CILP Inhibits hyaline cartilage fibrosis and chondrocyte ferroptosis via keap1-Nrf2 axis in early osteoarthritis exercise therapy

Abstract

Background

By analyzing the single-cell RNA-Seq libraries we established of OA joints during exercise therapy, we found cartilage intermediate zone might participate in early OA exercise therapy.

Methods

Early OA rat model was established by 4-week anterior cruciate ligament transection (ACLT). The radiomics was used to evaluate the relative damaged and undamaged area in OA patients’ cartilage. We overexpressed and knocked down CILP in early OA chondrocyte to explore its potential mechanism. The quantitative proteomics was used to examine the protein profiles of the CILP-treated chondorcyte. The Yeast One-Hybrid Assay, Co-Immunoprecipitation (Co-IP), Nrf2 cytosol-nuclei fractionation and ubiquitination assay were used to investigate the potential mechanism in CILP intervention. Western blot, ROS, JC-1, Ferrous ion, MDA and GSH detection, transmission electron microscopy (TEM) were used to explored the therapeutic effect of CILP on OA.

Results

Moderate exercise up-regulates CILP in the articular cartilage intermediate zone. CILP recovers the ratio of type II / I collagen, Sox9, α-SMA expression and competitively bind to Keap1 protein and reduce the stability of Keap1-Nrf2 dimer, thereby reducing the degree of Nrf2 ubiquitination and promoting Nrf2 nuclear translocation. Nrf2 nuclear translocation activated SLC7A11, HO-1, GPX4 and SOD-1 expression, then decreased the MDA contents, but increased GSH content, which ultimately inhibited chondrocytes ferroptosis and promoted hyalinization of fibrocartilage.

Conclusion

Exercise induced cartilage intermediate zone and CILP-Keap1-Nrf2 axis inhibits hyaline cartilage fibrosis and chondrocyte ferroptosis to alleviate early osteoarthritis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-026-06174-5.

Background

Osteoarthritis (OA) is the most common chronic joint disease, affecting approximately 530 million people worldwide [1]. Early OA may be a reversible process [2], and effective intervention has a profound impact on the prognosis of OA [3]. The World Health Organization recommends exercise therapy as the first-line treatment for early OA [4]. In our previous studies, we found that moderate intensity exercise therapy of early knee OA provides a window of opportunity to slow down the OA process [5–10]. In-depth study of exercise therapy can help to develop precision medicine for early OA.

In our previous work, cartilage and chondrocytes play important roles in the maintenance of biomechanical function of the knee joint during exercise therapy [5, 7, 11]. Articular cartilage exhibits great heterogeneity and complex microarchitecture, which is comprised of three anatomic zones, namely the superficial, intermediate and deep zones [12, 13], which endow the tissue with biphasic mechanical properties to withstand shearing force and compressional loading [14]. The chondrocytes in the intermediate zone showed a hypertrophic slightly round shape and clustered together. Type II collagen fibers are randomly distributed to withstand pressures from different directions, as this region is subjected to compressive and shear stresses [15]. Exercise-mediated mechanical stimuli are transmitted through the superficial zone to the intermediate zone, which plays the role of compression resistance and bearing the main load [16]. As a mechanosensitive zone, the intermediate zone plays a key role in the OA exercise therapy [17].

Using single-cell transcriptome profiling to determine differential gene expression within the OA exercise therapy [5], we identified a highly expressed gene, cartilage intermediate layer protein (CILP), which is expressed in the intermediate zone of articular cartilage and has been linked to cartilage degenerative diseases [18, 19]. In our preliminary experiment, we identified CILP as an important gene in the treatment of OA by exercise affecting the intermediate zone of articular cartilage through mechanical stimulation (Supplementary Fig. 1). However, the specific mechanism need be detailed.

In our pseudo-temporal analysis of the pathological process of OA cartilage, cartilage fibrosis plays an important role in early OA (Supplementary Fig. 2) [20]. Hyaline cartilage is the most common type of cartilage and has excellent mechanical properties, mainly acting as a load-bearing structure and lubricating joint movement [21]. In early OA, the articular cartilage surface is damaged to form fibrocartilage due to the loss of thick collagen fibers and the formation of type I collagen [22]. The ability of fibrocartilage to bear weight and resist mechanical wear is far less than that of hyaline cartilage, and fibrocartilage in joints will lead to further deterioration of OA [23, 24]. In view of the anti-fibrosis effect of CILP on other disease [25], it is necessary to investigate the in situ transformation of fibrocartilage into hyaline cartilage by exercise-induced CILP, namely “hyalinization of fibrocartilage”, as an important principle of cartilage repair in OA.

Chondrocyte death also can lead to excessive secretion and deposition of extracellular matrix proteins, and lead to hyaline cartilage fibrosis in early OA [26]. Ferroptosis is a programmed cell death that is driven by Fe2 + dependent lipid peroxidation [27, 28]. Our previous study reported ferroptosis is involved in OA pathogenesis and development [29]. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of the antioxidant response [30]. Under oxidative stress conditions, Nrf2 protein is stabilized and initiates a multistep pathway of activation that includes nuclear translocation [31]. The Keap1-Nrf2 pathway is critical in chondrocyte ferroptosis (Supplementary Fig. 3). It is necessary to investigate exercise induced CILP affects chondrocytes ferroptosis and cartilage fibrosis through the Keap1-Nrf2 pathway.

In this study, we determined the therapeutic effects of moderate intensity exercise on early OA. Then we found that exercise therapy acts on the intermediate zone of articular cartilage through mechanical stimulation. Using single-cell transcriptome sequencing and pseudo-temporal analysis of cartilage, we demonstrated the important role of exercise-induced CILP which expressed highly in cartilage intermediate zone in hyalinization of fibrocartilage. Through clinical samples, we further clarified the potential role of CILP in OA therapy. In vitro, we used proteomics to identify the molecular mechanism by which CILP inhibited Nrf2 ubiquitination and promoted Nrf2 nuclear translocation through Keap1-Nrf2 signaling pathway, which subsequently alleviated chondrocyte ferroptosis and promote hyalinization of fibrocartilage.

Method and materials

Participants

The protocol and experiments on joint specimen after human total knee arthroplasty were approved by the Ethics Committee of Shengjing Hospital of China Medical University (No: 2019PS629K) which abides the principles set out in the World Medical Association Helsinki Declaration. Inclusion/exclusion criteria of study participants had been described in previous literature [6, 8, 11]. We obtained informed consent from all of our patients.

Radiomics

An Avanto 3.0T MR Scanner (SIEMENS MAGNETON), was used to collect patients’ image with an 8-channel coil for the knee joint. Radiomics was used to predict and classify the cartilage damaged and undamaged areas in MRI images of OA patients. Following the protocol of the previous study [32, 33], we briefly stated the steps below. All pretreatment MRI images were acquired. Two experienced radiologists (7-year experience in bone and joint MRI reading) manually delineated the region of interest (ROI) using ITK-SNAP (version 3.8.0; http://www.itksnap.org/). The ROI cover the edge of articular cartilage on coronal T1WI and coronal fat-suppressed PDWI images to generate a three-dimensional ROI of articular cartilage. The feature extraction was performed using the Standardized Environment for Radiomics Analysis (SERA) [32]. All the features were normalized using Z-score normalization. The intraclass correlation coefficient (ICC) was used to evaluate the consistency of the extracted image features. Extractions of 623 features showed good reproducibilities (ICCs > 0.80). Using maximum relevance minimum redundancy (mRMR) and least absolute shrinkage and selection operator (LASSO) to reduce the dimensionality of the texture features in MRI. The rad-score was calculated according to the feature weight. Then, two support vector machine (SVM) models were built using the selected radiomics features to classify the damaged and undamaged regions. Different kernel functions of SVM were tested to find the best performance kernel functions, including linear, radial basis function (RBF), cubic and sigmoid kernel functions. A five-fold cross-validation method was used to train and validate the model.

