocytes treated with Ad-shCtrl and Ad-shLgr6. ROS contributes to changes of mitochondrial structure. Compared with those in Ad-shCtrl cells, mitochondrial area and cristae length in Ad-shLgr6 cells were significantly lower, consistent with aggravated ferroptosis (Fig. 6A, B). We sought to elucidate factors that induced ferroptosis. The chondrocytes treated with Ad-shLgr6 had higher ROS levels after being treated with IL-1β (Fig. 6C, D). Cartilage tissues of DMM mice treated with Ad-shLgr6 had higher ROS levels and higher LPO (Fig. 6E–G). Fatty acid is a key regulator of ferroptosis. We found that free fatty acid content was elevated in the cartilages of diabetic and LGR6-knockdown mice, and exogenous fatty acid treatment further enhanced ROS and LPO in LGR6-deficient chondrocytes (Fig. 6H–J). These data indicate that LGR6 deficiency creates a cellular environment conducive to ferroptosis, which is further aggravated by metabolic stressors such as elevated fatty acids.

FIG. 6.

LGR6 deficiency aggravates ferroptosis of chondrocytes. (A, B) Qualitative analysis of mitochondrial cristae length (A) and mitochondrial area (B) in chondrocytes treated with Ad-shCtrl and Ad-shLgr6. (C, D) Representative images (C) and qualitative analysis (D) of ROS in Ad-shCtrl and Ad-shLgr6 chondrocytes treated with IL-1β. (E, F) Representative images (E) and qualitative analysis (F) of ROS in Ad-shCtrl and Ad-shLgr6 cartilages treated with DMM. (G) Lipid peroxidation in Ad-shCtrl and Ad-shLgr6 chondrocytes treated with IL-1β. (H) FFA concentration in the sera and the fluids from cartilages from mice bearing DMM with Ad-shLgr6, Ad-Lgr6, or DM. (I and J) ROS levels (I) and lipid peroxidation (J) in the sera and the fluids from cartilages from mice bearing DMM with Ad-shLgr6. Significant p-values between groups are presented as **p < 0.01, ***p < 0.001, and ****p < 0.0001 by Student’s t-test, n = 5. FFA, free fatty acid; ROS, reactive oxygen species.

Discussion

This study identifies LGR6 as a novel and critical protective factor in DM-OA. We demonstrate that LGR6 expression is significantly downregulated in diabetic cartilage and that its loss exacerbates, while its OE ameliorates, OA progression by modulating chondrocyte ferroptosis. Our findings bridge a critical knowledge gap by positioning LGR6 as a molecular nexus that integrates hyperglycemic metabolic stress with iron-dependent cell death in chondrocytes, elucidating a mechanism previously unexplored in DM-OA pathogenesis.

The accelerated joint degeneration observed in patients with diabetes has been clinically recognized, yet the underlying molecular drivers remain incompletely defined. Our study provides a mechanistic explanation by linking hyperglycemia to the suppression of LGR6, a receptor whose role in cartilage biology has been obscure. While prior research has predominantly focused on LGR6’s functions in stem cell populations and organ development (Cui et al., 2018; King et al., 2024), its involvement in chondrocyte survival and OA pathophysiology has been entirely unreported. This distinct context underscores the novelty of our work. Furthermore, although ferroptosis has recently emerged as a contributor to OA (Sun et al., 2021; Yao et al., 2021), its specific regulation in the context of diabetes has been unclear. We now establish a direct link between a diabetes-compromised joint environment (characterized by LGR6 downregulation) and the potentiation of the ferroptotic cell death pathway, offering a unified framework connecting systemic metabolic dysregulation to localized structural joint damage.

