Intra-articular injection of Erastin induces OA pathological progression as an experimental model

Intra-articular injection of Erastin induces OA pathological progression as an experimental model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intra-articular injection of Erastin induces OA pathological progression as an experimental model Liangcai Hou, Kai Sun, Xiong Zhang, Fan Lu, Jingting Xu, Zhou Guo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6830580/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Developing optimal osteoarthritis (OA) models is critical for therapeutic advancement. While ferroptosis is linked to OA pathogenesis, validated models for studying ferroptosis in OA remain scarce. Methods: In vitro : Mouse/C28I2/human chondrocytes were treated with 10 μM Erastin to assess ferroptosis, inflammation, extracellular matrix degradation, senescence, and antioxidant responses. OA patient and neonatal mouse cartilage explants were cultured ± Erastin (48 h) for Safranin O/IHC analysis. In vivo : 72 C57BL/6J mice (8-week-old) were divided into: (1) Destabilized medial meniscus (DMM) surgery group (Sham: contralateral knee); (2) 1 mg/kg Erastin intra-articular injection; (3) 10 mg/kg Erastin injection. Tissues were collected at 4/8/12 weeks for micro-CT and histology analysis (n=8/group/timepoint). Results: Erastin triggered ferroptosis, senescence, inflammation, and extracellular matrix degradation in mouse/C28I2/human chondrocytes, alongside NRF2 pathway activation and suppressed extracellular matrix synthesis. In cartilage explants (mice/OA patients), Erastin reduced COL2A1 and elevated MMP13 (IHC). In vivo, OARSI scores (Safranin O) and synovitis scores (H&E) increased significantly in DMM and Erastin (1/10 mg/kg) groups vs. Sham. IHC confirmed GPX4/COL2A1 downregulation and MMP13 upregulation in treated groups. Conclusions: A single intra-articular Erastin injection induces chondrocyte degeneration and cartilage damage mimicking human OA, offering a stable, simplified model compared to surgically complex DMM. This approach directly targets ferroptosis pathways, enabling precise mechanistic studies and streamlined preclinical testing of anti-ferroptosis therapies. Cartilage Osteoarthritis Chondrocyte Erastin Ferroptosis Mouse Animal model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteoarthritis (OA), the most prevalent degenerative joint disorder, induces persistent pain and functional disability, with growing global incidence correlated to aging demographics[ 1 , 2 ]. Over 59% of OA patients exhibit comorbidities like cardiovascular/metabolic disorders[ 3 ], while current therapies only alleviate symptoms without halting progression[ 4 ]. Despite clinical/preclinical efforts, OA’s chronicity and interspecies translational gaps hinder mechanistic insights[ 5 , 6 ]. Reliance on a single animal model for OA research has inherent limitations. Established approaches—including aging models, surgical induction ( e.g ., anterior cruciate ligament transection [ACLT] and destabilized medial meniscus [DMM] [ 6 , 7 ]), and chemical induction (e.g., monoiodoacetate[ 8 ], papain[ 4 ], and collagenase[ 9 ])—collectively enhance mechanistic understanding of OA[ 7 , 10 ]. However, widely utilized DMM models exhibit limitations such as variable outcomes and technical complexity. These shortcomings necessitate complementary strategies to isolate specific molecular pathways and dissect OA mechanisms with precision. Ferroptosis, a lipid peroxidation-driven cell death pathway distinct from apoptosis/necrosis[ 11 ], is implicated in age-related pathologies including neurodegenerative disorders[ 12 ]. In OA, ferroptosis-related genes correlate with disease progression[ 13 ], and its activation in chondrocytes underlies ECM degradation and inflammation[ 14 ]. Targeting ferroptosis with inhibitors (Ferrostatin-1) or iron chelators (Deferoxamine) attenuates OA progression in mice[ 15 ]. Critically, glutathione peroxidase 4 (GPX4)—a lipid peroxidation suppressor—is downregulated in human OA cartilage[ 16 ], and its cartilage-specific knockout accelerates OA via ferroptosis[ 17 ], solidifying ferroptosis as a pivotal OA pathogenic mechanism. Erastin, a canonical ferroptosis inducer targeting mitochondrial voltage-dependent anion channels (VDACs)[ 11 , 18 ], drives chondrocyte dysfunction in OA. Our prior work demonstrated that Erastin-treated chondrocytes exhibit upregulated ferroptosis markers, extracellular matrix (ECM) degradation, and pro-inflammatory responses[ 14 ]. In vivo , biweekly intra-articular Erastin injections (5 mg/kg) over 8 weeks induced marked OA-like degeneration in murine knee joints, with histopathology confirming chondrocyte damage and ECM loss[ 15 ]. These findings establish Erastin as a potent tool for modeling ferroptosis-driven OA pathology. Current OA models exhibit critical constraints: aging models require prolonged induction periods, limiting their utility for rapid mechanistic studies; surgical approaches like DMM suffer from technical complexity and outcome variability, while collagenase-induced models demand meticulous dose optimization to achieve consistent pathology. Importantly, none directly interrogate ferroptosis—a central pathogenic mechanism bridging oxidative stress and metabolic dysfunction in OA. We developed a novel OA model through intra-articular Erastin administration, which bypasses surgical complexity by directly activating ferroptosis. Unlike conventional models that indiscriminately accelerate joint degeneration, our system synchronously induces OA phenotypes and ferroptosis activation, enabling precise interrogation of lipid peroxidation-mediated chondrocyte death and ECM collapse. Methods Ethics statement Animal experiments (Approval No. TJH-202112022): All procedures involving animals received approval from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) and were conducted in compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Eight-week-old male C57BL/61 mice, supplied by the Tongji Hospital Experimental Animal Center, were utilized to establish the animal models. Human samples (Approval No. TJIRB20210905): Studies involving human specimens were performed in accordance with the Helsinki Declaration and obtained ethical clearance from the Tongji Hospital Ethics Committee. Written informed consent was acquired from every participant. Osteoarthritis cartilage samples were obtained from patients (n = 4) undergoing total knee arthroplasty at Tongji Hospital. Detailed information regarding the OA patients is presented in Tables 1 and 2 (included at the end of the manuscript). Table 1 OA patients’ information Sample ID Age Gender Height(cm) Weight(kg) OA location 1 67 Male 170 84 Left 2 66 Female 158 60 Right 3 72 Male 172 72 Left 4 67 Female 163 58 Left Table 2 Amputees’ information Sample ID Age Gender Height(cm) Weight(kg) OA location 1 32 Male 175 79 Right 2 38 Male 173 71 Left 3 29 Female 163 52 Right 4 21 Femal 166 50 Right Reagents Erastin (S7242, Selleck) was dissolved in DMSO for experimental use. The Cell Counting Kit-8 (CCK-8) (HY-K0301) was acquired from MedChemExpress. RNA extraction was performed using a kit from Omega Biotek (R6834-01). Hifair® III 1st Strand cDNA Synthesis SuperMix (11141ES60) and SYBR Green Master Mix (11203ES03) were sourced from Yeasen. For reactive oxygen species (ROS) detection, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (#S0033) was obtained from Beyotime (Shanghai, China). Lipid peroxidation was assessed using C11-BODIPY (581/591) probe (D3861, Thermo Fisher). Cell proliferation was evaluated with an EdU staining kit (RiboBio, Guangzhou, China), and labile iron pool was detected with FerroOrange (F374, Dojindo). Animals and treatments To isolate estrogen's specific impact on OA pathogenesis, we utilized 8-week-old male C57BL/61 mice (n = 72), thereby eliminating confounding effects from cyclic estrogen fluctuations in females. Animals were randomized into three groups: the DMM model group, the intra-articular injection group receiving 1 mg/kg Erastin, and the intra-articular injection group receiving 10 mg/kg Erastin, with the right knee joint of the DMM model group serving as the Sham group. DMM surgery was conducted on the left knee per established protocol[ 19 ], initiating with intraperitoneal anesthesia (1% pentobarbital) followed by meniscal destabilization. The intra-articular Erastin injection groups received a single injection into the left knee joint. Mice were euthanized at 4, 8, and 12 weeks post-surgery or injection for micro-computed tomography and histological analysis. Cells and treatments Experimental cells included primary mouse chondrocytes, C28I2 cells, and human chondrocytes. Mouse chondrocytes were isolated from 5-day-old C57BL/6J mice through sequential enzymatic digestion: knee joint cartilage was minced, digested with 0.25% trypsin (30 min), then treated with 0.25% type II collagenase (4–6 h). Harvested cells were resuspended and maintained in DMEM/F12 medium (10% fetal bovine serum, 1% penicillin/streptomycin) at 37°C. Only early-passage chondrocytes (P1-P2) were utilized for subsequent assays. The C28/I2 cell line was generously provided by Dr. Huang Junmin from Zhongshan Hospital, Fudan University. Human chondrocytes were obtained from children undergoing polydactyly correction surgery. Cartilage specimens were subjected to sequential enzymatic digestion: initial treatment with 0.25% trypsin (30 min) followed by 0.25% type II collagenase (20 h). Isolated cells were resuspended in DMEM/F12 medium (10% fetal bovine serum, 1% penicillin/streptomycin) and maintained at 37°C. All experiments exclusively used primary human chondrocytes. Western Blot We extracted total protein using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The concentration of total protein was determined using a BCA Protein Assay Kit (Boster, AR1110). Equal amounts of total protein (8–10 µg) were combined with loading buffer and heated at 100°C (10 min). Subsequently, Samples were then electrophoresed on SDS-PAGE gels and electrotransferred onto PVDF membranes. Membranes underwent blocking with 5% skim milk (1 h, RT), followed by overnight incubation at 4°C with primary antibodies (listed in Table 3 , located at the end of this article). After four 7-minute TBST washes, membranes were exposed to HRP-conjugated secondary antibodies (1 h, RT). Protein signals were detected using a Bio-Rad ChemiDoc system (Hercules, CA). Table 3 primary antibodies used in the Western Blot experiment. ANTIBODIES SOURCE IDENTIFIER GAPDH Proteintech Group, Wuhan, Hubei, China 60004-1-Ig Collagen II Proteintech Group, Wuhan, Hubei, China 15943-1-AP NRF2 Proteintech Group, Wuhan, Hubei, China 16396-1-AP HO-1 Proteintech Group, Wuhan, Hubei, China 10701-1-AP P53 Proteintech Group, Wuhan, Hubei, China 60283-2-Ig ACSL4 Proteintech Group, Wuhan, Hubei, China 22401-1-AP Cyclin D1 Proteintech Group, Wuhan, Hubei, China 26939-1-AP P21 Proteintech Group, Wuhan, Hubei, China 10355-1-AP P16 Proteintech Group, Wuhan, Hubei, China 28416-1-AP Aggrecan ABclonal, Wuhan, Hubei, China A8536 NQO1 Abcam, Cambridge, UK #ab80588 MMP13 Abcam, Cambridge, UK #ab39012 SOX9 Abcam, Cambridge, UK #ab185966 GPX4 Abcam, Cambridge, UK #ab125066 MMP3 Boster, Wuhan, Hubei, China #BM4074 iNOS Cell Signaling Technology, Beverly, MA, USA #13120 COX2 Cell Signaling Technology, Beverly, MA, USA #12882 HRP Conjugated AffiniPure Goat Anti-mouse IgG Boster, Wuhan, Hubei, China BA1050 HRP Conjugated AffiniPure Goat Anti-rabbit IgG Boster, Wuhan, Hubei, China BA1054 FITC Conjugated AffiniPure Goat Anti-rabbit IgG Boster, Wuhan, Hubei, China BA1105 FITC Conjugated AffiniPure Goat Anti-mouse IgG Boster, Wuhan, Hubei, China BA1101 Quantitative real-time PCR Total RNA (1 µg) was isolated and quantified employing Omega Biotek's RNA Extraction Kit. cDNA synthesis utilized Hifair®III First Strand cDNA Synthesis SuperMix. qPCR amplification proceeded with SYBR Green Master Mix under optimized cycling: 95°C for 5 min (initial denaturation); 40 cycles of 95°C for 10 sec (denaturation) and 60°C for 30 sec (combined annealing/extension). Melting curves were generated by sequential incubation: 95°C (15 sec) → 60°C (60 sec) → 95°C (15 sec). GAPDH served as endogenous control. Target gene primers are listed in Table 4 , located at the end of this article. Table 4 Primer sequence used in the RT-qPCR experiment. Sequence Mmp3 Mouse Forward: 5′-GAAACGGGACAAGTCTGTGGAG-3′ Reverse: 5′-ATGAAAATGAAGGGTCTTCCGGTCC-3′ Inos Mouse Forward: 5′-CAGGGAGAACAGTACATGAACAC − 3′ Reverse: 5′-TTGGATACACTGCTACAGGGA − 3′ Ptgs2 Mouse Forward: 5′-TTCAACACACTCTATCACTGGC − 