Human OA articular cartilage and clinicopathological features collection

As mentioned in our previous study [6, 11], the cartilage and the clinicopathological features of patients with knee osteoarthritis after total knee replacement were collected and divided into damaged and undamaged groups according to radiomics result and the Osteoarthritis Research Society International (OARSI) scale, which uses a point system ranging from 0 to 24 [34]. We compared the damaged area with the undamaged area in the same donors.

Experimental animals and early osteoarthritis model

Male Sprague–Dawley (SD) rats (140 ± 5 g, 4-week-old, specific-pathogen-free, eight rats per group, and six rats per cage) were procured from HFK Bioscience Co. Ltd. (Beijing, China). The study was approved by the Ethics Committee of China Medical University (no. 2021PS130K(X1)), adhering to the regulations stipulated in the World Medical Association Helsinki Declaration. The upkeep and care of the experimental rats complied with the committee’s guidelines, as described in previous studies [6, 9].

The OA model was established by anterior cruciate ligament transection (ACLT) of both lower limbs [35]. The early OA model was established over a period of 4 weeks, following previous studies and confirmed by histology evaluation [35]. The 36 rats were segregated into three groups: the control group was sham-operated (CG), the osteoarthritis group (OA), and the osteoarthritis plus moderate exercise group (OAM), with 12 rats in each group. Post-modelling, the OAM group began treadmill training on the ZH-PT animal treadmill exercise platform (Zhongshidichuang Science & Technology Development Co. Ltd., Beijing, China) using appropriate light, acoustic, and electrical stimuli. The moderate treadmill exercise protocol (19.2 m/min) was based on our prior studies [5, 6, 9, 36, 37]. As our previous research demonstrated that moderate-intensity exercise has the optimal therapeutic effect on osteoarthritis [6, 7, 9, 10, 36, 38]. A total of 36 SD rats were used for single-cell transcriptome sequencing (n = 12) [5].

Tissue sampling and collection

After the final treadmill exercise session, rats were euthanized via a pentobarbital overdose, as per our established protocol [6, 9]. Tissue samples, including synovium, cartilage, and subchondral bone, were subsequently collected for single-cell transcriptome sequencing.

For histological and immunohistochemical evaluations, knee joints were fixed using a 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO, United States) for a duration of 7 days. Subsequently, the samples underwent decalcification in a 20% EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) for 6 weeks. Following decalcification, the samples were dehydrated using a graded series of ethanol and xylene (Sigma-Aldrich) and embedded in paraffin (Sigma-Aldrich) [6]. All sections were obtained from the medial tibial plateau.

Single-cell transcriptome sequencing and bioinformatics analysis

Synovium, cartilage, and subchondral bone were collected from the CG, OA, and OAM groups. Sequencing libraries were generated following a comprehensive transcriptome sequencing and single-cell gene-expression profiling protocol as previously reported [39, 40]. Single-cell RNA-Seq libraries were generated using the SeekOne^® Digital Droplet Single Cell 3’ library preparation kit (SeekGene, Beijing, China).

To identify exercise-related differentially expressed genes in chondrocyte, we compared the expression profiles of the chondrocyte of the OA, and OAM groups. Volcano plots of differentially expressed genes were generated using the Limma/R package (version 3.5.1). The primary parameters were set as log fold change, with a log fold change > 2 indicating differentially expressed genes. These differentially expressed genes were then submitted to the DAVID v6.8 tool (https://david.ncifcrf.gov/) for annotation, visualization, and integrated discovery [6].

Pseudo-time trajectory

Following by the previous study [41], the Monocle2 R package (v2.14.0) (http://cole-trapnell-lab.github.io/monocle-release/) was used to dissect gene expression changes associated with cell state transitions in chondrocyte clusters. The ‘DDRTree’ function in Monocle2 R package was used to reduce the dimensionality of the datasets, the ‘orderCells’ function was used to sorted cells in a pseudo-temporal order. Pseudo-time trajectories with default parameters were inferred using the ‘reduceDimension’ function, and the reconstructed trajectories were visualized using the ‘plot_cell_trajectory’ function. Significantly different marker genes were extracted from each module, where clustering was performed based on pseudo-temporal expression patterns and representative heat maps were generated.

Histology evaluation of articular cartilage of human and early OA animal models

We embedded cartilage of human and joints from SD rats in paraffin. Sagittal sections of 4.5-µm thickness were cut from the tibiofemoral joints and stained with hematoxylin and eosin (H&E), toluidine blue, for histological evaluation as described in our previous study [6, 7].

As detailed in our earlier research [6, 11], we categorized the cartilage and the clinicopathological characteristics of patients with knee osteoarthritis into damaged and undamaged area. This categorization was guided OARSI, which uses a point system ranging from 0 to 24 [34].

In animal models, knee joint images were captured by NRecon version 1.6 software (Bruker). As detailed in our previous study, both the tibial and femoral joints were evaluated based on OARSI score [6, 42]. Both the tibial and femoral joints were evaluated based on a maximum OARSI score of 48. Followed by the previous study [43], we use score 0–12 of 24 (tibial or femoral) to identify early OA.

Immunohistochemistry

Briefly, following the kit manufacturer’s instructions (CAT # SP-9001, Zhongshan Jinqiao, China), we performed immunostaining of proteins using a two-step method. Procedures were carried out as we have previously described [9, 10]. Antibodies including, anti-Collagen II (ab34712, 1:200, abcam, Cambridge, MA, USA ), anti-Collagen I (ab270993, 1:200, abcam), anti-Sox9 (ab185966, 1:200, abcam), anti-MMP13 (ab219620, 1:200, abcam), anti-CLIP (sc-12725, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GPX4 (ab125066, 1:200, abcam).

Open field test

The open field apparatus was obtained from Shanghai Xinruan Software Technology (China). The experimental animals were quickly and gently placed in the central area of the experimental box (100 cm × 100 cm × 60 cm black open-field box), and the video acquisition and analysis system was started at the same time to automatically record the activity of the experimental animals in the open field. The experimental time was usually set to 6 min. At the end of each test, the feces of the previous animal should be removed, and the odor should be removed with 75% alcohol, and the chamber should be dry and odorless before the next animal experiment.The data were exported for statistical analysis. The distance (cm) the rat moved within the cage during 6 min, average speed, and number of explorations in the central region was calculated.

Rat gait analysis

The CatWalk gait analysis system (Noldus Information Technology B.V., Netherlands) was used to evaluate the gait dynamics of SD rats. On the day after the end of the exercise treatment, the CatWalk system was used to test rats in each group. Gait analysis software (CatWalk XT 10.6) was used to calculate gait parameters automatically. This experiment mainly evaluated the effects of the following gait parameters after OA exercise treatment, including the regularity index (%), posture (stands) and hind limb fingerprint position (cm).

Multiple fluorescent immunohistochemical staining

After paraffin sections were deparaffinized, the slides were washed with sterilized water for 1 min and repeated three times. A five-color multiplex immunofluorescence histochemical kit (absin, abs50013, China) was used to evaluate SD rat joint section. Following the manufacturer’s instructions, enzymatic antigen retrieval (C1033, Solarbio Science & Technology Co., Ltd., Beijing, China) was performed at 37 °C for 30 min to repair antigens in the sections. Then, 3% H₂O₂ was used at 25 °C for 30 min to eliminate endogenous peroxidase activity, while blocking serum (15019, Cell Signaling Technology, Danvers, MA, United States) was used at 25 °C for 30 min to block nonspecific antigens. Then, sections were incubated with anti-Collagen II (ab34712, 1:50, abcam), anti-Collagen I (ab270993, 1:5, abcam), anti-Sox9 (ab185966, 1:50, abcam), anti-Aggrecan (abs121273, 1:50,absin). Fluorescence staining was used to amplify the signal (different colors) for the color display of the corresponding antibody. After each round of staining, the staining was confirmed by fluorescence microscope, then cleaned with TBST, and new primary antibodies were added. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min. Coverslips were visualized under a confocal microscope (Olympus).