A key question arising from our data is how LGR6, a cell surface receptor, transduces signals to suppress intracellular ferroptosis. We propose that LGR6 likely modulates ferroptosis through a multipronged signaling network, with the Wnt/β-catenin pathway serving as a central, though perhaps not exclusive, mechanism. As an established potentiator of Wnt signaling via R-spondin binding (Bennett et al., 2005), LGR6 activation would be expected to stabilize β-catenin and promote its nuclear translocation. This is highly relevant because β-catenin-dependent transcription can activate a suite of antioxidant genes. A prime candidate effector downstream of this cascade is NRF2, a master regulator of the antioxidant response. NRF2 stabilization induces the transcription of key ferroptosis defense genes, including GPX4, and suppresses proinflammatory and pro-oxidant mediators such as PTGS2 (Zhou et al., 2023). Our observations—that LGR6 OE restores GPX4 levels, suppresses PTGS2, and reestablishes redox homeostasis (GSH/ROS)—align perfectly with the activation of a β-catenin/NRF2 axis. Alternatively, or concurrently, LGR6 may influence iron metabolism independently of Wnt. Given its homology to LGR4/5, which can signal through ERK and mTOR pathways, LGR6 loss might dysregulate cellular iron flux. For instance, ferroptosis inducers often inhibit the cystine/glutamate antiporter (System Xc-) via ERK activation, whereas mTORC1 promotes the synthesis of iron storage proteins. Disruption of these pathways in LGR6-deficient chondrocytes could explain the elevated iron availability and subsequent LPO we observed. Downstream, the suppression of GPX4 emerges as the central terminal effector. Depleted GPX4 fails to detoxify phospholipid hydroperoxides, directly leading to membrane rupture and ferroptotic cell death.

The clinical relevance of our findings is substantial. Current OA therapies primarily manage symptoms rather than halting disease progression. For the large and growing population of patients with T2DM and OA, treatment options are particularly limited. Our work suggests that targeting the LGR6 pathway could address the root cause of accelerated cartilage loss in these individuals. Unlike generalized antioxidants, which face challenges of specificity and systemic delivery, LGR6 activation offers a strategy for cell-type-specific modulation of redox homeostasis within the joint. Therapeutic LGR6 agonism, perhaps via engineered R-spondin mimetics or small-molecule agonists, could potentially boost the endogenous GPX4-mediated defense system in chondrocytes, thereby disrupting the ferroptotic cascade. This approach holds promise for developing disease-modifying OA drugs specifically for patients with diabetes.

Despite the significant findings, our study has several limitations that should be acknowledged. While the streptozotocin (STZ)/high-fat diet (HFD) murine model is an established system for studying T2DM, it incompletely recapitulates the chronic, multisystemic complexity of human T2DM. Similarly, sustained HG exposure in vitro provides an incomplete surrogate for the dynamic metabolic and inflammatory stresses within the human DM-OA joint microenvironment. We also did not assess the potential contribution of synovitis or subchondral bone remodeling to the LGR6-mediated chondroprotection observed here. Although we strongly implicated GPX4 and PTGS2 in the protective mechanism, the precise downstream effectors, including the specific contributions of β-catenin and NRF2, require direct functional validation in future studies. Most critically, while our correlative evidence (e.g., GPX4/PTGS2 dysregulation, mitochondrial damage, and LPO) strongly supports a role for ferroptosis, we did not directly test the causal relationship. Future studies employing ferroptosis inhibitors (e.g., ferrostatin-1 and liproxstatin-1) in both WT and LGR6-KO diabetic OA models are essential to confirm that the exacerbated OA phenotype in LGR6-deficient mice is indeed rescued by the specific suppression of ferroptosis.

In conclusion, our data establish LGR6 as a guardian of chondrocyte health in diabetic conditions, orchestrating a protective axis against ferroptosis likely through Wnt/β-catenin-NRF2 signaling and the stabilization of GPX4. Therapeutic targeting of this axis represents a promising and novel strategy to mitigate diabetes-induced cartilage degeneration. Future investigations should prioritize evaluating LGR6-based therapeutics in human diabetic OA joints and delineating its precise mechanistic interactions using genetic models, patient-derived tissues, and targeted pathway modulation.

Materials and Methods

Study approval

The Institutional Review Board (IRB) of The First Affiliated Hospital of Wannan Medical College reviewed and approved all experimental procedures involving human participants (Approval Protocol Number: WMC-2023-IRB-045). All participants provided written informed consent before surgery.

We conducted all animal experiments in accordance with the Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of the Medical School of Yangzhou University approved the study protocol (Approval Protocol Number: 2024-DW-SB-016).