3′ Reverse: 5′-AGAAGCGTTTGCGGTACTCAT − 3′ Gapdh Mouse Forward: 5′-AACATCAAATGGGGTGAGGCC-3′ Reverse: 5′-GTTGTCATGGATGACCTTGGC-3′ Gpx4 Mouse Forward: 5′-GATGGAGCCCATTCCTGAACC-3′ Reverse: 5′-CCCTGTACTTATCCAGGCAGA-3′ Acsl4 Mouse Forward: 5′-CTCACCATTATATTGCTGCCTGT − 3′ Reverse: 5′-TCTCTTTGCCATAGCGTTTTTCT − 3′ MMP3 Human Forward: 5′-GGCAAGACAGCAAGGCATAGAGAC − 3′ Reverse: 5′-GACCAACATCAGGAACTCCACACC − 3′ COL2A1 Human Forward: 5′-GCAAGCAAGGAGACAGAGGAGAAG − 3′ Reverse: 5′-AGAAGGACCAGCAGGACCAGAAG − 3′ PTGS2 Human Forward: 5′-CCATTGACCAGAGCAGGCAGATG − 3′ Reverse: 5′-TGGCTTCCAGTAGGCAGGAGAAC − 3′ ACSL4 Human Forward: 5′-AACTGCCTTGGCTGTACTGCTATTC − 3′ Reverse: 5′-GTGTGACAGAGCGATATGGACTTCC − 3′ GPX4 Human Forward: 5′-CCCGATACGCTGAGTGTGGTTTG − 3′ Reverse: 5′-CCTTGCCCTTGGGTTGGATCTTC − 3′ GAPDH Human Forward: 5′-CAGGAGGCATTGCTGATGAT − 3′ Reverse: 5′-GAAGGCTGGGGCTCATTT-3′ CDKN1A Human Forward: 5′-TTGATTAGCAGCGGAACAAGGAGTC − 3′ Reverse: 5′-GAGAAACGGGAACCAGGACACATG − 3′ CDKN2A Human Forward: 5′-TCATCAGTCACCGAAGGTCCTACAG − 3′ Reverse: 5′-TGCTCACTCCAGAAAACTCCAACAC − 3′ Cell viability assays Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) to quantify cell proliferation. Mouse or human chondrocytes were seeded into 96-well plates at a density of approximately 5,000 cells/well and adhered for 24 h prior to treatment. Erastin exposure (10 µM) was initiated for 12, 24, or 48 h. Subsequently, each well's medium was substituted with 100 µL fresh medium containing 10 µL CCK-8 reagent. Following 1 h incubation, absorbance at 450 nm was quantified using a Leica microplate reader (Wetzlar, Germany). Edu Staining Mouse or human chondrocytes were seeded into 96-well plates at approximately 5,000 cells per well and allowed to adhere for 24 hours. Cells were then treated with 10 µM Erastin. After 12, 24, or 48 hours of treatment, chondrocytes were incubated with 10 µM EdU for 2 hours. Following incubation, the cells were fixed with paraformaldehyde and treated with 2 mg/mL glycine. Permeabilization was performed using 1% Triton X-100 for 5 minutes, after which the staining solution was added for 30 minutes. The cells were washed with 0.5% Triton X-100 and subsequently stained with Hoechst. Stained images were captured using an inverted fluorescence microscope (OLYMPUS, IX71). EdU staining was performed in triplicate, with the same field of view selected each time. The number of EdU-positive cells was quantified and expressed as a fraction of the total cell count in the selected area. Mouse/human chondrocytes (5,000 cells/well in 96-well plates) were adhered for 24 h prior to 10 µM Erastin exposure. At 12/24/48 h post-treatment, cells were fixed in 4% paraformaldehyde, quenched with 2 mg/mL glycine, and permeabilized using 1% Triton X-100 (5 min). EdU reaction cocktail was applied for 30 min, followed by three 0.5% Triton X-100 washes. Nuclei were counterstained with Hoechst. Images were acquired using an Olympus IX71 inverted fluorescence microscope. Triplicate assays quantified EdU + cells as proliferative index (vs. total Hoechst + cells in designated fields) Immunofluorescence staining After 24 h of 10 µM Erastin exposure, chondrocytes were washed thrice with PBS, fixed with 4% formaldehyde (15 min), and permeabilized with 1% Triton X-100 (5 min, RT). Blocking was performed with 5% BSA (1 h, RT). Primary antibodies were applied at optimal dilutions and incubated overnight at 4°C (humidified chamber). Fluorophore-conjugated secondary antibodies (1:200) were then added for 1 h at 37°C. Nuclei were counterstained with DAPI (5 min). Images were captured using an Olympus BX51 upright fluorescence microscope. Reactive Oxygen Species (ROS) and Lipid ROS Intracellular ROS generation was assayed with DCFH-DA, whereas lipid ROS accumulation was monitored using C11-BODIPY 581/591 [ 20 , 21 ]. Chondrocytes in six-well plates were first incubated for 24 h, then exposed to 10 µM C11-BODIPY or DCFH-DA under light-protected conditions for 30 min. After an additional PBS wash, cellular fluorescence was observed using a fluorescence microscope (OLYMPUS, IX71). FerroOrange staining We cultured chondrocytes in six-well plates and incubated them with 1 µM FerroOrange in HBSS for 30 minutes at 37°C, 21% oxygen, and 5% CO 2 after a 24-hour incubation period. Following incubation, we immediately imaged the cells using a fluorescence microscope. Histological and immunohistochemical staining Left knee joints were immersed in 4% paraformaldehyde (3 d), embedded in paraffin, and sectioned. Sections underwent Safranin O/fast green staining for blinded OARSI scoring by histopathologists[ 22 ]. Synovitis was quantified via H&E staining: synovial lining thickness scored 0–3 (medial/lateral), cumulative range 0–6 (0 = 1–2 layers; 1 = 3–4; 2 = 5–9; 3 ≥ 10) [ 23 ]. For IHC, dewaxed/rehydrated samples were blocked with BSA + 0.1% Triton X-100 (1 h), then incubated with primary antibodies (COL2A1/MMP13/GPX4). DAB-conjugated secondary antibodies were applied before hematoxylin counterstaining. Images were acquired using fluorescence microscopy. Micro-computed tomography (micro-CT) Left knee joints fixed in 4% paraformaldehyde underwent scanning with a Viva CT 80 system (Scanco Medical AG, Switzerland) under these parameters: 100 kV, 98 µA, 10.5 µm resolution. Three-dimensional (3D) images were reconstructed using Scanco Medical software. Evaluate the size of the osteophyte based on the transverse image of the tibial plateau. Subchondral bone in the medial femoral condyle was analyzed by two blinded observers across 30 consecutive layers (initiating 15 layers proximal to the tibial plateau edge). Quantitative parameters assessed using Scanco analysis software comprised bone volume fraction (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N), based on data obtained via the micro-CT system. RNA-sequencing Total RNA extraction, library preparation, and sequencing were contracted to Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China), adhering to their standardized protocols. Bioinformatics analysis was executed on their proprietary Cloud Platform. Statistical analysis All analyses were conducted in GraphPad Prism 8.0 (San Diego, CA, USA). Multiple group comparisons utilized one-way ANOVA with Tukey's post hoc testing. Inter-group differences for continuous variables were assessed by unpaired two-tailed Student's t-test. Non-parametric data (e.g., OARSI grades) underwent Mann-Whitney U test (two groups) or Kruskal-Wallis with Dunn's test (≥ 3 groups). Data represent mean ± SEM; P < 0.05 defined statistical significance. Results 1. Intra-articular injection of Erastin-induced cartilage degeneration in mice. To evaluate Erastin's effects on articular cartilage, destabilization of the medial meniscus (DMM) was established in mice (n=8/group) as an osteoarthritis model, with contralateral knees as sham controls. Animals received intra-articular Erastin (1 or 10 mg/kg). At 4/8/12 weeks post-intervention, knee joints were harvested for micro-CT and histology. Safranin O/fast green staining demonstrated that both DMM surgery and Erastin administration induced pathological hallmarks of cartilage degeneration, including: superficial cartilage fibrosis, Proteoglycan loss (reduced matrix staining) and structural integrity disruption (Fig. 1A). Cartilage degeneration severity was quantified via the OARSI scoring system. Relative to the Sham group, OARSI scores exhibited a significant increase in both the DMM model group and animals administered intra-articular Erastin (1 mg/kg and 10 mg/kg) (Fig. 1B). Nevertheless, comparisons between the DMM group and either Erastin dosage group (1 mg/kg or 10 mg/kg) revealed no statistically significant disparities in OARSI scores, demonstrating equivalent effects at both concentrations. Concurrently, there was no significant difference in OARSI scores between the intra-articular injection of Erastin at 1 mg/kg and 10 mg/kg groups, indicating that both concentrations of Erastin could induce stable modeling. Moreover, OARSI scores in the DMM model and intra-articular Erastin-administered groups (1 mg/kg and 10 mg/kg) exhibited parallel trends across 4-, 8-, and 12-week timepoints in mice, supporting the modeling stability of intra-articular Erastin injection. H&E staining revealed expansion of the synovial lining cell layer and heightened stromal cellular density in the DMM cohort and both Erastin-treated groups (Fig. 1C), with concomitant significant elevations in synovitis scores (Fig. 1D). Immunohistochemical analysis demonstrated reduced quantities of GPX4-positive cells within the knee joint cartilage of both DMM-induced and Erastin-treated groups (Fig. 1E and F). Relative to Sham controls, COL2A1 immunostaining intensity and positive cell counts in the cartilage matrix were markedly diminished in these experimental groups (Fig. 1G and H), concomitant with a significant rise in MMP13-positive cells (Fig. 1I and J). Osteophyte formation and subchondral bone alterations were quantified using micro-CT across experimental groups. Primary analysis focused on the medial tibial plateau subchondral region, where bone volume changes were assessed through parameters including bone volume fraction (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp). Data revealed decreased BV/TV, Tb.N, and Tb.Th values with elevated Tb.Sp in the DMM cohort relative to Sham controls. However, no significant differences were noted between the 1 mg/kg and 10 mg/kg Erastin groups when compared to the Sham group. These findings suggest that the DMM model induces a reduction in bone volume fraction, trabecular number, and trabecular thickness, leading to the remodeling of subchondral bone. Notably, intra-articular administration of 1 mg/kg or 10 mg/kg Erastin showed no significant modulatory effects on these indices or subchondral bone remodeling (Supplementary Fig. 1 A-D). Three-dimensional reconstructions and transverse tibial plateau scans confirmed distinct osteophytes at patellar cartilage margins and medial femoral condyle articular surfaces in both DMM-induced and Erastin-treated groups (1 mg/kg and 10 mg/kg), whereas the Sham group displayed negligible osteophyte formation (Fig. 1K and L). Collectively, intra-articular Erastin administration successfully induces murine cartilage degeneration. 2. Eukaryotic mRNA sequencing reveals that Erastin intervention promotes changes in the expression of OA-related genes in chondrocytes. To delineate the impact of Erastin on chondrocyte gene expression associated with OA pathogenesis and advancement, eukaryotic mRNA sequencing was performed on primary chondrocytes from Erastin-treated mice for systematic transcriptomic profiling. Fig. 2A displays the expression level distribution across samples via boxplots, while volcano plot and heatmap analyses depict the differentially expressed gene profiles between treatment cohorts (Fig. 2B and C). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that Erastin treatment resulted in the enrichment of numerous OA-related gene pathways within the transcriptome (Fig. 2D) . These pathways include ECM-receptor interaction, Glutathione metabolism, Ferroptosis, Inflammation-related pathways, Senescence-related pathways, and Antioxidant pathways. Collectively, these data support that Erastin intervention induces changes in the expression of genes associated with OA progression in chondrocytes. 3. Erastin increased ECM catabolism and decreased anabolism in chondrocytes Chondrocytes maintain ECM homeostasis via coordinated anabolic-catabolic balance[24]. Heatmap profiling screened significantly dysregulated OA-related gene signatures between cohorts (Fig. 3A). We designated Aggrecan, COL2A1, and SOX9 as biomarkers of ECM anabolism, with MMP3 and MMP13 serving as catabolism indicators. Western blotting confirmed that Erastin treatment significantly downregulated anabolic markers (Aggrecan, COL2A1, SOX9) while upregulating catabolic markers (MMP3, MMP13) in murine chondrocytes (Fig. 3B). Erastin-treated C2812 cells exhibited alterations aligned with murine chondrocyte responses (Fig. 3C). Western blotting confirmed significant downregulation of Aggrecan, COL2A1, and SOX9 proteins coupled with MMP3 upregulation in human chondrocytes following Erastin exposure (Fig. 3D). Furthermore, immunofluorescence staining revealed that Erastin intervention in human chondrocytes inhibited COL2A1 expression (Fig. 3E and F). Additionally, qRT-PCR results showed that Erastin promoted the expression of Mmp3 in mouse chondrocytes (Fig. 3G). Erastin promoted the expression of MMP3 and inhibited the expression of COL2A1 in human chondrocytes (Fig. 3H). Erastin-exposed murine articular explants exhibited diminished Safranin O/fast green staining intensity at cartilage surfaces (Fig. 3I). Immunohistochemistry detected reduced COL2A1-immunoreactive cells alongside elevated MMP13-positive cell populations (Fig. 3J). Human joint explant outcomes strongly concorded with murine data (Fig. 3K and L). Collectively, results from multiple cell types including mouse chondrocytes, C28I2 cell line, and human chondrocytes indicated that Erastin increased ECM catabolism and decreased anabolism in chondrocytes. 