Primary chondrocyte isolation, culture, passage and treatment

Rat primary chondrocytes were isolated from the knee articular cartilage of 4-week-old SD rats. The 3 mg/ml pronase K (V900887, Sigma-Aldrich, St. Louis, MO, United States) and 2.0 mg/ml collagenase D (C0130, Sigma-Aldrich) were used to cartilage digestion. The isolation and culture of chondrocyte were detailed in our previous study [6, 9]. Resuspended chondrocytes were transferred into culture flasks and cultured with DMEM/F-12 supplemented with 10% FBS (abs972, Absin Bioscience Inc., Shanghai, China) and 1% penicillin with streptomycin (Hyclone Laboratories Inc., Logan, UT, United States) at 37 °C in a 5% CO2 incubator. Then, the chondrocytes were passaged at a 1:4 ratio with 0.05% trypsin (C0202; Beyotime Biotech, Shanghai, China) until 70%–80% confluence. The primary, 3, 5, 7 and 9 passages chondrocytes were collected for characteristic phenotype (cell morphology, Collagen II/ I) analysis, and the most suitable early OA chondrocyte fibrosis model was established followed by previous study [44].

The role of CILP in OA treatment was assessed by transfecting G3 chondrocytes with recombinant adenovirus carrying either CILP knockdown or overexpression sequences (multiplicity of infection [MOI] = 30, polybrene = 1 µg/mL; Hanbio, China). CILP knockdown conditions were grouped as follows: sh-NC (KD-NC), HBAD-Adeasy-r- CILP shRNA1-EGFP (KD-01), and HBAD-Adeasy-r- CILP shRNA2-EGFP (KD-02). CILP overexpression conditions were divided into Ad-NC (OE-NC) and HBAD-Adeasy-r- CILP − 3xflag-EGFP (OE- CILP). In rescue experiment, ML385 (HY-100523, MedChem Expres, NJ, USA ) was used as ferroptosis acativator.

Exposure of chondrocytes to cyclic tensile strain (CTS)

Chondrocytes were seeded onto collagen I-coated Bioflex 6-well culture plates (Flexcell International, Hillsborough, NC) and cultured until they reached 70%-80% confluence. Cyclic tensile strain (CTS) stimulation was then applied using an FX-5000 Flexcell system (Flexcell International, McKeesport, PA). Following our established protocol [8, 10], cells were subjected to a 4-hour regimen of low (5% elongation, 0.5 Hz), moderate (10% elongation, 0.5 Hz), or high (15% elongation, 0.5 Hz) CTS.

Western blot analysis

As detailed in our previous study [6, 7, 9, 11], both cartilage and chondrocytes were lysed using RIPA (9806 S, Cell Signaling Technology). The antibodies including: anti-Collagen II (ab34712, 1:1500, abcam), anti-Collagen I (ab270993, 1:1500, abcam), anti-Sox9 (ab185966, 1:1500, abcam), anti-α-SMA (SAB5500002, 1:1500, Sigma-Aldrich, St.Louis, MO, USA), anti-SLC7A11(ab307601, 1:1500, abcam ), anti-CEMIP (21129-1-AP, 1:1500, proteintech, Wuhan, China), anti-GPX4(ab125066, 1:1500, abcam), anti-Aggrecan (13880-1-AP, 1:1500, proteintech), anti-CILP (sc-12725, 1:1500, Santa Cruz Biotechnology), anti-HO-1(ab189491, 1:1500, abcam), anti-SOD1(10269-1-AP, 1:1500, proteintech), anti-Nrf2 (ab62352, 1:1500, abcam), anti-β-actin(ab8227, 1:1500, abcam).

Quantitative proteomics of CILP-treated chondrocyte and related bioinformatics analysis

To examine the protein profiles of the CILP-treated chondorcyte, we employed the data-independent quantitative proteomics analysis [45]. All analyses were conducted using a Q-Exactive HFX mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). Chromatographic separation was carried out on the EASY-nLC 1000 HPLC System (Thermo, USA). The parameters were followed by previous study [46], all of the Q Exactive raw data was searched using DIA-NN (v1.8.1). A global false discovery rate (FDR) was set to 0.01, and protein groups were considered for quantification if they had at least 2 peptides. Finally, the differences between each group were analyzed using bioinformatics analysis.

Yeast one-hybrid assay

The Keap1 enhancer was used as bait to screen for its binding factors from a cDNA library derived from chondrocytes using the BD Matchmaker One-Hybrid System (Clontech, K1617-1) and the manufacturer suggested protocol with modifications. The coding sequences of CILP and Keap1 were inserted into the XhoI and BamHI restriction sites of the pGADT7-Rec2 vector (Supplementary Data Table S1).

The constructs were co-transformed into yeast strain Y187. Yeast colonies were grown at 30 °C for 3–5 days on SD/−Trp/−Leu medium (DDO). The stock solution and bacterial solution diluted 10, 100 and 1000 times (1, 10^− 1, 10^− 2,10^− 3) were then screened on SD/−Trp/−Leu/−His medium (TDO) supplemented with 30 mM 3-amino-1,2,4-triazole (3-AT) for 3 days. p53HIS2 and pGADT7-p53 were used as positive controls. p53HIS2 and pGADT7-SmMYBs were used as the corresponding negative controls.

Validation of the CILP-keap1 interaction site

The Keap1-R483A point mutant was generated by site-directed mutagenesis (A14604, Thermo Fisher Scientific Inc., ) on a Myc-tagged rat Keap1 plasmid and verified by sequencing. HEK293T cells were co-transfected with Flag-tagged CILP and either Myc-tagged Keap1-WT or Keap1-R483A. After 48 h, cells were lysed, and proteins were immunoprecipitated using anti-Myc beads. Protein interactions were analyzed by Western blotting with anti-Flag and anti-Myc antibodies.

Co-immunoprecipitation (Co-IP)

For Co-Immunoprecipitation (Co-IP), cells were lysed using IP/Co-IP lysis buffer (PC102, Epizyme, Shanghai, China). The lysates were subsequently immunoprecipitated using anti-KEAP1 antibody (4678, 1:50, Cell Signaling Technology, Danvers, MA, USA), anti-Nrf2 (ab62352, 1:50, abcam), anti-CILP (sc-12725, 1:50, Santa Cruz Biotechnology). A total of 2 µg rat IgG antibody (A7031, Beyotime, Shanghai, China) was used as an internal control. These components were mixed with Protein A/G magnetic beads (HY-K0202, MedChemExpress, Monmouth Junction, NJ, USA) and incubated at 4℃ with rotation overnight. The following steps were the same as those used in western blot analysis.

Nrf2 ubiquitination assay

The chondrocyte lysates were immunoprecipitated using anti-Nrf2 (ab62352, 1:50, abcam), and then incubated with ubiquitylation assay kit (ab139467, abcam) at 37 °C for 1–4 h followed by manufacturer’s instructions. Then analyze by SDS-PAGE and Western blot with anti-ubiquitination antibody.

Cytosol-nuclei fractionation

We used the nuclear and cytoplasmic protein extraction kit to dissociate the cytoplasmic and nuclear proteins (PK10014, Proteintech, China) according to the manufacturer’s instructions. Fractions were analyzed by SDS-PAGE and western blot with specific antibodies.