Mouse models and experimental OA induction

Animals

We purchased LGR6-KO and WT C57BL/6 mice from Gempharmatech Co., Ltd. (Nanjing, China). We generated homozygous LGR6-KO mice by crossing heterozygous breeders. We validated KO efficiency via quantitative reverse transcription (qRT)-PCR and Western blotting of articular cartilage, which confirmed a >90% reduction in Lgr6 mRNA and protein levels compared with WT controls.

We housed all mice under standard conditions with a 12-h light/dark cycle and provided food and water ad libitum. After a 2-week acclimatization period, we randomly assigned 8-week-old male mice to experimental groups using a random number table.

Diabetic OA model

We induced T2DM by feeding mice an HFD for 4 weeks, followed by a single intraperitoneal injection of STZ (30 mg/kg in citrate buffer, pH 4.5). We considered mice with random blood glucose levels exceeding 11.1 mmol/L to be diabetic. We then induced OA 4 weeks postdiabetes confirmation via surgical DMM under anesthesia. For the sham group, we performed a sham surgery involving joint capsule incision without meniscal destabilization. We structured the experimental groups as follows (n = 5 per group): sham group, DMM group, and DM+DMM group.

Genetic modulation in vivo

For LGR6 OE, we administered an AAV9 vector expressing mouse Lgr6 under the control of the COL2A1 promoter (AAV9-LGR6; HanBio, Shanghai, China; 1 × 10⁹ plaque-forming units [PFU]) via intra-articular injection 4 weeks after diabetes induction. We injected control mice with an AAV9-null vector. We confirmed successful OE by a >3-fold increase in cartilage LGR6 expression versus controls. For knockdown studies, we injected an adenovirus encoding short hairpin RNA against Lgr6 (Ad-shLGR6; HanBio) or a scrambled control (Ad-shCtrl) intra-articularly (1 × 10⁹ PFU). We euthanized mice 8 weeks post-DMM surgery or 2 weeks postviral injection for analysis.

Cell culture and in vitro treatments

Primary chondrocyte isolation and culture

We isolated primary murine articular chondrocytes from the knee joints of C57BL/6 mice. We cultured primary chondrocyte cells in 1 × Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37°C in a 5% CO₂ atmosphere. We transferred cells to a culture plate and subcultured them when cell density reached 80%, using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) for detachment. To preserve cellular phenotype, we utilized chondrocytes from passages 2–4 for experiments.

In vitro models

We seeded human articular chondrocytes at 5 × 10⁵ cells per well into 65 mm plates containing 1X DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and cultured at 37°C under 5% CO₂ for 24 h. To mimic a diabetic environment in vitro, we treated chondrocytes with 25 mM HG. To induce an inflammatory response, we stimulated cells with 10 ng/mL recombinant mouse IL-1β (R&D Systems, USA). For genetic modulation, we transfected chondrocytes with adenoviral shLGR6 (Ad-shLGR6; HanBio; sequence validated) or LGR6 OE virus (Ad-LGR6; HanBio), using an empty adenovirus (Ad-null) as a control. We confirmed knockdown and OE efficiencies by Western blotting, typically achieving >70% knockdown and >3-fold OE. Following 24 h of incubation, we extracted total RNA and total protein from chondrocytes and synoviocytes using TRIzol reagent and radioimmunoprecipitation assay (RIPA) buffer, respectively. We performed in vitro studies with five biological replicates per condition, repeated independently five times.

Histological and immunostaining analysis

Tissue processing and staining

We fixed mouse knee joints in 4% paraformaldehyde, decalcified them in EDTA, and embedded them in paraffin. We prepared sagittal sections (5-μm thickness). We evaluated cartilage degeneration using Safranin O/Fast Green staining and scored them in a blinded manner by two independent observers according to the OARSI scoring system.

Immunohistochemistry and immunofluorescence

For IHC, we deparaffinized sections, performed antigen retrieval, and incubated them with primary antibodies against LGR6, MMP3, and MMP13 overnight at 4°C, followed by appropriate secondary antibodies. For IF, we fixed, permeabilized, and stained chondrocytes grown on coverslips or tissue sections with primary antibodies against COL2A1, MMP13, NOS2, or LGR6, followed by fluorophore-conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole for nuclear counterstaining. We captured images using an Olympus IX73 microscope. For all histological scoring, we blinded the investigators to the group allocation.