4. Erastin aggravated inflammation in chondrocytes. Inflammatory processes constitute a key driver of osteoarthritis pathogenesis. [25-27]. A heatmap analysis identified several inflammation-related genes that demonstrated significant differential expression between the two groups (Fig. 4A). Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were designated as inflammatory biomarkers in this study. Western blotting demonstrated significant upregulation of iNOS and COX2 protein levels in murine chondrocytes following Erastin exposure (Fig. 4B), C28I2 cells (Fig. 4C), and human chondrocytes (Fig. 4D). Additionally, qRT-PCR results further confirmed that Erastin intervention elevated the expression of iNOS and Ptgs2 in mouse chondrocytes (Fig. 4E) and promoted PTGS2 expression in human chondrocytes as well (Fig. 4F). Collectively, these findings suggest that Erastin enhances inflammatory responses in chondrocytes. 5. Erastin aggravated ferroptosis in chondrocytes As a specific ferroptosis inducer, Erastin modulates pathways that our prior work established as significant contributors to OA pathogenesis [14]. Heatmap profiling identified markedly dysregulated ferroptosis-associated gene signatures across cohorts (Fig. 5A). We designated acyl-CoA synthetase long-chain family member 4 (ACSL4) and p53 as ferroptosis-activation biomarkers, with GPX4 functioning as a ferroptosis-inhibitory indicator. Western blot analysis demonstrated that Erastin treatment in mouse chondrocytes (Fig. 5B), C28I2 cells (Fig. 5C), and human chondrocytes (Fig. 5D) resulted in increased expression of ACSL4 and p53, alongside a decrease in GPX4 expression. The accumulation of lipid ROS and ROS is characteristic of ferroptosis; To assess this, we employed C11-BODIPY to detect lipid ROS and DCFH-DA to measure ROS levels. Data revealed that Erastin exposure triggered robust accumulation of lipid peroxides and reactive oxygen species within murine chondrocytes (Fig. 5E-H). Similar patterns were observed in human chondrocytes following Erastin treatment (Fig. 5I-L). Ferroptosis represents an iron-dependent programmed cell death modality driven predominantly by ferrous ions (Fe²⁺) [11]. To further verify the occurrence of ferroptosis, we used the FerroOrange probe to detect Fe 2+ accumulation in chondrocytes. Our findings revealed that Erastin induced significant cytoplasmic Fe 2+ accumulation in mouse chondrocytes (Fig. 5M and N). To assess the impact of Erastin on chondrocyte proliferation, we employed EdU staining, which demonstrated that Erastin inhibited the proliferation of both mouse chondrocytes (Fig. 5O and P) and human chondrocytes (Fig. 5Q and R). Additionally, the CCK8 assay revealed that Erastin significantly reduced the viability of mouse chondrocytes (Fig. 5S), with this inhibitory effect on cell viability becoming more pronounced over time. A similar reduction in viability was observed in human chondrocytes (Fig. 5T). Moreover, qRT-PCR analysis further confirmed significant upregulation of Acsl4 transcripts coupled with Gpx4 downregulation in both murine (Fig. 5U) and human chondrocytes (Fig. 5V) following Erastin exposure. Taken together, these findings demonstrate Erastin effectively potentiates ferroptosis in chondrocytes. 6. Erastin aggravated senescence in chondrocytes OA progression correlates with accumulated senescent cells in articular tissues [28, 29]. Heatmap profiling revealed markedly dysregulated senescence-associated gene signatures across experimental cohorts (Fig. 6A). Cyclin D1, P21, and P16 were employed as senescence hallmark indicators. Immunoblotting demonstrated Erastin-mediated downregulation of Cyclin D1 concomitant with P21 upregulation in murine chondrocytes (Fig. 6B). In C28I2 cells, Erastin inhibited Cyclin D expression while promoting the expression of P21 and P16 (Fig. 6C). Similar results were consistently observed in human chondrocytes (Fig. 6D). Furthermore, qRT-PCR analysis confirmed Erastin provoked marked upregulation of CDKN1A (P21) and CDKN2A (P16) transcript levels in human-derived chondrocytes. (Fig. 6E). Collectively, these findings indicated that Erastin promotes chondrocyte senescence. 7. Erastin activated the NRF2 antioxidant pathway in chondrocytes Heatmap profiling identified markedly dysregulated antioxidant-associated genes across cohorts (Fig. 7A). We designated NRF2 signaling effectors heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) as biomarkers of the NRF2 pathway. Immunoblotting demonstrated concomitant upregulation of NRF2, HO-1, and NQO1 proteins in murine chondrocytes following Erastin exposure (Fig. 7B). Consistent results were observed in C28I2 cells treated with Erastin (Fig. 7C). Additionally, in human chondrocytes, Erastin also promoted the expression of HO-1 and NQO1 (Fig. 7D). These findings indicated that Erastin activated the NRF2 antioxidant pathway. Discussion This study pioneers a novel C57BL/6J mouse osteoarthritis model through single-dose intra-articular Erastin administration. Comprehensive evaluations were conducted across three dimensions: For in vivo assessment, 8-week-old male C57BL/6J mice (n = 8/group) were randomized into: 1) DMM-induced OA model, 2) intra-articular Erastin (1 mg/kg), and 3) intra-articular Erastin (10 mg/kg) groups. Contralateral knee joints served as sham-operated controls. Longitudinal assessments (4, 8, 12 weeks post-intervention) integrated micro-CT with histopathological analyses (Safranin O/fast green, H&E, IHC) to quantify cartilage degeneration, synovitis, subchondral remodeling, ECM homeostasis, and ferroptosis markers. Micro-CT imaging revealed distinct osteophyte formation and subchondral bone remodeling across experimental groups. Three-dimensional reconstructions and transverse cartilage scans demonstrated pronounced tibial osteophytes in the DMM, 1 mg/kg Erastin, and 10 mg/kg Erastin groups, whereas no osteophyte formation was detected in sham controls. OARSI scoring via Safranin O/fast green staining highlighted progressive cartilage degeneration in all treatment groups (DMM and both Erastin doses) at 4, 8, and 12 weeks, characterized by superficial fibrillation, proteoglycan loss, and structural disorganization. Notably, OARSI scores in Erastin-treated groups were comparable to those in the DMM group, confirming equivalent cartilage damage severity. Synovial inflammation, a hallmark of OA progression, was evident in H&E -stained sections[ 30 ], showing synovial lining thickening and elevated synovitis scores in DMM and Erastin groups compared to sham. Synovitis severity remained consistent between Erastin and DMM groups across all timepoints. Immunohistochemical analysis revealed a significant reduction in COL2A1 (ECM anabolism marker) and GPX4 (ferroptosis inhibitor) expression, alongside increased MMP13 (ECM catabolism marker) levels in DMM and Erastin-treated groups. These findings collectively indicate that intra-articular Erastin injection disrupts ECM homeostasis by suppressing synthesis, enhancing degradation, and activating ferroptosis signaling pathways. Our study demonstrated that intra-articular injection of Erastin at different concentrations did not show significant differences compared to the DMM group in terms of cartilage integrity, synovial inflammation, and gene expression in chondrocytes. This suggests that both low-dose (1 mg/kg) and high-dose (10 mg/kg) Erastin intra-articular injections can achieve the desired modeling effect, allowing flexibility in determining the concentration based on experimental needs. Histological evaluations across all timepoints (4, 8, 12 weeks) demonstrated consistent phenotypic progression, confirming the model’s temporal consistency. This dual flexibility—dose independence and timepoint stability—enables researchers to tailor experimental designs to specific needs while maintaining robust OA pathology induction. Ex Vivo Validation: Erastin-treated mouse and human cartilage explants (48 h) exhibited proteoglycan loss (Safranin O staining) and disrupted ECM balance, as shown by immunohistochemistry: reduced COL2A1 expression and elevated MMP13 levels. These ex vivo outcomes aligned with in vivo and cellular findings, reinforcing Erastin’s ability to drive OA-like ECM remodeling and validating its pathological consistency across experimental models. Cellular Level: Erastin induced OA-like pathological changes across mouse, C28I2, and human chondrocytes by disrupting ECM homeostasis. Key synthesis markers (Aggrecan, COL2A1, SOX9) were suppressed, while catabolic enzymes (MMP3, MMP13) were elevated, mimicking the ECM imbalance characteristic of OA. Pro-inflammatory responses, validated via Western blot and qRT-PCR, further aligned Erastin-treated cells with OA pathophysiology. Ferroptosis was robustly confirmed through upregulated ACSL4 and P53, downregulated GPX4, iron overload (FerroOrange), and lipid/ROS accumulation (C11-BODIPY and DCFH-DA). Functional assays (EdU, CCK8) demonstrated reduced chondrocyte proliferation and viability, consistent with ferroptosis. Concurrently, Erastin amplified senescence phenotypes and activated the NRF2 antioxidant pathway, reflecting stress adaptations seen in human OA. The MIA injection model[ 8 , 31 ] inhibits glyceraldehyde-3-phosphate dehydrogenase in chondrocytes to disrupt glycolysis, inducing apoptosis, ECM degradation, acute inflammation, and cartilage destruction. Its simplicity makes it ideal for studying OA pain mechanisms and early pathology. The DMM model[ 7 , 32 ] induces post-traumatic OA via surgical knee instability, causing mechanical imbalance, cartilage wear, subchondral remodeling, and osteophytosis—suitable for mechanosensitive pathway research. However, it involves complex surgery with infection risks and high inter-individual variability due to surgical precision dependence. In contrast, the Erastin model circumvents DMM’s technical challenges and variability, offering a ferroptosis-targeted, streamlined approach to OA pathogenesis. With single intra-articular administration, it recapitulates core OA features (ECM dysregulation, synovitis, subchondral changes), establishing it as a superior preclinical tool for mechanistic and therapeutic studies. However, this study has several limitations. Firstly, our animal experiments exclusively utilized C57BL/6J mice, and further validation of the modeling effects in other mammalian species has yet to be conducted. Secondly, ferroptosis represents only one of the multiple molecular mechanisms involved in the onset and progression of OA. Consequently, while this animal model may be suitable for investigating the significance of the ferroptosis pathway in OA mechanisms, it may not be applicable for studying other molecular mechanisms. Conclusions This study pioneers a single intra-articular Erastin-induced C57 murine osteoarthritis model, recapitulating DMM-equivalent cartilage degeneration, synovitis, subchondral remodeling, and ferroptosis activation over 12 weeks with dose-independent efficacy. Multi-level validation confirmed Erastin-driven ECM imbalance (reduced COL2A1, elevated MMP13), iron overload, and NRF2 pathway activation. Cross-species consistency in human explants and chondrocytes highlighted conserved ferroptosis mechanisms via GPX4 suppression. While distinct from natural OA progression, this model offers a simplified, temporally stable tool for investigating ferroptosis-specific contributions to OA pathogenesis, bridging translational gaps through mechanistic clarity and experimental flexibility. Abbreviations ACLT: Anterior cruciate ligament transection; ACSL4: Acyl-CoA synthetase long-chain family member 4; CCK-8: Cell Counting Kit-8; COX2: Cyclooxygenase-2; DMM: Destabilized Medial Meniscus; GPX4: Glutathione peroxidase 4; iNOS: Inducible nitric oxide synthase; KEGG: Kyoto Encyclopedia of Genes and Genomes; micro-CT: Micro-computed tomography; OA: Osteoarthritis; ROS: Reactive Oxygen Species; Tb.N: Trabecular number; Tb.Sp: Trabecular separation; Tb.Th: Trabecular thickness; VDAC: Voltage-dependent anion channel; Declarations Declarations of interest none. Ethics statement The collection of human cartilage was approved by the Ethics Committee of Tongji Hospital (TJ-IRB20210905) after obtaining informed consent from the patients. The animal experiment was approved by the Ethics Committee of Tongji Hospital (TJH202212051). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Consent for publication All authors read and approved the final manuscript. Funding This study was supported by the National Natural Science Foundation of China (Grants No. 82372475, 82172498, 11602155) and the Hainan Provincial Natural Science Foundation of China (Grant No. 821MS164). Author Contribution All authors have participated sufficiently in this work to assume public accountability for relevant portions. Study conception and design: LH, KS, JQ, FG. Data acquisition, analysis, and interpretation: LH, KS, XZ, FL, JX, ZG, GW, ZZ, YH, ZR, JH, XL. Manuscript drafting: LH, KS. Critical revision of intellectual content: All authors. Acknowledgement The authors express gratitude to Dr. Huang Junmin (Zhongshan Hospital, Fudan University) for providing the C28/I2 cell line and acknowledge the Experimental Center of Tongji Hospital for technical assistance in performing immunofluorescence experiments. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Yan H, Guo J, Zhou W, Dong C, Liu J: Health-related quality of life in osteoarthritis patients: a systematic review and meta-analysis . Psychol Health Med 2022, 27 (8):1859-1874. Moreno-Ligero M, Moral-Munoz JA, Salazar A, Failde I: mHealth Intervention for Improving Pain, Quality of Life, and Functional Disability in Patients With Chronic Pain: Systematic Review . 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Osteoarthritis Cartilage 2011, 19 (7):864-873. Komori T: Molecular Processes in Chondrocyte Biology . Int J Mol Sci 2020, 21 (11). Liu-Bryan R: Inflammation and intracellular metabolism: new targets in OA . Osteoarthritis Cartilage 2015, 23 (11):1835-1842. Nedunchezhiyan U, Varughese I, Sun AR, Wu X, Crawford R, Prasadam I: Obesity, Inflammation, and Immune System in Osteoarthritis . Front Immunol 2022, 13 :907750. Sanchez-Lopez E, Coras R, Torres A, Lane NE, Guma M: Synovial inflammation in osteoarthritis progression . Nat Rev Rheumatol 2022, 18 (5):258-275. Coryell PR, Diekman BO, Loeser RF: Mechanisms and therapeutic implications of cellular senescence in osteoarthritis . Nat Rev Rheumatol 2021, 17 (1):47-57. Liu Y, Zhang Z, Li T, Xu H, Zhang H: Senescence in osteoarthritis: from mechanism to potential treatment . Arthritis Res Ther 2022, 24 (1):174. Scanzello CR: Role of low-grade inflammation in osteoarthritis . Curr Opin Rheumatol 2017, 29 (1):79-85. Sudo T, Akeda K, Kawaguchi K, Hasegawa T, Yamada J, Inoue N, Masuda K, Sudo A: Intradiscal injection of monosodium iodoacetate induces intervertebral disc degeneration in an experimental rabbit model . Arthritis Res Ther 2021, 23 (1):297. McCulloch K, Huesa C, Dunning L, Litherland GJ, Van 't Hof RJ, Lockhart JC, Goodyear CS: Accelerated post traumatic osteoarthritis in a dual injury murine model . Osteoarthritis Cartilage 2019, 27 (12):1800-1810. Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.pdf Supplementaryfig1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6830580","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475239425,"identity":"aa47d902-4b26-4d72-90bc-b08c782ea1d6","order_by":0,"name":"Liangcai Hou","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liangcai","middleName":"","lastName":"Hou","suffix":""},{"id":475239426,"identity":"f382a434-5228-4a63-bc95-c0affdcc2c7d","order_by":1,"name":"Kai Sun","email":"","orcid":"","institution":"Huazhong University of Science and 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Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYJCCDxJsDAz8zMyHHxCrg3EGSItkO1uaAfFaGIBaDM7zKEgQpV4+Ivlgg0WZjbzxYR4GA4Yam2iCWgxvpCU2SJxLM9x2mPfAA4ZjabkNBLXMyDF/INl2OMHsMF+CAWPDYaK0GDaAtBg38xhIEKVFXgKqxYCZWC0GPM8gfplxGBjICcT4Rb49+WCzBDDE+PsPH37wocaGCFsOMDAww+MjgZBysC1AQxk/EKNyFIyCUTAKRi4AABNQPVJ7VjPyAAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Fengjing","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2025-06-05 15:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6830580/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6830580/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85643226,"identity":"f6a66f32-4a48-4c07-8568-fbf937b7c38e","added_by":"auto","created_at":"2025-06-30 08:07:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntra-articular injection of Erastin induced cartilage degeneration in mice. \u003c/strong\u003eFollowing DMM surgery or intra-articular injections, mice (n=8/group/timepoint) were euthanized at 4-, 8-, and 12-week intervals. (A,B) Cartilage degeneration was quantified via Safranin O/fast green staining, with OA progression graded using the OARSI scoring system (scale bar: 200 µm). (C,D) Synovitis severity was assessed by H\u0026amp;E staining-based scoring (scale bar: 200 µm). (E-J) Immunohistochemical evaluation of GPX4, COL2A1, and MMP13 expression included quantification of immunopositive cells (scale bar: 100 µm). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/85e7deb80ceb03cb38f99cd5.png"},{"id":85644887,"identity":"2378ca9a-ea10-45be-92d0-c19331997141","added_by":"auto","created_at":"2025-06-30 08:23:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEukaryotic mRNA sequencing reveals that Erastin intervention promotes changes in the expression of OA-related genes in chondrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEukaryotic mRNA sequencing was performed on the primary chondrocytes of the mouse, which were treated with Erastin. The concentration of Erastin used in this study was 10 μM. (A) Boxplot showing the expression levels for each sample. (B) Volcano plot and (C) heatmap analyses illustrate proteins with significantly different levels in mouse primary chondrocytes under the indicated treatments (n = 3 samples per group). (D) KEGG analysis highlights enriched pathways related to the treatments.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/aeefb76c4f53cdd99af4aaba.png"},{"id":85643231,"identity":"d6e12f4e-50c1-4fb1-b346-716a64960330","added_by":"auto","created_at":"2025-06-30 08:07:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":590861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErastin increased ECM catabolism and decreased anabolism in chondrocytes.\u003c/strong\u003e Primary murine chondrocytes (C57BL/6), C28I2 human chondrocyte line, and human primary chondrocytes were exposed to 10 μM Erastin for 24 h. (A) Heatmap displaying significantly dysregulated OA-associated genes in Erastin-treated versus control groups. (B-D) Immunoblotting detected protein profiles of ECM markers (Aggrecan, COL2A1, SOX9) and catabolic enzymes (MMP3, MMP13) in: (B) murine, (C) C28I2, and (D) human chondrocytes. (E,F) Immunofluorescence visualized COL2A1 distribution in human chondrocytes (scale bar: 50 µm). (G,H) qRT-PCR quantified Col2a1 and Mmp3 transcripts in (G) murine and (H) human chondrocytes. (I,K) Safranin O/fast green staining demonstrating cartilage degeneration in (I) murine and (K) human articular explants post-Erastin. (J,L) IHC enumeration of COL2A1+ and MMP13+ cells in (J) murine and (L) human explants. Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/c91eb1591a1198d2a10ca1c3.png"},{"id":85643232,"identity":"e2d18643-b39c-41fb-9d26-a7673ed91c3a","added_by":"auto","created_at":"2025-06-30 08:07:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":207324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErastin aggravated inflammation in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57 mouse chondrocytes, the C28I2 cell line, and human chondrocytes were treated with 10 μM Erastin for 24 h. (A) The heat map shows several inflammation-related genes significantly differentially expressed between Ctrl and Erastin-treated groups. (B) Western Blot revealed the representative protein expression levels of iNOS and COX2 in C57 mouse chondrocytes. (C) Western Blot revealed the representative protein expression levels of iNOS and COX2 in C28I2 cell line. (D) Immunoblotting demonstrated upregulated protein profiles of iNOS and COX2 in human-derived chondrocytes. (E,F) iNOS and Ptgs2 expression in mouse chondrocytes (E) and human chondrocytes (F) was examined by qRT-PCR. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/97841e6c1909545bb0e96908.png"},{"id":85643234,"identity":"4bd66996-adea-4b70-9993-bb76450ef687","added_by":"auto","created_at":"2025-06-30 08:07:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":571750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErastin aggravated ferroptosisin chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary murine chondrocytes (C57BL/6), C28I2 human chondrocyte line, and human primary chondrocytes were treated with 10 μM Erastin for 24h. (A) Heatmap of significantly dysregulated ferroptosis-related genes (Erastin vs. Ctrl). (B-D) Immunoblotting of ferroptosis markers ACSL4, p53, and GPX4 in: (B) murine, (C) C28I2, and (D) human chondrocytes. (E,G) Lipid peroxide visualization and quantification in murine chondrocytes (scale bar: 200 µm). (F,H) Intracellular ROS assessment in murine chondrocytes (scale bar: 200 µm). (I,K) Lipid peroxide levels in human chondrocytes (scale bar: 100 µm). (J,L) ROS quantification in human chondrocytes (scale bar: 100 µm). (M,N) FerroOrange-stained Fe²⁺ accumulation and quantification in murine chondrocytes (scale bar: 100 µm). (O-R) EdU assay for proliferative capacity in (O,P) murine and (Q,R) human chondrocytes (scale bar: 100 µm). (S) CCK-8 viability of murine chondrocytes exposed to Erastin (12/24/48h). (T) CCK-8 results in human chondrocytes (24h). (U,V) qRT-PCR analysis of Acsl4 and Gpx4 transcripts in (U) murine and (V) human chondrocytes. Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/5f30c00a260652d7dae2fac2.png"},{"id":85644603,"identity":"368ab8a2-d2cc-4732-bd68-e2809dd1cc90","added_by":"auto","created_at":"2025-06-30 08:15:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":236084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErastin aggravated senescence in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57 mouse chondrocytes, the C28I2 cell line, and human chondrocytes were treated with 10 μM Erastin for 24 h.(A) The heat map shows several senescence-related genes significantly differentially expressed between Ctrl and Erastin-treated groups. (B) Western Blot revealed the representative protein expression levels of Cyclin D and P21 in C57 mouse chondrocytes. (C) Western Blot revealed the representative protein expression levels of Cyclin D, P21, and P16 in C28I2 cell line. (D) Western Blot revealed the representative protein expression levels of Cyclin D, P21, and P16 in human chondrocytes. (E) Cdkn1a and Cdkn2a expression in human chondrocytes was examined by qRT-PCR. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/6c25fdd808fb0282d5f2ed22.png"},{"id":85644601,"identity":"636a5265-12e8-4ed3-acf3-27822f0ba8a4","added_by":"auto","created_at":"2025-06-30 08:15:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":205654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErastin activated the NRF2 antioxidant pathway in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary murine chondrocytes (C57BL/6), C28I2 human chondrocyte line, and human primary chondrocytes underwent 24h treatment with 10 μM Erastin. (A) Heatmap displaying significantly dysregulated senescence-associated genes (Erastin vs. Ctrl). (B-D) Immunoblotting detected Cyclin D1, P21, and P16 protein dynamics in: (B) murine, (C) C28I2, and (D) human chondrocytes. (E) qRT-PCR quantification of CDKN1A (p21) and CDKN2A (p16) transcripts in human chondrocytes. Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/e13f56f1eebe8636381b7b06.png"},{"id":87187816,"identity":"ab345671-7a80-41cf-a23b-1de0eabc7a9f","added_by":"auto","created_at":"2025-07-21 10:46:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5325143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/f3172e8b-ac58-4972-b87c-a67b57acc10f.pdf"},{"id":85644595,"identity":"b4bc3958-997b-41ce-8775-1aeb316e59cd","added_by":"auto","created_at":"2025-06-30 08:15:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":604710,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/842051a9fbdb739984795026.pdf"},{"id":85643228,"identity":"9016c3bb-9bce-4cdd-bf0c-0dc63cd17786","added_by":"auto","created_at":"2025-06-30 08:07:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":217455,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6830580/v1/208a273757bcc80d5714e2f8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intra-articular injection of Erastin induces OA pathological progression as an experimental model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA), the most prevalent degenerative joint disorder, induces persistent pain and functional disability, with growing global incidence correlated to aging demographics[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Over 59% of OA patients exhibit comorbidities like cardiovascular/metabolic disorders[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], while current therapies only alleviate symptoms without halting progression[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite clinical/preclinical efforts, OA\u0026rsquo;s chronicity and interspecies translational gaps hinder mechanistic insights[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReliance on a single animal model for OA research has inherent limitations. Established approaches\u0026mdash;including aging models, surgical induction (\u003cem\u003ee.g\u003c/em\u003e., anterior cruciate ligament transection [ACLT] and destabilized medial meniscus [DMM] [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]), and chemical induction (e.g., monoiodoacetate[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], papain[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and collagenase[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e])\u0026mdash;collectively enhance mechanistic understanding of OA[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, widely utilized DMM models exhibit limitations such as variable outcomes and technical complexity. These shortcomings necessitate complementary strategies to isolate specific molecular pathways and dissect OA mechanisms with precision.\u003c/p\u003e \u003cp\u003eFerroptosis, a lipid peroxidation-driven cell death pathway distinct from apoptosis/necrosis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], is implicated in age-related pathologies including neurodegenerative disorders[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In OA, ferroptosis-related genes correlate with disease progression[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and its activation in chondrocytes underlies ECM degradation and inflammation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Targeting ferroptosis with inhibitors (Ferrostatin-1) or iron chelators (Deferoxamine) attenuates OA progression in mice[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Critically, glutathione peroxidase 4 (GPX4)\u0026mdash;a lipid peroxidation suppressor\u0026mdash;is downregulated in human OA cartilage[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and its cartilage-specific knockout accelerates OA via ferroptosis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], solidifying ferroptosis as a pivotal OA pathogenic mechanism.\u003c/p\u003e \u003cp\u003eErastin, a canonical ferroptosis inducer targeting mitochondrial voltage-dependent anion channels (VDACs)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], drives chondrocyte dysfunction in OA. Our prior work demonstrated that Erastin-treated chondrocytes exhibit upregulated ferroptosis markers, extracellular matrix (ECM) degradation, and pro-inflammatory responses[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e, biweekly intra-articular Erastin injections (5 mg/kg) over 8 weeks induced marked OA-like degeneration in murine knee joints, with histopathology confirming chondrocyte damage and ECM loss[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings establish Erastin as a potent tool for modeling ferroptosis-driven OA pathology.\u003c/p\u003e \u003cp\u003eCurrent OA models exhibit critical constraints: aging models require prolonged induction periods, limiting their utility for rapid mechanistic studies; surgical approaches like DMM suffer from technical complexity and outcome variability, while collagenase-induced models demand meticulous dose optimization to achieve consistent pathology. Importantly, none directly interrogate ferroptosis\u0026mdash;a central pathogenic mechanism bridging oxidative stress and metabolic dysfunction in OA.\u003c/p\u003e \u003cp\u003eWe developed a novel OA model through intra-articular Erastin administration, which bypasses surgical complexity by directly activating ferroptosis. Unlike conventional models that indiscriminately accelerate joint degeneration, our system synchronously induces OA phenotypes and ferroptosis activation, enabling precise interrogation of lipid peroxidation-mediated chondrocyte death and ECM collapse.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eAnimal experiments (Approval No. TJH-202112022): All procedures involving animals received approval from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) and were conducted in compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Eight-week-old male C57BL/61 mice, supplied by the Tongji Hospital Experimental Animal Center, were utilized to establish the animal models.\u003c/p\u003e \u003cp\u003eHuman samples (Approval No. TJIRB20210905): Studies involving human specimens were performed in accordance with the Helsinki Declaration and obtained ethical clearance from the Tongji Hospital Ethics Committee. Written informed consent was acquired from every participant. Osteoarthritis cartilage samples were obtained from patients (n\u0026thinsp;=\u0026thinsp;4) undergoing total knee arthroplasty at Tongji Hospital. Detailed information regarding the OA patients is presented in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (included at the end of the manuscript).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOA patients\u0026rsquo; information\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeight(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWeight(kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOA location\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeft\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRight\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeft\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeft\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmputees\u0026rsquo; information\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeight(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWeight(kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOA location\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRight\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeft\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRight\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRight\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eErastin (S7242, Selleck) was dissolved in DMSO for experimental use. The Cell Counting Kit-8 (CCK-8) (HY-K0301) was acquired from MedChemExpress. RNA extraction was performed using a kit from Omega Biotek (R6834-01). Hifair\u0026reg; III 1st Strand cDNA Synthesis SuperMix (11141ES60) and SYBR Green Master Mix (11203ES03) were sourced from Yeasen. For reactive oxygen species (ROS) detection, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (#S0033) was obtained from Beyotime (Shanghai, China). Lipid peroxidation was assessed using C11-BODIPY (581/591) probe (D3861, Thermo Fisher). Cell proliferation was evaluated with an EdU staining kit (RiboBio, Guangzhou, China), and labile iron pool was detected with FerroOrange (F374, Dojindo).\u003c/p\u003e\n\u003ch3\u003eAnimals and treatments\u003c/h3\u003e\n\u003cp\u003eTo isolate estrogen's specific impact on OA pathogenesis, we utilized 8-week-old male C57BL/61 mice (n\u0026thinsp;=\u0026thinsp;72), thereby eliminating confounding effects from cyclic estrogen fluctuations in females. Animals were randomized into three groups: the DMM model group, the intra-articular injection group receiving 1 mg/kg Erastin, and the intra-articular injection group receiving 10 mg/kg Erastin, with the right knee joint of the DMM model group serving as the Sham group. DMM surgery was conducted on the left knee per established protocol[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], initiating with intraperitoneal anesthesia (1% pentobarbital) followed by meniscal destabilization. The intra-articular Erastin injection groups received a single injection into the left knee joint. Mice were euthanized at 4, 8, and 12 weeks post-surgery or injection for micro-computed tomography and histological analysis.\u003c/p\u003e\n\u003ch3\u003eCells and treatments\u003c/h3\u003e\n\u003cp\u003eExperimental cells included primary mouse chondrocytes, C28I2 cells, and human chondrocytes. Mouse chondrocytes were isolated from 5-day-old C57BL/6J mice through sequential enzymatic digestion: knee joint cartilage was minced, digested with 0.25% trypsin (30 min), then treated with 0.25% type II collagenase (4\u0026ndash;6 h). Harvested cells were resuspended and maintained in DMEM/F12 medium (10% fetal bovine serum, 1% penicillin/streptomycin) at 37\u0026deg;C. Only early-passage chondrocytes (P1-P2) were utilized for subsequent assays. The C28/I2 cell line was generously provided by Dr. Huang Junmin from Zhongshan Hospital, Fudan University. Human chondrocytes were obtained from children undergoing polydactyly correction surgery. Cartilage specimens were subjected to sequential enzymatic digestion: initial treatment with 0.25% trypsin (30 min) followed by 0.25% type II collagenase (20 h). Isolated cells were resuspended in DMEM/F12 medium (10% fetal bovine serum, 1% penicillin/streptomycin) and maintained at 37\u0026deg;C. All experiments exclusively used primary human chondrocytes.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e \u003cp\u003eWe extracted total protein using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The concentration of total protein was determined using a BCA Protein Assay Kit (Boster, AR1110). Equal amounts of total protein (8\u0026ndash;10 \u0026micro;g) were combined with loading buffer and heated at 100\u0026deg;C (10 min). Subsequently, Samples were then electrophoresed on SDS-PAGE gels and electrotransferred onto PVDF membranes. Membranes underwent blocking with 5% skim milk (1 h, RT), followed by overnight incubation at 4\u0026deg;C with primary antibodies (listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, located at the end of this article). After four 7-minute TBST washes, membranes were exposed to HRP-conjugated secondary antibodies (1 h, RT). Protein signals were detected using a Bio-Rad ChemiDoc system (Hercules, CA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eprimary antibodies used in the Western Blot experiment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eANTIBODIES\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSOURCE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIDENTIFIER\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60004-1-Ig\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollagen II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15943-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNRF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16396-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHO-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10701-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60283-2-Ig\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACSL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22401-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclin D1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26939-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10355-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech Group, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28416-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggrecan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABclonal, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA8536\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNQO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ab80588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ab39012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOX9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ab185966\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGPX4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ab125066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBoster, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#BM4074\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiNOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology, Beverly, MA, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#13120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOX2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology, Beverly, MA, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#12882\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHRP Conjugated AffiniPure Goat Anti-mouse IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBoster, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBA1050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHRP Conjugated AffiniPure Goat Anti-rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBoster, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBA1054\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFITC Conjugated AffiniPure Goat Anti-rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBoster, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBA1105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFITC Conjugated AffiniPure Goat Anti-mouse IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBoster, Wuhan, Hubei, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBA1101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA (1 \u0026micro;g) was isolated and quantified employing Omega Biotek's RNA Extraction Kit. cDNA synthesis utilized Hifair\u0026reg;III First Strand cDNA Synthesis SuperMix. qPCR amplification proceeded with SYBR Green Master Mix under optimized cycling: 95\u0026deg;C for 5 min (initial denaturation); 40 cycles of 95\u0026deg;C for 10 sec (denaturation) and 60\u0026deg;C for 30 sec (combined annealing/extension). Melting curves were generated by sequential incubation: 95\u0026deg;C (15 sec) \u0026rarr; 60\u0026deg;C (60 sec) \u0026rarr; 95\u0026deg;C (15 sec). GAPDH served as endogenous control. Target gene primers are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, located at the end of this article.