Cellular ROS production

Chondrocytes were seeded in 6-well plates (1.5 × 10⁶ cells/well). ROS production was measured using 2′, 7′ -dichlorodihydrofluorescein diacetate (DCFH-DA) (S0033, Beyotime), which is directly oxidized by ROS (e.g., superoxide ion, hydrogen peroxide, and hydroxyl). Chondrocytes were incubated with 10µM DCFH-DA for 35 min at 37 °C in the dark. Fluorescence was detected by fluorescent microscopy and measured with the BD FACSCalibur (488 nm, excitation; 525 nm, emission).

JC-1 staining

The mitochondrial membrane potential in chondrocyte was determined via a JC-1 fluorescent probe (abs50016, absin), and incubated with JC-1 working solution for 20 min at 37 °C. Then, JC-1 buffer solution was used to wash the cells at least three times. The fluorometric ratio of JC-1 aggregates to JC-1 monomers was used as an indicator of mitochondrial dysfunction.

Ferrous ion detection

The level of cellular iron was detected using FerroOrange (Dojindo, Kumamoto, Japan) following the manufacturer’s instructions and imaged using a fluorescence microscope. We measured the fluorescence intensity using ImageJ.

Malondialdehyde (MDA) and GSH detection

MDA content and GSH content were detected using the Malondialdehyde assay kit (abx257171, Abbexa Ltd, Cambridge, UK), reduced glutathione assay kit (abx150350, Abbexa Ltd) following the manufacturer’s instructions. The level of GSH was measured using a microplate reader at the absorption wavelengths of 405 nm. The values for the levels of MDA was measured using a spectrophotometer at the absorption wavelengths of 532 nm.

Transmission electron microscopy (TEM)

Following by our previous study [6, 8, 9], the cellular morphology and subcellular structures were then observed using a Hitachi 800 transmission electron microscope (TEM) (Tokyo, Japan).

Statistical analysis

Results are presented as means ± 95% confidence interval for difference (mean ± 95% CI), analyzed using GraphPad Prism 5 (GraphPad Software Inc, San Diego, California, USA). Statistical evaluations were conducted using Student’s t-test and one-way ANOVA, facilitated by IBM SPSS Statistics 25.0. A p-value of < 0.05 was considered statistical significant.

Result

CILP was Up-regulated in the cartilage of OA-affected SD rats after exercise therapy

The synovium, cartilage, and subchondral bone were analyzed using single-cell transcriptome sequencing. The t-distributed stochastic neighbor embedding (t-SNE) projection (Supplementary Fig. 1B) identified 8 distinct cell clusters, including chondrocytes, fibroblasts, B cells, macrophages, NK cells, among others. This study, however, primarily focused on the differential gene expression in chondrocytes. In the OAM group, 170 genes were downregulated and 222 genes were upregulated compared to the OA group. The top 9 differentially expressed genes (DEGs) between the OAM and OA groups were highlighted in supplementary Fig. 1C. Single cell RNA sequencing of the whole joint identified CILP as the significantly upregulated gene in chondrocytes following moderate exercise, highlighting its role as a key mechanoresponsive factor in early OA therapy.

Hyaline cartilage fibrosis is a pathological change of chondrocytes in the early stage of osteoarthritis

To further investigate the trajectory of chondrocytes in OA, pseudo-temporal analysis was used to simulate the pathological process of OA chondrocytes. Starting from node 2, the chondrocytes showed some phenotypic changes (Supplementary Fig. 2A). We identified seven chondrocyte subtypes throughout OA pathology, such as RegC, Fc, Homc, Htc, preFC and so on. As shown in supplementary Fig. 2, RegC and preHTC subtype was in the early stage of OA chondrocytes (node 2). As shown in supplementary Fig. 2D, the type I and III collagen content of RegC subgroup was increased which indicated cartilage fibrosis. Taken together, hyaline cartilage fibrosis is a pathological change of chondrocytes in the early stage of osteoarthritis.

CILP is down-regulated in the damaged areas of articular cartilage in clinical patients

The damaged area and relatively undamaged cartilage area of OA patients were divided by radiomics. The red ROI area to the blue area is the change trend of cartilage thickness from thick to thin and from intact to damaged (Fig. 1A). Histological analysis of the damaged and undamaged areas was performed in the corresponding patients undergoing TKA. The OARSI score was used to further confirm that the undamaged area had more intact cartilage than the damaged area. Using western blot (Fig. 1B) and immunohistochemistry (Fig. 1D) to detect the proteins in the cartilage U and D regions, we found that the ratio of typeⅡcollagen / typeⅠcollagen, Sox9 expression in the U region were significantly higher than those in the D region ( Fig. 1C, E). As shown in Fig. 1F and G, CILP is down-regulated in the damaged areas of cartilage in clinical patients.

Fig. 1.

CILP is down-regulated in the damaged areas of articular cartilage in clinical patients. (A) Digital radiograph and radiomics of patients with knee osteoarthritis (OA) (n = 16). The red ROI area to the blue area is the change trend of cartilage thickness from thick to thin and from intact to damage. Anterior–posterior (A-P). (B) Western blot of CILP and typeⅡcollagen, typeⅠcollagen, Sox9, MMP13 in undamaged (U) and damaged (D) areas. (C) Relative protein level of CILP and type II collagen, type I collagen, Sox9, MMP13 in undamaged and damaged areas, with β-actin as control. (D) Gross image, H&E, toluidine blue and IHC staining of knee articular cartilage. Blue box indicate undamaged area, red box indicate undamaged area. (E) OARSI score of histology evaluation. Statistical analysis of CILP, collagen II / I, MMP13, Sox9. *p < 0.001). (F) Quantification with heat maps for relative protein level of CILP in undamaged (U) and damaged (D) areas, with β-actin as the endogenous control (n = 16). (G) Pearson r: correlation of CILP data

Moderate intensity treadmill exercise can increase the expression of CILP in the cartilage intermediate zone of SD rats, and can restore the imaging, histological, subjective and objective function of the knee joint in SD rats.

We confirmed the occurrence of early OA in our rat model using histology evaluation. The OA group showed superficial zone oedema and/or superficial fibrillation (abrasion), focal superficial matrix condensation or little discontinuity (Fig. 2A). In Toluidine Blue staining, a more intense basophilic (bluish-purple) hue is a direct histological indicator of higher proteoglycan content, a hallmark of healthy hyaline cartilage [43].Histological analysis based on the OARSI scores showed that OA group each tibia or femoral joint was between 0 and 12 score which indicated early OA, and the OAM group had therapeutic effect in tibiofemoral joints (Fig. 2E).

Fig. 2.

Moderate intensity treadmill exercise can restore the imaging, histological, subjective and objective function of the knee joint in early OA models. (A) H&E, toluidine blue and (B) IHC evaluation of rats. Macroscopic and OARSI score of rats. (C) Track and heatmap of open field test of rats. Gait analysis of rats, including step sequence, footprint-time an footprint-pressure figure. LF: Left forelimb; LH: Left hindlimb; RF: Right forelimb; RH: Right hindlimb. (D) Open field test of rats. Left axis including distance and average speed. Right Y axis including central zone exploration times. (E) Statistical analysis of gait analysis. Significant differences were found between the CG and OA groups (*P < 0.001; ANOVA), and OA and OAM groups (#P < 0.001, #a P = 0.0213; ANOVA)

We observed surface roughening, fibrillation, fissures which were typically early OA pathologic change in the OA group compared with CG. However, OAM group alleviates the OA group symptoms. The macroscopic score also shows the occurrence of alleviations in the OAM group compared with OA (P < 0.001) (Fig. 2A). We further evaluated SD rats using H&E and toluidine blue staining histological observations. We found that the OA showed cartilage damage and hypocellularity compared with the CG; OAM displayed a relatively complete and smoother cartilage surface compared with that in OA. Our IHC (Fig. 2B) staining revealed that the optical density of type II / I collagen was higher in CG and OAM groups than in OA group. We further noticed that the Sox9, GPX4, and CILP was higher in OAM than in OA.