Western blotting

We homogenized cartilage tissues or chondrocytes in RIPA lysis buffer (Beyotime, China) supplemented with 1% phenylmethylsulfonyl fluoride. We determined protein concentrations using a bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, USA, or Beyotime, China). We separated equal amounts of protein (10–20 μg) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred them to polyvinylidene fluoride membranes. We blocked the membranes and then incubated them overnight at 4°C with primary antibodies against MMP3, MMP13, NOS2, COL2A1, SOX9, GPX4, PTGS2, LGR6, and GAPDH. After incubating with horseradish peroxidase-conjugated secondary antibodies, we visualized protein bands using enhanced chemiluminescence reagents and quantified them using Image Lab 3.0 software (Bio-Rad, USA).

Quantitative reverse transcription polymerase chain reaction

We extracted total RNA from cartilage tissues or chondrocytes using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. We synthesized cDNA using a reverse transcription kit (Takara, Japan). We performed quantitative PCR using Power SYBR® Green PCR Master Mix (Applied Biosystems, USA) on a QuantStudio real-time PCR system. We calculated gene expression levels using the 2^–ΔΔCt method, with GAPDH serving as the endogenous control. We will provide primer sequences upon request.

Transmission electron microscopy

We collected fresh cartilage tissues from mouse knees or human OA samples and cut them into 1 mm³ pieces. We immediately fixed the samples in 2.5% glutaraldehyde. We then postfixed them in 1% osmium tetroxide, dehydrated them in a graded ethanol and acetone series, and embedded them in epoxy resin. We stained ultrathin sections (70 nm) with uranyl acetate and lead citrate. We examined mitochondrial morphology using a Talos L120C G2 transmission electron microscope (Thermo Fisher Scientific, USA) at 120 kV. We assessed and quantified mitochondrial cristae integrity in a blinded manner.

Ferroptosis-associated assays

Reactive oxygen species

We measured intracellular ROS levels using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Beyotime, China). We incubated chondrocytes, or cartilage sections, with 10 μM DCFH-DA for 30 min at 37°C in the dark. We observed fluorescence intensity under a fluorescence microscope (Olympus IX73) and quantified it.

Glutathione

We seeded chondrocytes in a 6-well plate. We treated cells as described above for 4 h. For tissue sample, we added 500 µL protein removal reagent to 30–50 mg cartilage tissue. Subsequently, we homogenized the tissue with a homogenizer in an ice bath, placed it at 4°C for 10 min, and centrifuged it at 10,000 g at 4°C for 10 min. We collected the supernatant and determined total GSH levels using the GSH Assay Kit (Beyotime, Jiangsu, China) following the manufacturer’s instructions.

Malondialdehyde

We measured MDA content using the LPO MDA Assay Kit (Beyotime, Jiangsu, China). We seeded chondrocytes in a 6-well plate. We treated cells as described above for 4 h. We harvested protein lysates using cell lysis buffer supplemented with protease inhibitor. We added 200 mL MDA detection working buffer into 100 mL samples or standard solution, followed by heating in a 100°C hot iron block for 15 min and water bath cooling to room temperature. Then, we centrifuged the tubes at 1000 g at room temperature for 10 min and measured the absorbance of A532. We quantified protein concentrations using a BCA Protein Assay Kit (Thermo, Waltham, USA) to normalize the MDA content.

Lipid hydroperoxide

We quantified LPO levels using the LPO Assay Kit (Beyotime, Jiangsu, China), following the manufacturer’s protocol. Briefly, we homogenized cartilage tissues (30–50 mg) or chondrocyte lysates (from 1 × 10⁶ cells) in ice-cold phosphate-buffered saline (PBS). After centrifugation at 12,000 g for 10 min at 4°C, we mixed 200 µL of supernatant with 600 µL of FOX reagent (100 µM xylenol orange, 250 µM ammonium ferrous sulfate, 25 mM H₂SO₄, and 4 mM BHT in methanol). We incubated the mixture at 37°C for 30 min protected from light. We measured absorbance at 500 nm using a microplate reader (BioTek, USA). We normalized LPO concentration to total protein content determined by the BCA assay.