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequence used in the RT-qPCR experiment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMmp3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMouse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-GAAACGGGACAAGTCTGTGGAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-ATGAAAATGAAGGGTCTTCCGGTCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eInos\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMouse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-CAGGGAGAACAGTACATGAACAC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-TTGGATACACTGCTACAGGGA \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ePtgs2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMouse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-TTCAACACACTCTATCACTGGC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-AGAAGCGTTTGCGGTACTCAT \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eGapdh\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMouse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-AACATCAAATGGGGTGAGGCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-GTTGTCATGGATGACCTTGGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eGpx4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMouse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-GATGGAGCCCATTCCTGAACC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-CCCTGTACTTATCCAGGCAGA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eAcsl4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMouse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-CTCACCATTATATTGCTGCCTGT \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-TCTCTTTGCCATAGCGTTTTTCT \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMMP3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-GGCAAGACAGCAAGGCATAGAGAC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-GACCAACATCAGGAACTCCACACC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCOL2A1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-GCAAGCAAGGAGACAGAGGAGAAG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-AGAAGGACCAGCAGGACCAGAAG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ePTGS2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-CCATTGACCAGAGCAGGCAGATG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-TGGCTTCCAGTAGGCAGGAGAAC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eACSL4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-AACTGCCTTGGCTGTACTGCTATTC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-GTGTGACAGAGCGATATGGACTTCC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eGPX4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-CCCGATACGCTGAGTGTGGTTTG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-CCTTGCCCTTGGGTTGGATCTTC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-CAGGAGGCATTGCTGATGAT \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-GAAGGCTGGGGCTCATTT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCDKN1A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-TTGATTAGCAGCGGAACAAGGAGTC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-GAGAAACGGGAACCAGGACACATG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCDKN2A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eHuman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward: 5\u0026prime;-TCATCAGTCACCGAAGGTCCTACAG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse: 5\u0026prime;-TGCTCACTCCAGAAAACTCCAACAC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell viability assays\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8) to quantify cell proliferation. Mouse or human chondrocytes were seeded into 96-well plates at a density of approximately 5,000 cells/well and adhered for 24 h prior to treatment. Erastin exposure (10 \u0026micro;M) was initiated for 12, 24, or 48 h. Subsequently, each well's medium was substituted with 100 \u0026micro;L fresh medium containing 10 \u0026micro;L CCK-8 reagent. Following 1 h incubation, absorbance at 450 nm was quantified using a Leica microplate reader (Wetzlar, Germany).\u003c/p\u003e\n\u003ch3\u003eEdu Staining\u003c/h3\u003e\n\u003cp\u003eMouse or human chondrocytes were seeded into 96-well plates at approximately 5,000 cells per well and allowed to adhere for 24 hours. Cells were then treated with 10 \u0026micro;M Erastin. After 12, 24, or 48 hours of treatment, chondrocytes were incubated with 10 \u0026micro;M EdU for 2 hours. Following incubation, the cells were fixed with paraformaldehyde and treated with 2 mg/mL glycine. Permeabilization was performed using 1% Triton X-100 for 5 minutes, after which the staining solution was added for 30 minutes. The cells were washed with 0.5% Triton X-100 and subsequently stained with Hoechst. Stained images were captured using an inverted fluorescence microscope (OLYMPUS, IX71). EdU staining was performed in triplicate, with the same field of view selected each time. The number of EdU-positive cells was quantified and expressed as a fraction of the total cell count in the selected area.\u003c/p\u003e \u003cp\u003eMouse/human chondrocytes (5,000 cells/well in 96-well plates) were adhered for 24 h prior to 10 \u0026micro;M Erastin exposure. At 12/24/48 h post-treatment, cells were fixed in 4% paraformaldehyde, quenched with 2 mg/mL glycine, and permeabilized using 1% Triton X-100 (5 min). EdU reaction cocktail was applied for 30 min, followed by three 0.5% Triton X-100 washes. Nuclei were counterstained with Hoechst. Images were acquired using an Olympus IX71 inverted fluorescence microscope. Triplicate assays quantified EdU\u003csup\u003e+\u003c/sup\u003e cells as proliferative index (vs. total Hoechst\u003csup\u003e+\u003c/sup\u003e cells in designated fields)\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eAfter 24 h of 10 \u0026micro;M Erastin exposure, chondrocytes were washed thrice with PBS, fixed with 4% formaldehyde (15 min), and permeabilized with 1% Triton X-100 (5 min, RT). Blocking was performed with 5% BSA (1 h, RT). Primary antibodies were applied at optimal dilutions and incubated overnight at 4\u0026deg;C (humidified chamber). Fluorophore-conjugated secondary antibodies (1:200) were then added for 1 h at 37\u0026deg;C. Nuclei were counterstained with DAPI (5 min). Images were captured using an Olympus BX51 upright fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReactive Oxygen Species (ROS) and Lipid ROS\u003c/h2\u003e \u003cp\u003eIntracellular ROS generation was assayed with DCFH-DA, whereas lipid ROS accumulation was monitored using C11-BODIPY 581/591 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Chondrocytes in six-well plates were first incubated for 24 h, then exposed to 10 \u0026micro;M C11-BODIPY or DCFH-DA under light-protected conditions for 30 min. After an additional PBS wash, cellular fluorescence was observed using a fluorescence microscope (OLYMPUS, IX71).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFerroOrange staining\u003c/h2\u003e \u003cp\u003eWe cultured chondrocytes in six-well plates and incubated them with 1 \u0026micro;M FerroOrange in HBSS for 30 minutes at 37\u0026deg;C, 21% oxygen, and 5% CO\u003csub\u003e2\u003c/sub\u003e after a 24-hour incubation period. Following incubation, we immediately imaged the cells using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistological and immunohistochemical staining\u003c/h2\u003e \u003cp\u003eLeft knee joints were immersed in 4% paraformaldehyde (3 d), embedded in paraffin, and sectioned. Sections underwent Safranin O/fast green staining for blinded OARSI scoring by histopathologists[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Synovitis was quantified via H\u0026amp;E staining: synovial lining thickness scored 0\u0026ndash;3 (medial/lateral), cumulative range 0\u0026ndash;6 (0\u0026thinsp;=\u0026thinsp;1\u0026ndash;2 layers; 1\u0026thinsp;=\u0026thinsp;3\u0026ndash;4; 2\u0026thinsp;=\u0026thinsp;5\u0026ndash;9; 3\u0026thinsp;\u0026ge;\u0026thinsp;10) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For IHC, dewaxed/rehydrated samples were blocked with BSA\u0026thinsp;+\u0026thinsp;0.1% Triton X-100 (1 h), then incubated with primary antibodies (COL2A1/MMP13/GPX4). DAB-conjugated secondary antibodies were applied before hematoxylin counterstaining. Images were acquired using fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicro-computed tomography (micro-CT)\u003c/h2\u003e \u003cp\u003eLeft knee joints fixed in 4% paraformaldehyde underwent scanning with a Viva CT 80 system (Scanco Medical AG, Switzerland) under these parameters: 100 kV, 98 \u0026micro;A, 10.5 \u0026micro;m resolution. Three-dimensional (3D) images were reconstructed using Scanco Medical software. Evaluate the size of the osteophyte based on the transverse image of the tibial plateau. Subchondral bone in the medial femoral condyle was analyzed by two blinded observers across 30 consecutive layers (initiating 15 layers proximal to the tibial plateau edge). Quantitative parameters assessed using Scanco analysis software comprised bone volume fraction (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N), based on data obtained via the micro-CT system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA extraction, library preparation, and sequencing were contracted to Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China), adhering to their standardized protocols. Bioinformatics analysis was executed on their proprietary Cloud Platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were conducted in GraphPad Prism 8.0 (San Diego, CA, USA). Multiple group comparisons utilized one-way ANOVA with Tukey's post hoc testing. Inter-group differences for continuous variables were assessed by unpaired two-tailed Student's t-test. Non-parametric data (e.g., OARSI grades) underwent Mann-Whitney U test (two groups) or Kruskal-Wallis with Dunn's test (\u0026ge;\u0026thinsp;3 groups). Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 defined statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.