The open field test was used to investigate the effect of moderate intensity exercise on the recovery of subjective knee function (the spontaneous activity) in SD rats. The results including total distance, average speed, and central zone exploration times, and the motion trail were recorded (Fig. 2C). Compared with the CG group, OA meaningfully reduced the pain thresholds and spontaneous activity. Compared with the OA group, OAM significantly increased total distance, average speed (P < 0.01), and central zone exploration times (P < 0.01) (Fig. 2D).

The gait analysis was used to investigate the effect of moderate intensity exercise on the recovery of objective knee function (Fig. 2C). The regularity index, rat stand time of left and right hind limb were lower in the OA group compared to the CG, while OAM recover these index compare with OA group (P < 0.001). OAM group also showed a smaller paw print position when compared to OA group (P < 0.05). The results of gait analysis showed that the rear limb coordination of the OAM group was greatly improved (Fig. 2E).

In western blot, OAM recover the ratio of type II / I collagen, Sox9, α-SMA expression, and also increase anti-ferroptosis protein SLC7A11, HO-1, GPX4 and SOD-1 expression (Fig. 3A, B).

Fig. 3.

Moderate intensity treadmill exercise can increase the expression of CILP and cartilage-specific protein.(A) Western blot of rat cartliage, including CILP and typeⅡcollagen, typeⅠcollagen, Sox9,α-SMA expression, and anti-ferroptosis protein SLC7A11, HO-1, GPX4 and SOD-1 expression. (B) Statistical analysis of rat cartliage protein. CG and OA groups (*a P = 0.0066; *b P = 0.0178; *c P = 0.0468; *d P = 0.0022; ANOVA); OA and OAM groups (#a P = 0.0073; #b P = 0.0065; #c P = 0.0387; #d P = 0.0425; #e P = 0.0354; #f P = 0.0043; #g P = 0.0026; ANOVA). Data are expressed as the mean ± 95% confidence interval; n = 3 per group. (C) The IF and IHC evaluation of CILP expresion in superficial zone, intermediate zone and deep zone in CG, OA OAM groups. Significant differences were found between the superficial zone and intermediate zone groups (*P < 0.001; ANOVA), and intermediate zone and deep zone groups (#P < 0.001; ANOVA)

The IF was used to investigate CILP location which zone in cartilage, we found intermediate zone had the highest CILP relative fluorescence intensity (P < 0.001) (Fig. 3C). In IHC evaluation, the expression of CILP in intermediate zone was highest in CG, OA, OAM groups. In addition, intermediate zone of OAM group was highest in CILP expression and OA group was the lowest.

The cartilage intermediate zone acts as a mechanical stimulation-sensitive zone in exercise therapy

By multiple fluorescent immunohistochemical staining (Fig. 4A and B), we compared the differences between the zones (superficial zone, intermediate zone and deep zone) and between groups (CG, OA and OAM) of each zone. When compared within the groups, the ratio of type II / I collagen (P < 0.001) and aggrecan (P < 0.05) were highest in the intermediate zone. The Sox9 differences were not significant (P > 0.05). As for differences of each zones between group, the ratio of type II / I collagen, aggrecan and Sox9 of superficial zone, intermediate zone and deep zone were all decreased in OA group, but recover in OAM (P < 0.05).

Fig. 4.

The cartilage intermediate zone acts as a mechanical stimulation-sensitive zone in exercise therapy. (A) By multiple fluorescent immunohistochemical staining, we divided the cartilage into three zones (superficial zone, intermediate zone and deep zone). (B) Multiple fluorescent immunohistochemical staining of CG, OA, OAM groups; green: collagen II; yellow: collagen I; aggrecan: orange; red: sox9; blue: DAPI. (C) Statistical analysis of the relative fluorescence intensity differences between the zones (superficial zone, intermediate zone and deep zone). Significant differences were found between the superfcial and intermediate zones (*P < 0.001; *a P = 0.033; ANOVA), and intermediate and deep zones (#P < 0.001, #a P = 0.045; ANOVA). (D) Statistical analysis of the relative fluorescence intensity of superficial zone between groups. Significant differences comparison was same with Fig. 2. #a P = 0.0041. (E) Statistical analysis of the relative fluorescence intensity of intermediate zone between groups. (F) Statistical analysis of the relative fluorescence intensity of deep zone between groups. Significant differences were found between the CG and OA groups (*P < 0.001; ANOVA), and OA and OAM groups (#P < 0.001, #a P = 0.0014; ANOVA). Data are expressed as the mean ± 95% confidence interval; n = 3 per group

Generation 3 chondrocytes (G3) could mimic early fibrosis of articular cartilage with early OA

During chondrocyte passage, the adherent morphology of chondrocytes gradually changed from completely irregular polygon to slender spindle shape. G3 showed an early fibrochondrocyte appearance (Fig. 5A). Cell density varies during dedifferentiation, but all comparisons used cells at similar confluence. Thus, the observed fibrotic changes reflect dedifferentiation status, not density effects. The ratio of type II / I collagen, aggrecan, sox9 decreased during passage (Fig. 5B). Compared with primary chondrocytes, the ratio of type II /type I collagen decreased from 5.85 to 1.63 folds in Generation 3 chondrocyte (G3) (Fig. 5D). As shown in Fig. 5C and E, the relative fluorescence intensity of type II collagen in G3 was significantly decreased compared with G0 (P < 0.001). By comparing the cell modeling using the inducer TGF-β1 for cartilage fibrosis, we found that type II/I collagen, Sox9, and α-SMA of G3 cells were not statistically significantly different from the early cartilage fibrosis model induced by low concentrations of TGF-β1 (10ng/mL) (Fig. 5F, H).

Fig. 5.

Generation 3 chondrocytes (G3) could mimic early fibrosis of articular cartilage with early OA and moderate CTS alleviates early fibrosis of chondrocytes by upregulating CILP. (A) Gross image of the adherent morphology of chondrocytes during passage. (B) Western blot of the hyaline cartilage versus fibrocartilage phenotype in different passage chondrocytes. G0: primary chondrocyte; G3: Generation 3 chondrocyte and so on. (C) Immunofluorescence of collagen II in different passage chondrocytes. (D) Statistical analysis of the hyaline cartilage versus fibrocartilage phenotype in different passage chondrocytes. Significant differences were found between the G0 and G3 groups (*P < 0.001; ANOVA), G3 and G5 (#P < 0.001, #a P = 0.019; #b P = 0.021; #c P = 0.004; ANOVA), G3 and G7 (+ P < 0.001; +a P = 0.002; +b P = 0.003; ANOVA), G3 and G9 (^P < 0.001; ANOVA). (E) Statistical analysis of the relative fluorescence intensity in different passage chondrocytes (#a P = 0.015; ANOVA). (F) Western blot of the expression of hyaline cartilage versus fibrocartilage phenotype in G3 chondrocytes and TGF-β1-induced chondrocyte fibrosis models. (G) Western blot analysis of phenotypic proteins such as typeⅡcollagen, typeⅠcollagen, CEMIP, CILP, Sox9, GPX4 of G3 chondrocytes after CTS treatment. (H) Statistical analysis of G3 chondrocytes and TGF-β1-induced chondrocyte fibrosis models. G0 and G3 groups (*P < 0.001; *a P = 0.0052; ANOVA); G3 and TGF-β1 10ng groups (#P < 0.001; #a P = 0.0122; ANOVA); G3 and TGF-β1 20ng group (+ P < 0.001; +a P = 0.0293; ANOVA). (I) Statistical analysis of G3 chondrocytes after CTS treatment. G0 and G3 groups (*P < 0.001; *a P = 0.006; ANOVA); G3 and G3 + CTS (low) groups (#P < 0.001; #a P = 0.007; #a P = 0.008; ANOVA); G3 and CTS (medium) group (+ P < 0.001; +a P = 0.0015; ANOVA); G3 and CTS (high) group (^P < 0.001; ANOVA). Data are expressed as the mean ± 95% confidence interval; n = 3 per group

Moderate CTS alleviates early fibrosis of chondrocytes in vitro by upregulating CILP

The up-regulation of CILP was most significant in G3 cells after moderate CTS treatment (P < 0.001). Moreover, the protein expression levels of type II/I collagen, Sox9, GPX4 showed significant recovery effect compared with G3’s early fibrosis phenotype (P < 0.05). The fibrosis-characteristic protein CEMIP was higher in G3 and especially in G3 + high CTS group (P < 0.001) (Fig. 5G, I).