Flow cytometry (PE/FITC) analysis

For surface marker or apoptosis analysis, we harvested chondrocytes and washed them twice with PBS. We resuspended cells in staining buffer (PBS with 2% FBS) and incubated them with PE-conjugated anti-ACSL4 (1:100, Abcam) or FITC-conjugated Annexin V (1:50, BD Biosciences) for 30 min at 4°C in the dark. For Annexin V/propidium iodide (PI) apoptosis assays, we dual-stained cells with FITC-Annexin V and PI according to the manufacturer’s protocol (BD Pharmingen). We analyzed samples within 1 h using a BD FACSCanto II flow cytometer (BD Biosciences). We processed data with FlowJo v10.8 software (TreeStar), gating based on unstained and single-stained controls.

Blinding procedures

We implemented rigorous blinding protocols throughout the study to minimize bias in data acquisition and analysis.

For histological scoring, all joint sections prepared for Safranin O/Fast Green staining and OARSI scoring were coded with anonymous numerical identifiers by a researcher not involved in the subsequent evaluation. The two independent observers who performed the histological scoring were completely blinded to the group assignments throughout the analysis.

For morphometric and image analysis, all analyses of IF intensity, TEM images, and other quantitative image-based data were performed on anonymized digital files. The analyst was unaware of the experimental group associated with each image.

For in vitro experiments, cell culture treatments and subsequent assays were conducted with plates and samples labeled with coded identifiers. The researchers performing the assays and quantifying the results were blinded to the treatment groups.

During data processing, the blinding codes were only broken after all quantitative measurements and analyses were completed and the raw data dataset was finalized.

Statistical analysis

We present all data as the mean ± standard deviation. We performed statistical analyses using SPSS 28.0 (IBM, USA) and GraphPad Prism 9.0 (GraphPad Software, USA). We verified the normality of data distribution using the Shapiro–Wilk test. We prespecified nonparametric alternatives (Mann–Whitney) for nonnormal data. For comparisons between two groups, we used an unpaired two-tailed Student’s t-test. For comparisons among multiple groups, we performed one-way or two-way analysis of variance, followed by Tukey’s post hoc test for multiple comparisons. We considered a p value <0.05 statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). We performed all in vitro experiments with at least five biological replicates per condition and repeated them independently five times.

An electronic laboratory notebook was not used.

Footnotes

Author Disclosure Statement

Study data were deidentified, stored on encrypted servers, and accessible only by the research team. The authors declare no conflict of interest.

Funding Information

No funding was received for this article.

Abbreviations

References

Bennett

, Longo

, Wright

, et al. Regulation of Osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A, 2005; 102(9):3324–3329; doi: 10.1073/pnas.0408742102

Centers for Disease and Prevention. Prevalence of doctor-diagnosed arthritis and arthritis-attributable activity limitation—United States, 2007-2009. MMWR Morb Mortal Wkly Rep, 2010; 59:1261–1265.

Courties

. Osteoarthritis and diabetes: Is there a true link? Joint Bone Spine, 2024; 91(3):105684.

Cui

, Huang

, Wang

, et al. Down-regulation of LGR6 promotes bone fracture recovery using bone marrow stromal cells. Biomed Pharmacother, 2018; 99:629–637.

Cui

, Niziolek

, MacDonald

, et al. Lrp5 functions in bone to regulate bone mass. Nat Med, 2011; 17(6):684–691.

de Jonge

, Rozenberg

, Vieyra

, et al. Achilles tendons in people with type 2 diabetes show mildly compromised structure: An ultrasound tissue characterisation study. Br J Sports Med, 2015; 49(15):995–999.

Derry

, Hegde

, Jackson

, et al. Revisiting the intersection of amyloid, pathologically modified tau and iron in Alzheimer’s disease from a Ferroptosis perspective. Prog Neurobiol, 2020; 184:101716.

Elder

, Filiberto

, Su

, et al. Maresin 1 activates LGR6 signaling to inhibit smooth muscle cell activation and attenuate murine abdominal aortic aneurysm formation. Faseb J, 2021; 35(8):e21780.