\u0026nbsp; \u0026nbsp;Intra-articular injection of Erastin-induced cartilage degeneration in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate Erastin\u0026apos;s effects on articular cartilage, destabilization of the medial meniscus (DMM) was established in mice (n=8/group) as an osteoarthritis model, with contralateral knees as sham controls. Animals received intra-articular Erastin (1 or 10 mg/kg). At 4/8/12 weeks post-intervention, knee joints were harvested for micro-CT and histology. Safranin O/fast green staining demonstrated that both DMM surgery and Erastin administration induced pathological hallmarks of cartilage degeneration, including: superficial cartilage fibrosis, Proteoglycan loss (reduced matrix staining) and structural integrity disruption (Fig. 1A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCartilage degeneration severity was quantified via the OARSI scoring system. Relative to the Sham group, OARSI scores exhibited a significant increase in both the DMM model group and animals administered intra-articular Erastin (1 mg/kg and 10 mg/kg) (Fig. 1B). Nevertheless, comparisons between the DMM group and either Erastin dosage group (1 mg/kg or 10 mg/kg) revealed no statistically significant disparities in OARSI scores, demonstrating equivalent effects at both concentrations. \u0026nbsp;Concurrently, there was no significant difference in OARSI scores between the intra-articular injection of Erastin at 1 mg/kg and 10 mg/kg groups, indicating that both concentrations of Erastin could induce stable modeling. Moreover, OARSI scores in the DMM model and intra-articular Erastin-administered groups (1 mg/kg and 10 mg/kg) exhibited parallel trends across 4-, 8-, and 12-week timepoints in mice, supporting the modeling stability of intra-articular Erastin injection. H\u0026amp;E staining revealed expansion of the synovial lining cell layer and heightened stromal cellular density in the DMM cohort and both Erastin-treated groups (Fig. 1C), with concomitant significant elevations in synovitis scores (Fig. 1D).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis demonstrated reduced quantities of GPX4-positive cells within the knee joint cartilage of both DMM-induced and Erastin-treated groups (Fig. 1E and F). Relative to Sham controls, COL2A1 immunostaining intensity and positive cell counts in the cartilage matrix were markedly diminished in these experimental groups (Fig. 1G and H), concomitant with a significant rise in MMP13-positive cells (Fig. 1I and J).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOsteophyte formation and subchondral bone alterations were quantified using micro-CT across experimental groups. Primary analysis focused on the medial tibial plateau subchondral region, where bone volume changes were assessed through parameters including bone volume fraction (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp). Data revealed decreased BV/TV, Tb.N, and Tb.Th values with elevated Tb.Sp in the DMM cohort relative to Sham controls. \u0026nbsp;However, no significant differences were noted between the 1 mg/kg and 10 mg/kg Erastin groups when compared to the Sham group. These findings suggest that the DMM model induces a reduction in bone volume fraction, trabecular number, and trabecular thickness, leading to the remodeling of subchondral bone. Notably, intra-articular administration of 1 mg/kg or 10 mg/kg Erastin showed no significant modulatory effects on these indices or subchondral bone remodeling (Supplementary Fig. 1 A-D). Three-dimensional reconstructions and transverse tibial plateau scans confirmed distinct osteophytes at patellar cartilage margins and medial femoral condyle articular surfaces in both DMM-induced and Erastin-treated groups (1 mg/kg and 10 mg/kg), whereas the Sham group displayed negligible osteophyte formation (Fig. 1K and L). Collectively, intra-articular Erastin administration successfully induces murine cartilage degeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Eukaryotic mRNA sequencing reveals that Erastin intervention promotes changes in the expression of OA-related genes in chondrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate the impact of Erastin on chondrocyte gene expression associated with OA pathogenesis and advancement, eukaryotic mRNA sequencing was performed on primary chondrocytes from Erastin-treated mice for systematic transcriptomic profiling. Fig. 2A displays the expression level distribution across samples via boxplots, while volcano plot and heatmap analyses depict the differentially expressed gene profiles between treatment cohorts (Fig. 2B and C). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that Erastin treatment resulted in the enrichment of numerous OA-related gene pathways within the transcriptome (Fig. 2D) . These pathways include ECM-receptor interaction, Glutathione metabolism, Ferroptosis, Inflammation-related pathways, Senescence-related pathways, and Antioxidant pathways. Collectively, these data support that Erastin intervention induces changes in the expression of genes associated with OA progression in chondrocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eErastin increased ECM catabolism and decreased anabolism in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChondrocytes maintain ECM homeostasis via coordinated anabolic-catabolic balance[24]. Heatmap profiling screened significantly dysregulated OA-related gene signatures between cohorts (Fig. 3A). We designated Aggrecan, COL2A1, and SOX9 as biomarkers of ECM anabolism, with MMP3 and MMP13 serving as catabolism indicators. Western blotting confirmed that Erastin treatment significantly downregulated anabolic markers (Aggrecan, COL2A1, SOX9) while upregulating catabolic markers (MMP3, MMP13) in murine chondrocytes (Fig. 3B). Erastin-treated C2812 cells exhibited alterations aligned with murine chondrocyte responses (Fig. 3C). Western blotting confirmed significant downregulation of Aggrecan, COL2A1, and SOX9 proteins coupled with MMP3 upregulation in human chondrocytes following Erastin exposure (Fig. 3D). Furthermore, immunofluorescence staining revealed that Erastin intervention in human chondrocytes inhibited COL2A1 expression (Fig. 3E and F). Additionally, qRT-PCR results showed that Erastin promoted the expression of Mmp3 in mouse chondrocytes (Fig. 3G). Erastin promoted the expression of MMP3 and inhibited the expression of COL2A1 in human chondrocytes (Fig. 3H). Erastin-exposed murine articular explants exhibited diminished Safranin O/fast green staining intensity at cartilage surfaces (Fig. 3I). Immunohistochemistry detected reduced COL2A1-immunoreactive cells alongside elevated MMP13-positive cell populations (Fig. 3J). Human joint explant outcomes strongly concorded with murine data (Fig. 3K and L). Collectively, results from multiple cell types including mouse chondrocytes, C28I2 cell line, and human chondrocytes indicated that Erastin increased ECM catabolism and decreased anabolism in chondrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e \u003cstrong\u003eErastin aggravated inflammation in chondrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInflammatory processes constitute a key driver of osteoarthritis pathogenesis. [25-27]. A heatmap analysis identified several inflammation-related genes that demonstrated significant differential expression between the two groups (Fig. 4A). Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were designated as inflammatory biomarkers in this study. Western blotting demonstrated significant upregulation of iNOS and COX2 protein levels in murine chondrocytes following Erastin exposure (Fig. 4B), C28I2 cells (Fig. 4C), and human chondrocytes (Fig. 4D). Additionally, qRT-PCR results further confirmed that Erastin intervention elevated the expression of iNOS and Ptgs2 in mouse chondrocytes (Fig. 4E) and promoted PTGS2 expression in human chondrocytes as well (Fig. 4F). Collectively, these findings suggest that Erastin enhances inflammatory responses in chondrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Erastin aggravated ferroptosis in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a specific ferroptosis inducer, Erastin modulates pathways that our prior work established as significant contributors to OA pathogenesis [14]. Heatmap profiling identified markedly dysregulated ferroptosis-associated gene signatures across cohorts (Fig. 5A). We designated acyl-CoA synthetase long-chain family member 4 (ACSL4) and p53 as ferroptosis-activation biomarkers, with GPX4 functioning as a ferroptosis-inhibitory indicator. Western blot analysis demonstrated that Erastin treatment in mouse chondrocytes (Fig. 5B), C28I2 cells (Fig. 5C), and human chondrocytes (Fig. 5D) resulted in increased expression of ACSL4 and p53, alongside a decrease in GPX4 expression. The accumulation of lipid ROS and ROS is characteristic of ferroptosis; To assess this, we employed C11-BODIPY to detect lipid ROS and DCFH-DA to measure ROS levels. Data revealed that Erastin exposure triggered robust accumulation of lipid peroxides and reactive oxygen species within murine chondrocytes (Fig. 5E-H).\u0026nbsp;Similar patterns were observed in human chondrocytes following Erastin treatment (Fig. 5I-L).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFerroptosis represents an iron-dependent programmed cell death modality driven predominantly by ferrous ions (Fe\u0026sup2;⁺) [11]. To further verify the occurrence of ferroptosis, we used the FerroOrange probe to detect Fe\u003csup\u003e2+\u003c/sup\u003e accumulation in chondrocytes. Our findings revealed that Erastin induced significant cytoplasmic Fe\u003csup\u003e2+\u003c/sup\u003e accumulation in mouse chondrocytes (Fig. 5M and N). To assess the impact of Erastin on chondrocyte proliferation, we employed EdU staining, which demonstrated that Erastin inhibited the proliferation of both mouse chondrocytes (Fig. 5O and P) and human chondrocytes (Fig. 5Q and R). Additionally, the CCK8 assay revealed that Erastin significantly reduced the viability of mouse chondrocytes (Fig. 5S), with this inhibitory effect on cell viability becoming more pronounced over time. A similar reduction in viability was observed in human chondrocytes (Fig. 5T). Moreover, qRT-PCR analysis further confirmed significant upregulation of Acsl4 transcripts coupled with Gpx4 downregulation in both murine (Fig. 5U) and human chondrocytes (Fig. 5V) following Erastin exposure. Taken together, these findings demonstrate Erastin effectively potentiates ferroptosis in chondrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Erastin aggravated senescence in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOA progression correlates with accumulated senescent cells in articular tissues [28, 29]. Heatmap profiling revealed markedly dysregulated senescence-associated gene signatures across experimental cohorts (Fig. 6A). Cyclin D1, P21, and P16 were employed as senescence hallmark indicators. Immunoblotting demonstrated Erastin-mediated downregulation of Cyclin D1 concomitant with P21 upregulation in murine chondrocytes (Fig. 6B). In C28I2 cells, Erastin inhibited Cyclin D expression while promoting the expression of P21 and P16 (Fig. 6C). Similar results were consistently observed in human chondrocytes (Fig. 6D). Furthermore, qRT-PCR analysis confirmed Erastin provoked marked upregulation of CDKN1A (P21) and CDKN2A (P16) transcript levels in human-derived chondrocytes. (Fig. 6E). Collectively, these findings indicated that Erastin promotes chondrocyte senescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Erastin activated the NRF2 antioxidant pathway in chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeatmap profiling identified markedly dysregulated antioxidant-associated genes across cohorts (Fig. 7A). We designated NRF2 signaling effectors heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) as biomarkers of the NRF2 pathway. Immunoblotting demonstrated concomitant upregulation of NRF2, HO-1, and NQO1 proteins in murine chondrocytes following Erastin exposure (Fig. 7B). Consistent results were observed in C28I2 cells treated with Erastin (Fig. 7C). Additionally, in human chondrocytes, Erastin also promoted the expression of HO-1 and NQO1 (Fig. 7D). These findings indicated that Erastin activated the NRF2 antioxidant pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study pioneers a novel C57BL/6J mouse osteoarthritis model through single-dose intra-articular Erastin administration. Comprehensive evaluations were conducted across three dimensions:\u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e assessment, 8-week-old male C57BL/6J mice (n\u0026thinsp;=\u0026thinsp;8/group) were randomized into: 1) DMM-induced OA model, 2) intra-articular Erastin (1 mg/kg), and 3) intra-articular Erastin (10 mg/kg) groups. Contralateral knee joints served as sham-operated controls. Longitudinal assessments (4, 8, 12 weeks post-intervention) integrated micro-CT with histopathological analyses (Safranin O/fast green, H\u0026amp;E, IHC) to quantify cartilage degeneration, synovitis, subchondral remodeling, ECM homeostasis, and ferroptosis markers.\u003c/p\u003e \u003cp\u003eMicro-CT imaging revealed distinct osteophyte formation and subchondral bone remodeling across experimental groups. Three-dimensional reconstructions and transverse cartilage scans demonstrated pronounced tibial osteophytes in the DMM, 1 mg/kg Erastin, and 10 mg/kg Erastin groups, whereas no osteophyte formation was detected in sham controls.