CILP competitively bind to the sites of keap1 protein and reduce the stability of Keap1-Nrf2 dimer, decrase Nrf2 ubiquitination and promoting Nrf2 nuclear translocation

To investigate the molecular mechanisms underlying the effects of CILP on chondrocytes, we performed proteomic analysis of intracellular protein molecules after CILP overexpression. Volcano plots showed the differentially expressed proteins (Supplementary Fig. 3A), selected according to the criteria of (log2 |fold-change| ≥ 1.2 and P < 0.05). The Keap1 protein was found to be enriched in the OE-CILP group compared with the G3 group (Supplementary Fig. 3B). Subcellular location showed Keap1 enriched in chondrocyte cytoplasm (Supplementary Fig. 3C). Following quantitative proteomics, we utilized GO functional annotation analysis and KEGG pathway enrichment analysis to explore potential signaling pathways interacting with Keap1. Based on the protein function cluster (Supplementary Fig. 3D), we found that CILP interacted with Keap1 and affected Nrf2, thereby affecting ferroptosis to promote early OA cartilage fibrosis (Supplementary Fig. 3E and F).

In our study, potential binding sites for Keap1 and CILP were identified by yeast one-hybrid assays. CILP promoted Keap1/Nrf2 dissociation by competing with Nrf2 to bind the Arg483 site of Keap1 (Fig. 6A). Co-IP results showed that there was a more positive interaction between CILP and Keap1 in the OE-CILP group than in the KD-CILP group. To directly validate the specific binding of CILP to the Arg483 residue of Keap1, we performed site-directed mutagenesis. We constructed Myc-tagged wild-type Keap1 (Keap1-WT-Myc) and a mutant where Arg483 was substituted with alanine (Keap1-R483A-Myc), and co-transfected them with Flag-tagged CILP (CILP-Flag) into cells. Co-immunoprecipitation using anti-Myc beads revealed that while CILP robustly interacted with Keap1-WT, its binding to the Keap1-R483A mutant was severely impaired, providing direct genetic evidence that the Arg483 site is essential for this physical interaction (Fig. 6B). As shown in Fig. 6C, the Keap1 protein was detected in the IP-CILP in OE-CILP group, and no Keap1 protein was observed in the band of IgG binding protein. Additionally, IB- CILP was detected in the IP- Keap1 group (Fig. 6C). Co-IP results also showed that the interaction between Keap1 and Nrf2 was decreased in OE-CILP group. As shown in Fig. 6D, the Nrf2 protein was detected in the IP-Keap1 in KD-CILP group, and less Nrf2 protein was observed in the band of OE-CILP group binding protein (Fig. 6D). Furthermore, the trend of IB-Keap1 detected in the IP-Nrf2 group was the same as described above (Fig. 6E). As shown in Fig. 6F, Nrf2 ubiquitination decreased in the OE-CILP group, while Nrf2 ubiquitination increased in the KD group. In addition, OE-CILP accumulated Nrf2 translocation to the nucleus. However, KD-CILP reverse this trend (Fig. 6G).

Fig. 6.

CILP competitively bind to the sites of Keap1 protein and reduce the stability of Keap1-Nrf2 dimer, decrase Nrf2 ubiquitination and promoting Nrf2 nuclear translocation. (A) Yeast one-hybrid assay to identify potential binding sites between Keap1 and CILP. (B) Point mutation validation: Co-immunoprecipitation (Co-IP) shows that mutation of the Arg483 site in Keap1 (Keap1-R483A) severely impairs its interaction with CILP, confirming Arg483 as critical for CILP-Keap1 complex formation.(C, D) Interaction between CILP and Keap1: Co-IP was performed in chondrocytes using anti-Keap1 or anti-CILP antibodies, respectively, followed by immunoblotting to confirm their binding.(E) Interaction between Keap1 and Nrf2: Co-IP demonstrates that CILP overexpression impairs the interaction between Keap1 and Nrf2. (F) Nrf2 ubiquitination level: CILP overexpression reduces the ubiquitination of Nrf2. (G) Nuclear and cytoplasmic distribution of Nrf2: CILP overexpression promotes nuclear translocation of Nrf2. Data are presented as mean ± 95% confidence interval. *P < 0.001, *a P = 0.0099 vs. negative control; #P < 0.001, OE-CILP vs. KD-02 group; ANOVA. β-actin and Histone H3 were used as loading controls for cytoplasmic and nuclear proteins, respectively

CILP ameliorate chondrocyte ferroptosis through Keap1-Nrf2 pathway in vitro

Using western blot, we found OE-CILP group increase the ratio of type II / I collagen, Sox9, α-SMA expression, and also increase anti-ferroptosis protein SLC7A11, HO-1, GPX4 and SOD-1 expression (Fig. 7A, B). However, KD-CILP group reverse these therapeutic effect (Fig. 7C, D). Confocal microscope showed a decrease in intracellular ROS levels in OE-CILP group. Flow cytometry analysis supported this result cellular ROS (Fig. 7E). There also were an increase in mitochondrial membrane potential (Fig. 7G, H) and a decrease in intracellular Fe2 + levels in OE-CILP group (Fig. 7I, J). In addition, TEM also showed ferroptosis in the CILP-knockdown group, mainly characterized by the presence of smaller mitochondria, increased membrane density, and reduced mitochondrial cristae (Fig. 8A). The content of MDA (Fig. 8C) and GSH (Fig. 8D) in chondrocyte also significantly decreased by CILP (P < 0.05). Collectively, these results suggest that CILP overexpression reversed the progression of cartilage fibrosis and chondrocyte ferroptosis in early OA. However, KD-CILP reverse this therapeutic effect.

Fig. 7.