Englund

. Osteoarthritis, part of life or a curable disease? A bird’s-eye view. J Intern Med, 2023; 293(6):681–693.

10.

Friedmann Angeli

, Schneider

, Proneth

, et al. Inactivation of the Ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol, 2014; 16(12):1180–1191.

11.

Gui

, Lin

, Huan

, et al. Elevated expression of ICAM-1 in synovium is associated with early inflammatory response for cartilage degeneration in type 2 diabetes mellitus. J Cell Biochem, 2019; 120(8):13177–13186.

12.

, Lin

, Chen

. Specificity protein 1-mediated ACSL4 transcription promoted the osteoarthritis progression through suppressing the Ferroptosis of chondrocytes. J Orthop Surg Res, 2023; 18(1):188.

13.

, Chen

, Zhao

, et al. Lipoxin A4 ameliorates knee osteoarthritis progression in rats by antagonizing Ferroptosis through activation of the ESR2/LPAR3/Nrf2 axis in synovial fibroblast-like Synoviocytes. Redox Biol, 2024; 73:103143.

14.

Jeon

, Noh

, Lee

, et al. TRIM24-RIP3 axis perturbation accelerates osteoarthritis pathogenesis. Ann Rheum Dis, 2020; 79(12):1635–1643.

15.

Khedgikar

, Charles

, Lehoczky

. Mouse LGR6 regulates osteogenesis in vitro and in vivo through differential ligand use. Bone, 2022; 155:116267.

16.

King

, Wan

, Wagley

, et al. Signaling pathways associated with Lgr6 to regulate osteogenesis. Bone, 2024; 187:117207.

17.

Lai

FTT

, Yip

BHK

, Hunter

, et al. Metformin use and the risk of total knee replacement among diabetic patients: A propensity-score-matched retrospective cohort study. Sci Rep, 2022; 12(1):11571.

18.

Laiguillon

, Courties

, Houard

, et al. Characterization of diabetic osteoarthritic cartilage and role of high glucose environment on chondrocyte activation: Toward pathophysiological delineation of diabetes mellitus-related osteoarthritis. Osteoarthritis Cartilage, 2015; 23(9):1513–1522.

19.

Leroy

, Cai

, Tu

, et al. Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling. J Heart Lung Transplant, 2023; 42(5):562–574.

20.

, Li

, Hao

, et al. Maresin 1 intervention reverses experimental pulmonary arterial hypertension in mice. Br J Pharmacol, 2022; 179(22):5132–5147.

21.

, Cao

, Yin

, et al. Ferroptosis: Past, present and future. Cell Death Dis, 2020a;11(2):88.

22.

, Zhang

, Liu

, et al. Metformin limits osteoarthritis development and progression through activation of AMPK signalling. Ann Rheum Dis, 2020b;79(5):635–645.

23.

Louati

, Vidal

, Berenbaum

, et al. Association between diabetes mellitus and osteoarthritis: Systematic literature review and meta-analysis. RMD Open, 2015; 1(1):e000077.

24.

, Huang

, Zhang

, et al. Global hotspots and prospective trends for chondrocyte metabolic changes and oxidative stress in osteoarthritis: A bibliometric analysis. Adv Redox Res, 2025; 15:100130.

25.

Luo

, Shi

, Chen

, et al. Expression and significance of MMPs in synovial fluid, serum and PBMC culture supernatant stimulated by LPS in osteoarthritis patients with or without diabetes. Exp Clin Endocrinol Diabetes, 2019; 127(4):195–202.

26.

Maheu

, Bannuru

, Herrero-Beaumont

, et al. Why we should definitely include intra-articular hyaluronic acid as a therapeutic option in the management of knee osteoarthritis: Results of an extensive critical literature review. Semin Arthritis Rheum, 2019; 48(4):563–572.

27.

Mahoney-Sanchez

, Bouchaoui

, Ayton

, et al. Ferroptosis and its potential role in the physiopathology of Parkinson’s Disease. Prog Neurobiol, 2021; 196:101890.

28.