\u003c/p\u003e \u003cp\u003eOARSI scoring via Safranin O/fast green staining highlighted progressive cartilage degeneration in all treatment groups (DMM and both Erastin doses) at 4, 8, and 12 weeks, characterized by superficial fibrillation, proteoglycan loss, and structural disorganization. Notably, OARSI scores in Erastin-treated groups were comparable to those in the DMM group, confirming equivalent cartilage damage severity. Synovial inflammation, a hallmark of OA progression, was evident in H\u0026amp;E -stained sections[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], showing synovial lining thickening and elevated synovitis scores in DMM and Erastin groups compared to sham. Synovitis severity remained consistent between Erastin and DMM groups across all timepoints.\u003c/p\u003e \u003cp\u003eImmunohistochemical analysis revealed a significant reduction in COL2A1 (ECM anabolism marker) and GPX4 (ferroptosis inhibitor) expression, alongside increased MMP13 (ECM catabolism marker) levels in DMM and Erastin-treated groups. These findings collectively indicate that intra-articular Erastin injection disrupts ECM homeostasis by suppressing synthesis, enhancing degradation, and activating ferroptosis signaling pathways.\u003c/p\u003e \u003cp\u003eOur study demonstrated that intra-articular injection of Erastin at different concentrations did not show significant differences compared to the DMM group in terms of cartilage integrity, synovial inflammation, and gene expression in chondrocytes. This suggests that both low-dose (1 mg/kg) and high-dose (10 mg/kg) Erastin intra-articular injections can achieve the desired modeling effect, allowing flexibility in determining the concentration based on experimental needs. Histological evaluations across all timepoints (4, 8, 12 weeks) demonstrated consistent phenotypic progression, confirming the model\u0026rsquo;s temporal consistency. This dual flexibility\u0026mdash;dose independence and timepoint stability\u0026mdash;enables researchers to tailor experimental designs to specific needs while maintaining robust OA pathology induction.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEx Vivo\u003c/em\u003e Validation: Erastin-treated mouse and human cartilage explants (48 h) exhibited proteoglycan loss (Safranin O staining) and disrupted ECM balance, as shown by immunohistochemistry: reduced COL2A1 expression and elevated MMP13 levels. These \u003cem\u003eex vivo\u003c/em\u003e outcomes aligned with in vivo and cellular findings, reinforcing Erastin\u0026rsquo;s ability to drive OA-like ECM remodeling and validating its pathological consistency across experimental models.\u003c/p\u003e \u003cp\u003eCellular Level: Erastin induced OA-like pathological changes across mouse, C28I2, and human chondrocytes by disrupting ECM homeostasis. Key synthesis markers (Aggrecan, COL2A1, SOX9) were suppressed, while catabolic enzymes (MMP3, MMP13) were elevated, mimicking the ECM imbalance characteristic of OA. Pro-inflammatory responses, validated via Western blot and qRT-PCR, further aligned Erastin-treated cells with OA pathophysiology.\u003c/p\u003e \u003cp\u003eFerroptosis was robustly confirmed through upregulated ACSL4 and P53, downregulated GPX4, iron overload (FerroOrange), and lipid/ROS accumulation (C11-BODIPY and DCFH-DA). Functional assays (EdU, CCK8) demonstrated reduced chondrocyte proliferation and viability, consistent with ferroptosis. Concurrently, Erastin amplified senescence phenotypes and activated the NRF2 antioxidant pathway, reflecting stress adaptations seen in human OA.\u003c/p\u003e \u003cp\u003eThe MIA injection model[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] inhibits glyceraldehyde-3-phosphate dehydrogenase in chondrocytes to disrupt glycolysis, inducing apoptosis, ECM degradation, acute inflammation, and cartilage destruction. Its simplicity makes it ideal for studying OA pain mechanisms and early pathology.\u003c/p\u003e \u003cp\u003eThe DMM model[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] induces post-traumatic OA via surgical knee instability, causing mechanical imbalance, cartilage wear, subchondral remodeling, and osteophytosis\u0026mdash;suitable for mechanosensitive pathway research. However, it involves complex surgery with infection risks and high inter-individual variability due to surgical precision dependence.\u003c/p\u003e \u003cp\u003eIn contrast, the Erastin model circumvents DMM\u0026rsquo;s technical challenges and variability, offering a ferroptosis-targeted, streamlined approach to OA pathogenesis. With single intra-articular administration, it recapitulates core OA features (ECM dysregulation, synovitis, subchondral changes), establishing it as a superior preclinical tool for mechanistic and therapeutic studies.\u003c/p\u003e \u003cp\u003eHowever, this study has several limitations. Firstly, our animal experiments exclusively utilized C57BL/6J mice, and further validation of the modeling effects in other mammalian species has yet to be conducted. Secondly, ferroptosis represents only one of the multiple molecular mechanisms involved in the onset and progression of OA. Consequently, while this animal model may be suitable for investigating the significance of the ferroptosis pathway in OA mechanisms, it may not be applicable for studying other molecular mechanisms.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study pioneers a single intra-articular Erastin-induced C57 murine osteoarthritis model, recapitulating DMM-equivalent cartilage degeneration, synovitis, subchondral remodeling, and ferroptosis activation over 12 weeks with dose-independent efficacy. Multi-level validation confirmed Erastin-driven ECM imbalance (reduced COL2A1, elevated MMP13), iron overload, and NRF2 pathway activation. Cross-species consistency in human explants and chondrocytes highlighted conserved ferroptosis mechanisms via GPX4 suppression. While distinct from natural OA progression, this model offers a simplified, temporally stable tool for investigating ferroptosis-specific contributions to OA pathogenesis, bridging translational gaps through mechanistic clarity and experimental flexibility.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACLT: Anterior cruciate ligament transection; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eACSL4: Acyl-CoA synthetase long-chain family member 4; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCCK-8: Cell Counting Kit-8; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCOX2: Cyclooxygenase-2; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDMM: Destabilized Medial Meniscus; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGPX4: Glutathione peroxidase 4; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eiNOS: Inducible nitric oxide synthase; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes and Genomes; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003emicro-CT: Micro-computed tomography; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOA: Osteoarthritis; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROS: Reactive Oxygen Species; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTb.N: Trabecular number; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTb.Sp: Trabecular separation; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTb.Th: Trabecular thickness; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVDAC: Voltage-dependent anion channel; \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclarations of interest\u003c/h2\u003e \u003cp\u003enone.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eThe collection of human cartilage was approved by the Ethics Committee of Tongji Hospital (TJ-IRB20210905) after obtaining informed consent from the patients. The animal experiment was approved by the Ethics Committee of Tongji Hospital (TJH202212051).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Grants No. 82372475, 82172498, 11602155) and the Hainan Provincial Natural Science Foundation of China (Grant No. 821MS164).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors have participated sufficiently in this work to assume public accountability for relevant portions. Study conception and design: LH, KS, JQ, FG. Data acquisition, analysis, and interpretation: LH, KS, XZ, FL, JX, ZG, GW, ZZ, YH, ZR, JH, XL. Manuscript drafting: LH, KS. Critical revision of intellectual content: All authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express gratitude to Dr. Huang Junmin (Zhongshan Hospital, Fudan University) for providing the C28/I2 cell line and acknowledge the Experimental Center of Tongji Hospital for technical assistance in performing immunofluorescence experiments.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYan H, Guo J, Zhou W, Dong C, Liu J: \u003cstrong\u003eHealth-related quality of life in osteoarthritis patients: a systematic review and meta-analysis\u003c/strong\u003e. \u003cem\u003ePsychol Health Med \u003c/em\u003e2022, \u003cstrong\u003e27\u003c/strong\u003e(8):1859-1874.\u003c/li\u003e\n\u003cli\u003eMoreno-Ligero 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\u003cstrong\u003e29\u003c/strong\u003e(1):79-85.\u003c/li\u003e\n\u003cli\u003eSudo T, Akeda K, Kawaguchi K, Hasegawa T, Yamada J, Inoue N, Masuda K, Sudo A: \u003cstrong\u003eIntradiscal injection of monosodium iodoacetate induces intervertebral disc degeneration in an experimental rabbit model\u003c/strong\u003e. \u003cem\u003eArthritis Res Ther \u003c/em\u003e2021, \u003cstrong\u003e23\u003c/strong\u003e(1):297.\u003c/li\u003e\n\u003cli\u003eMcCulloch K, Huesa C, Dunning L, Litherland GJ, Van \u0026apos;t Hof RJ, Lockhart JC, Goodyear CS: \u003cstrong\u003eAccelerated post traumatic osteoarthritis in a dual injury murine model\u003c/strong\u003e. \u003cem\u003eOsteoarthritis Cartilage \u003c/em\u003e2019, \u003cstrong\u003e27\u003c/strong\u003e(12):1800-1810.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cartilage, Osteoarthritis, Chondrocyte, Erastin, Ferroptosis, Mouse, Animal model","lastPublishedDoi":"10.21203/rs.3.rs-6830580/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6830580/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Developing optimal osteoarthritis (OA) models is critical for therapeutic advancement. While ferroptosis is linked to OA pathogenesis, validated models for studying ferroptosis in OA remain scarce.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e: Mouse/C28I2/human chondrocytes were treated with 10 μM Erastin to assess ferroptosis, inflammation, extracellular matrix degradation, senescence, and antioxidant responses. OA patient and neonatal mouse cartilage explants were cultured ± Erastin (48 h) for Safranin O/IHC analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e: 72 C57BL/6J mice (8-week-old) were divided into: (1) Destabilized medial meniscus (DMM) surgery group (Sham: contralateral knee); (2) 1 mg/kg Erastin intra-articular injection; (3) 10 mg/kg Erastin injection. Tissues were collected at 4/8/12 weeks for micro-CT and histology analysis (n=8/group/timepoint).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eErastin triggered ferroptosis, senescence, inflammation, and extracellular matrix degradation in mouse/C28I2/human chondrocytes, alongside NRF2 pathway activation and suppressed extracellular matrix synthesis. In cartilage explants (mice/OA patients), Erastin reduced COL2A1 and elevated MMP13 (IHC). In vivo, OARSI scores (Safranin O) and synovitis scores (H\u0026amp;E) increased significantly in DMM and Erastin (1/10 mg/kg) groups vs. Sham. IHC confirmed GPX4/COL2A1 downregulation and MMP13 upregulation in treated groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e A single intra-articular Erastin injection induces chondrocyte degeneration and cartilage damage mimicking human OA, offering a stable, simplified model compared to surgically complex DMM. This approach directly targets ferroptosis pathways, enabling precise mechanistic studies and streamlined preclinical testing of anti-ferroptosis therapies.\u003c/p\u003e","manuscriptTitle":"Intra-articular injection of Erastin induces OA pathological progression as an experimental model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 08:06:57","doi":"10.21203/rs.3.rs-6830580/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d729885c-a8f3-4752-89d5-db31c4e1cb83","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T10:38:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-30 08:06:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6830580","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6830580","identity":"rs-6830580","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}