CILP ameliorate chondrocyte ferroptosis through Keap1-Nrf2 pathway in vitro. (A) Western blot of OE –CILP G3 chondrocyte, including CILP and typeⅡcollagen, typeⅠcollagen, Sox9, α-SMA expression, and anti-ferroptosis protein SLC7A11, HO-1, GPX4 and SOD-1 expression. (B) Statistical analysis of proteins of G3 chondrocytes after OE-CILP treatment. G0 and G3 groups (*P < 0.001; ANOVA); G3 and OE-CILP groups (#P < 0.001; #a P = 0.041; ANOVA). (C) Western blot of KD –CILP (KD-01, KD-02) G3 chondrocyte, including CILP and typeⅡcollagen, typeⅠcollagen, Sox9, α-SMA expression, and anti-ferroptosis protein SLC7A11, HO-1, GPX4 and SOD-1 expression. (D) Statistical analysis of G3 chondrocytes after KD-01, KD-02 treatment. G0 and G3 groups (*P < 0.001; *a P = 0.0271; *b P = 0.0013; ANOVA); G3 and KD-01 group (+ P < 0.001; +a P = 0.002; +b P = 0.0146; ANOVA); G3 and KD-02 group (^P < 0.001; ^a P = 0.0313; ^b P = 0.0498; ^c P = 0.0068; ^d P = 0.0015; ANOVA). (F) Confocal microscope showed intracellular ROS levels and flow cytometry analysis showed the result of cellular ROS. (F) Statistical analysis of G3 chondrocytes after OE-CILP, KD-01, KD-02 treatment. G0 and G3 groups (*P < 0.001; ANOVA); G3 and OE-CILP group (#P < 0.001; #a P = 0.011; ANOVA); G3 and KD-01, KD-02 groups (+ P < 0.001; ANOVA). (G) Results of incubation with the JC-1 fluorescent probe, FerroOrange probe (I), and corresponding statistical analysis are shown. (H) Statistical analysis of the relative fluorescence intensity of JC-1 (+ a P = 0.041; ANOVA). (I) Statistical analysis of the relative fluorescence intensity of Fe2+ (*a P = 0.008; #a P = 0.0015; +a P = 0.0012; ANOVA). Data are expressed as the mean ± 95% confidence interval; n = 3 per group

Fig. 8.

Exercise-CILP-Keap1-Nrf2 axis ameliorate early OA cartilage fibrosis through inhibiting chondrocyte ferroptosis. (A) Transmission electron microscopy of the mitochondrial morphology changes in chondrocytes. Blue arrows indicate normal mitochondria, red arrows indicate ferroptosis changed mitochondria (a reduction in mitochondrial volume, the disappearance of cristae, and membrane disruption). (B) Western blot of ML385 as ferroptosis activator to antagonize the anti-fibrotic and anti-ferroptosis effects of CILP. (C) The content of GSH in chondrocyte. Significant differences were found between the G0 and G3 groups (*P < 0.001; *a P = 0.006; ANOVA); the OE-CILP and G3 groups (#P < 0.001; #a P = 0.0023; ANOVA); G3 and KD-02 groups (+ P < 0.001; +a P = 0.0056; ANOVA). (D) The content of Malondialdehyde (MDA) in chondrocyte. Significant differences were found between the G0 and G3 groups (*P < 0.001; *a P = 0.0018; ANOVA); the OE-CILP and G3 groups (#P < 0.001; #a P = 0.0016; ANOVA); G3 and KD-02 groups (+ P < 0.001; ANOVA). (E) Statistical analysis of relative protein expression after ML385 treatment. Significant differences were found between NC and OE-CILP group (*P < 0.001; *a P = 0.0018; ANOVA); the OE-CILP and OE-CILP +ML385 groups (#P < 0.001; #a P = 0.0264; #a P = 0.042; ANOVA)

Exercise-CILP-Keap1-Nrf2 axis ameliorate early OA cartilage fibrosis through inhibiting chondrocyte ferroptosis

To investigate the importance of exercise-CILP-Keap1-Nrf2 axis and ferroptosis in the therapy of early OA cartilage fibrosis. We used ML385 as Nrf2 inhibitor to antagonize the anti-fibrotic and anti-ferroptosis effects of CILP. The ratio of type II/I collagen (P < 0.001) and α-SMA (P < 0.05) were sigificantly decreased by ML385 intervenue. There was no statistically significant difference in the level of Sox9, CILP and SOD-1. The levels of anti-ferroptosis protein such as Nrf2, GPX4, HO-1, SLC7A11 were all decreased significantly (P < 0.05) (Fig. 8B, E).

Discussion

This study confirms the therapeutic effect of moderate-intensity exercise on early OA, as demonstrated through histology, animal behavior, and comprehensive functional assessments. A key finding is the identification of the articular cartilage intermediate zone as a positive, mechanosensitive responder in exercise therapy. We further show that moderate-intensity exercise upregulates CILP within this zone. At the molecular level, CILP competitively binds to Keap1, destabilizes the Keap1-Nrf2 dimer, reduces Nrf2 ubiquitination, and promotes its nuclear translocation. This in turn activates the expression of downstream targets SLC7A11, HO-1, GPX4, and SOD-1, decreases MDA content, increases GSH levels, and ultimately inhibits chondrocyte ferroptosis. In parallel, this pathway promotes the phenotypic reversal of fibrocartilage toward hyaline cartilage. Therefore, we conclude that the exercise-induced CILP-Keap1-Nrf2 axis alleviates early OA by simultaneously suppressing chondrocyte ferroptosis and promoting fibrocartilage hyalinization.

Early OA is the focus of this study. The early symptoms of OA are relatively insidious, but their OA symptoms gradually worsen with the development of the disease [47]. Reduced patient compliance due to pain will lead to limited treatment strategies. Early osteoarthritis is a reversible process, less affected by pain, and personalized exercise prescriptions can be developed [2]. To establish the early OA model, we followed the Pritzker.et.al and F. Maier et al. studies [43, 48] which identify early OA with OARSI score (0–12 each tibia or femoral side), matrix staining depletion within the upper one-third of cartilage and reduction of the cartilage thickness and collagen content. The 4-week ACLT was used to establish an early OA rat model, which followed the above criteria and showed the decrease of cartilage type II collagen and the loss of cartilage aggrecan [49–51].

Serial passaging induces a gradual, intrinsic dedifferentiation of chondrocytes [52]. We used passaged chondrocytes that were dedifferentiated through cultivation, which was evident by a loss of proteoglycans, decreased COL-II deposition, and increased synthesis of COL-I compared to primary chondrocytes (G0) [53–55]. Chondrocytes shift from round toward fibroblastic shape with passage number. Several molecular markers of dedifferentiation have been identified. The decreasing Col II/I ratio provides a quantitative measure of the pathological shift from a hyaline to a fibrotic cartilage phenotype [21] (Fig. 5). Compared with primary chondrocytes, the ratio of type II /type I collagen decreased from 5.85 to 1.63 folds in generation 3 chondrocyte (G3). By comparing the cell modeling using the inducer TGF-β1 [56–58] for joint fibrosis, we found that G3 cells were close to the mild cartilage fibrosis model induced by low concentration TGF-β1. The G3 chondrocyte model was selected because it recapitulates the gradual, intrinsic dedifferentiation of early OA [44], enabling the study of phenotype reversal, whereas the TGF-β1 model [59]induces an acute fibrogenic signal more suitable for studying the inhibition of established fibrosis [60].

Decreased expression of type II collagen and increased expression of type I collagen are the main features of dedifferentiation and fibrosis of articular chondrocytes [21, 23]. Moreover, chondrocyte death and abnormal catabolism and metabolism of cartilage matrix caused by ferroptosis can lead to cartilage defects and damage [61]. In the process of cartilage repair that occurs after injury, the appearance of fibrotic cartilage can further aggravate the symptoms of OA. In this study, we used ferroptosis activator to interfere with the therapeutic effect of CILP and also found that the activation of ferroptsis led to aggravation of cartilage fibrosis (Fig. 7C). Combined with the previous study [26, 62], the joint study of chondrocyte ferroptosis and cartilage fibrosis after cartilage injury is the focus of this study.