Marshall

, Peat

, Nicholls

, et al. Metabolic risk factors and the incidence and progression of radiographic hand osteoarthritis: A population-based cohort study. Scand J Rheumatol, 2019; 48(1):52–63.

29.

Miao

, Chen

, Xue

, et al. Contribution of Ferroptosis and GPX4’s dual functions to osteoarthritis progression. EBioMedicine, 2022; 76:103847.

30.

Mobasheri

, Rayman

, Gualillo

, et al. The role of metabolism in the pathogenesis of osteoarthritis. Nat Rev Rheumatol, 2017; 13(5):302–311.

31.

Park

, Kim

, et al. Osteoclast-associated receptor blockade prevents articular cartilage destruction via chondrocyte apoptosis regulation. Nat Commun, 2020; 11(1):4343.

32.

, Li

, Xie

, et al. Fighting age-related orthopedic diseases: Focusing on Ferroptosis. Bone Res, 2023; 11(1):12.

33.

Schett

, Kleyer

, Perricone

, et al. Diabetes is an independent predictor for severe osteoarthritis: Results from a longitudinal cohort study. Diabetes Care, 2013; 36(2):403–409.

34.

Shi

, Lu

, Dai

, et al. Advanced glycation end productions and tendon stem/progenitor cells in pathogenesis of diabetic tendinopathy. World J Stem Cells, 2021; 13(9):1338–1348.

35.

Siu

, Zheng

, Ko

, et al. Increased interleukin 1beta levels in the Subacromial fluid in diabetic patients with rotator cuff lesions compared with nondiabetic patients. J Shoulder Elbow Surg, 2013; 22(11):1547–1551.

36.

Stockwell

, Friedmann Angeli

, Bayir

, et al. Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease. Cell, 2017; 171(2):273–285.

37.

Sun

, Guo

, Hou

, et al. Iron homeostasis in Arthropathies: From pathogenesis to therapeutic potential. Ageing Res Rev, 2021; 72:101481.

38.

Tuo

, Lei

, Jackman

, et al. Tau-mediated iron export prevents Ferroptotic damage after ischemic stroke. Mol Psychiatry, 2017; 22(11):1520–1530.

39.

Vanden Berghe

, Linkermann

, Jouan-Lanhouet

, et al. Regulated necrosis: The expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol, 2014; 15(2):135–147.

40.

Wang

, Hussain

, Wluka

, et al. Association between metformin use and disease progression in obese people with knee osteoarthritis: Data from the osteoarthritis initiative-a prospective cohort study. Arthritis Res Ther, 2019; 21(1):127.

41.

Williams

, London

, Husni

, et al. Type 2 diabetes and osteoarthritis: A systematic review and meta-analysis. J Diabetes Complications, 2016; 30(5):944–950.

42.

, Liyanage

, Plan

, et al. Dysregulated energy metabolism impairs chondrocyte function in osteoarthritis. Osteoarthritis Cartilage, 2023; 31(5):613–626.

43.

Yao

, Sun

, Yu

, et al. Chondrocyte Ferroptosis contribute to the progression of osteoarthritis. J Orthop Translat, 2021; 27:33–43.

44.

Zhang

, Yuan

, Wang

. LGR4: A new receptor member in endocrine and metabolic diseases. Endocr Rev, 2023a;44(4):647–667.

45.

Zhang

, Randell

, Sun

, et al. Hyperglycemia-related advanced glycation end-products is associated with the altered phosphatidylcholine metabolism in osteoarthritis patients with diabetes. PLoS One, 2017; 12(9):e0184105.

46.

Zhang

, Hou

, Guo

, et al. Lipid peroxidation in osteoarthritis: Focusing on 4-hydroxynonenal, malondialdehyde, and Ferroptosis. Cell Death Discov, 2023b;9(1):320.

47.

Zhao

, Shen

, Zheng

, et al. Cardiomyocyte LGR6 alleviates Ferroptosis in diabetic cardiomyopathy via regulating mitochondrial biogenesis. Metabolism, 2024; 159:155979.

48.

Zhou

, Jia

, Wang

, et al. Curcumin reverses Erastin-induced chondrocyte Ferroptosis by upregulating Nrf2. Heliyon, 2023; 9(10):e20163.