In this study, cartilage was found to be heterogeneous and anisotropic, with three regions having different composition and structural mechanical properties. Chondrocytes in the superficial zone were more likely to exhibit a fibrocartilage phenotype due to the higher amount of frictional type I collagen caused by frequent joint activity (Fig. 4). The chondrocytes in the superficial area were oblate and scattered on the cartilage surface. Type II collagen fibers are laterally distributed to withstand wear and stress [63]. Lubrication and painless sliding of the joint originate in the superficial zone, while the intermediate zone is responsible for transferring the load applied to the superficial zone to the deep zone of the cartilage [13, 64]. Collagen fibers in the deep zone are arranged perpendicular to the articular surface (Fig. 4B) [63, 65]. The intermediate zone accounts for about 40–60% of the articular cartilage thickness, the chondrocytes are rounded and medium in shape, and the collagen fibers are arranged randomly and perpendicularly to the articular surface. The main matrix components in intermediate zone are type II collagen (Col2a1) and aggrecan (Fig. 4B) [14]. Moreover, aggrecan content in the MZ reaches the maximum level and collagen type-II are highly expressed and randomly distributed to withstand pressures from different directions, as this region is subjected to compressive and shear stresses.

We established a moderate-intensity treadmill exercise protocol for exercise therapy of early OA, according to our previous study [6, 7, 9, 66]. We used histological analysis, open field test and gait analysis to confirm the therapeutic effect of moderate exercise on early OA in terms of physiological and psychological, subjective and objective knee function recovery (Fig. 2). Then, single-cell transcriptome sequencing was performed on the entire knee joint after exercise, and it was found that exercise may have an effect on early OA through the CILP gene in the intermediate zone of cartilage. In the CILP localization experiment, we confirmed that CILP is mainly enriched in the intermediate zone and upregulated by exercise. In intermediate zone, the ratio of type II / I collagen and aggrecan were also increased significantly (P < 0.05). As a mechanical stress-sensitive zone, the intermediate zone receives the mechanical stimulation signals from exercise therapy.

The observed upregulation of CILP specifically within early OA‑associated subpopulations (RegC, PreHTC) following exercise intervention provides compelling single‑cell evidence that moderate exercise exerts its therapeutic effect by restoring CILP expression across multiple fibrogenic and hypertrophic precursor states. This selective elevation aligns precisely with our focus on early OA pathology, as these subgroups represent key transitional cell states involved in initial matrix degradation and phenotypic shift. The restoration of CILP in these subsets—but not in the proliferation‑oriented RepC cluster—underscores its role as a mechanosensitive regulator of early chondrocyte fate rather than a generic marker of cell activity. Together, these data establish a direct molecular link between exercise and the attenuation of early OA‑associated cellular reprogramming, reinforcing that CILP‑mediated signaling is a central pathway through which mechanical stimulation preserves cartilage homeostasis at the onset of disease (Supplementary Fig. 2).

CILP inhibits the ubiquitination degradation of Nrf2 by binding to the Arg483 site of Keap1 (Fig. 6A), disrupting the stability of the Keap1-Nrf2 dimer (Fig. 6B-E). We found that the Keap1 increased after CILP-OE and decreased after CILP-KD. Together with previous studies that Nrf2 activates and upregulates Keap1 to maintain homeostasis [67, 68], we further confirm that Keap1 is a target gene of Nrf2 and its upregulation is a key negative feedback mechanism limiting Nrf2 hyperactivation. This is consistent with the dysregulation of Nrf2 signaling in OA reported in our previous study [29]and the reduction of Keap1 after CILP-KD in this study.

Our current study indicates that CILP-Keap1-Nrf2 antioxidative signaling pathway is involved in the hyalinization of fibrocartilage. Hyaline cartilage phenotypes such as the ratio of type II collagen to type I collagen, Sox9 and α-SMA were up-regulated after CILP intervention. In addition, recent evidence suggests that oxidative stress and the antioxidant system may also be critical to fibrosis [69–71]. We also demonstrate that Nrf2-mediated anti-ferroptosis activity depends on the induction of SLC7A11, GPX4, HO-1 and SOD1 which are currently recognized as central repressor of ferroptosis [72, 73]. Then alleviated the ferroptosis of chondrocytes by reducing the accumulation of ROS, inhibiting iron overload and lipid peroxidation in mitochondria (Fig. 6).

The role of CILP in OA pathogenesis exhibits apparent contradictions in recent literature [74]. While our study demonstrates that exercise-induced CILP upregulation inhibits chondrocyte ferroptosis and cartilage fibrosis via the Keap1-Nrf2 axis, work by Ma, F. et al., reports miR-140-3p-mediated suppression of CILP as a therapeutic strategy to ameliorate OA-associated ferroptosis [74]. Critically, our data reveal CILP as a mechanoresponsive protector in early OA. We observed CILP upregulation specifically in the cartilage intermediate zone following moderate exercise (Fig. 2J), coinciding with Nrf2 activation and reduced ferroptosis markers (GPX4↑, MDA↓; Fig. 2D A). This aligns with CILP’s established role in maintaining cartilage matrix integrity under physiological loading. We identified that CILP competitively disrupts Keap1-Nrf2 binding (Fig. 5B-E), reducing Nrf2 ubiquitination and enabling antioxidant gene transcription (SLC7A11, HO-1; Fig. 6A). This pathway is suppressed in human OA-damaged cartilage where CILP is downregulated (Fig. 1F-G). Our findings are consistent with those studies of other disease models [25, 75–77] .Conversely, Ma, F. et al.,’s observation of CILP as a ferroptosis promoter may reflect that CILP overexpression in late-stage OA or static cultures (without mechanical cues) could exhibit pathological effects, as suggested by elevated CILP in advanced OA synovial fluid.These divergent findings likely reflect context-dependent duality in CILP functionality, shaped by disease stage, mechanical microenvironment, and transcriptional regulation.

Our study has several limitations. First, the G3 chondrocyte passage model, while useful for studying dedifferentiation, cannot fully recapitulate the intricate multicellular and inflammatory microenvironment of the joint in vivo. Second, the histopathological assessment of early OA remains subjective; we used the OARSI grading system to minimize bias, yet definitive diagnostic criteria are still evolving. Third, while our data suggest an association between cartilage fibrosis and chondrocyte ferroptosis, further studies are needed to establish causality and detailed mechanism. Additionally, the use of TKA-derived tissues—comparing early and advanced lesions from the same joint—was a practical approach given the difficulty in obtaining pure early OA human cartilage, but it does not fully overcome the translational constraints shared with rodent models. Finally, the lack of a standardized preclinical exercise protocol highlights the need for validation in large animal models and human interventional cohorts to confirm the clinical relevance of the CILP–Keap1–Nrf2 axis.

Conclusion

In conclusion, exercise alleviates ferroptosis of chondrocytes and promotes hyalinization of fibrocartilage by mechanically stimulating intermediate zone of cartilage and regulating CILP-Keap1-Nrf2 axis in this region. We propose that early OA + mechanical stimulation: CILP induction by exercise activates Nrf2, inhibiting ferroptosis and fibrosis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization: SJ, YY, ZH; Methodology: SJ, ZH, CN, YS, GY, ZZ; Formal analysis and investigation: ZL, WZ, ZZ; Writing - original draft preparation: SJ, ZH; Writing - review and editing: SJ, YY, YW; Funding acquisition: YY; Resources: YW, YY; Supervision: YY.

Funding statement

This study was supported by the National Natural Science Foundation of China (Grant No. 82102613), the Liaoning Provincial Science and Technology Plan Project (Doctoral Research Initiation Project) (Grant No. 2023012135-JH3/4500) and the Technology Talent Project of Liaoning Provincial Education Department (Grant No. LJKQZ2021028).

Data availability

Data will be made available on request.

Declarations

Ethics approval statement

The studies involving human participants were reviewed and approved by the protocol and experiments on joint specimen after human replacement were approved by the Ethics Committee of Shengjing Hospital of China Medical University (No: 2021PS265K). The animal study was reviewed and approved by the Ethics Committee of China Medical University (no. 2021PS220K).

Patient consent statement

Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Conflict of interest disclosure

The authors have no conflicts of interest to declare that are relevant to the content of this article.