A total of three male and six female Wistar rats (age, 8-10 weeks; male weight, 280-320 g; female weight, 210-240 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and bred overnight in a 1:2 ratio to obtain 0-day-old neonatal pups (weight, 5-6 g). Wistar rats and neonatal rats were maintained in a specific pathogen-free facility, which was operated at 22±1°C temperature and 50±5% humidity, under a 12-h light/dark cycle with free access to food and water. A swaddling-induced DDH rat model was established, with the construction of this model strictly performed according to a previously reported protocol (19). Briefly, the 0-day-old neonatal rats (n=31) were randomly divided into the Control group (n=12; seven male and five female) and the DDH group (n=19; 10 male and nine female). The rats in the DDH group were subjected to straight-leg swaddling that restricted hip abduction and extension, as well as knee extension, with medical bandages for 10 postnatal days. The bandages for swaddling in the DDH group were changed every 2 days, and the rats were allowed to move freely for 4 h. Rats in the control group received no interventions. Subsequently, all rats were raised until they reached 4 weeks of age, anesthetized by isoflurane inhalation (3% for induction and 2% for maintenance), and euthanized via exsanguination. The hip joint was isolated for further analyses.

Before histological examination, the rat hip joints were macroscopically evaluated. Specifically, the hip joints were visually inspected with the naked eye to assess for obvious abnormalities, including dislocation, deformity and cartilage wear. Subsequently, the hip joints were fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and cut into 5-μm sections. Dewaxed sections were stained with hematoxylin for 5 min and eosin for 3 min, or Safranin O (SO) for 5 min and Fast Green (FG) for 1 min, all at room temperature, to evaluate the severity of cartilage lesions. Following dehydration with ethanol, permeabilization in xylene and mounting with neutral gum, images of the histological sections were captured under a light microscope at a magnification of ×40.

The genomic distribution and chromosomal localization of the DUSP26 gene were derived from NCBI (https://www.ncbi.nlm.nih.gov/gene). The protein structure of DUSP26-N was acquired from the Protein Data Bank with the accession code 5GTJ (https://www.rcsb.org/).

Acetabular cartilage samples were meticulously harvested from rat hip joints with the aid of microsurgical tools under microscopic guidance. Subsequently, the acetabular cartilage was fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and sliced into 5-μm sections. The sections were deparaffinized, rehydrated through a descending ethanol series (95, 85 and 75%) and treated with boiling antigen retrieval solution for 10 min followed by incubation with 3% H₂O₂ for 15 min at room temperature. After blocking with 1% bovine serum albumin (BSA; cat. no. A602440-0050; Sangon Biotech Co., Ltd.) for 15 min at room temperature, the sections were incubated with primary antibodies overnight at 4°C, each diluted to a ratio of 1:200. These antibodies included those specific for DUSP26 (cat. no. bs-7910R; BIOSS), COL2A1 (cat. no. AF0135; Affinity Biosciences), histone deacetylase (HDAC)1 (cat. no. AF6433; Affinity Biosciences), phosphorylated (p)-HDAC1 (Ser421) (cat. no. PA5-36810; Thermo Fisher Scientific, Inc.), HDAC2 (cat. no. AF6470; Affinity Biosciences), p-HDAC2 (Ser394) (cat. no. bs-5389R; BIOSS), HDAC8 (cat. no. AF6481; Affinity Biosciences) and p-HDAC8 (Ser39) (cat. no. AF3481; Affinity Biosciences). Following an extensive washing process in PBS, the sections were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) at dilution of 1:100 in PBS at 37°C for 45 min followed by a staining with 3,3'-diaminobenzidine. The sections then underwent counterstaining with hematoxylin for 3 min at room temperature, dehydration with ethanol and xylene successively, and were finally mounted with neutral gum. To assess the expression levels of the target protein, Image-Pro Plus 6.0 (Media Cybernetics, Inc.) was used to assess integrated optical density (IOD) of the positively stained region. The mean optical density of the target protein in each image was calculated as IOD/stained area. Each sample was analyzed on a single slice by imaging three representative fields of view at ×400 magnification.

Total RNA was extracted from rat acetabular cartilage using TRIpure Total RNA Extraction Reagent (cat. no. RP1001; BioTeke Corporation). cDNA was synthesized from 1 μg RNA using an All-in-One First-Strand SuperMix Kit according to the manufacturer's instructions (cat. no. MD80101; Guangzhou Magen Biotechnology Co., Ltd.). Subsequently, qPCR was performed in a 20-μl reaction mixture containing 1 μl cDNA, 0.5 μl SYBR green I (cat. no. SR4110; Beijing Solarbio Science & Technology Co., Ltd.), 10 μl 2X Fast Taq plus PCR Master Mix (cat. no. BL1014; Biosharp Life Sciences) and 0.5 μl each primer. qPCR was initiated with a 5-min denaturation step at 95°C, followed by 40 consecutive three-step cycles: Denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and extension at 72°C for 15 sec. Relative mRNA levels of target genes were calculated using the 2^−ΔΔCq method (20) and normalized to the housekeeping gene β-actin. The primer sequences of target genes are listed in Table SI.

mRNA-seq was performed by Novogene Co., Ltd. Briefly, total RNA from rat acetabular cartilage was extracted using TRNzol (cat. no. DP424; Tiangen Biotech Co., Ltd.). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (cat. no. N401-01/02; Vazyme Biotech Co., Ltd.). Standard RNA-seq library generation was performed using the Fast RNA-seq Lib Prep Kit V2 (cat. no. RK20306; ABclonal Biotech Co., Ltd.), according to the manufacturer's instructions, with an insert size of 250-300 bp, and subjected to paired-end 150 bp sequencing on the Illumina HWI-ST1276 instrument (Illumina, Inc.). Paired-end reads were filtered via: i) Removal of read pairs if either read contained adapter contamination; ii) exclusion of read pairs if uncertain bases constituted >10% of either read; and iii) elimination of read pairs if low-quality bases (Phred score <5) exceeded 50% of the total length in either read. Genes with significantly upregulated expression in the DDH group relative to the control group were identified using stringent criteria: Log₂ fold change (FC) >4 and P.adj<0.01. Principal component analysis of significantly upregulated genes was performed using the DESeq2 (version 1.46.0) (21) and ggplot2 (version 3.5.1; https://ggplot2.tidyverse.org) in R package (version 4.4.2; https://www.r-project.org). A heatmap of the significantly upregulated genes was constructed using pheatmap (version 1.0.12; https://cran.r-project.org/package=pheatmap) in R package (version 4.4.2). For Gene Ontology (GO) annotations, the significantly upregulated genes were analyzed using the R packages ggplot2 and patchwork (https://patchwork.data-imaginist.com).

For cartilage collection, another cohort of 75 0-day-old neonatal Wistar pups (weight, 5-6 g; 40 male and 35 female rats; housed as aforementioned) was raised to 5 days of age; subsequently, they were anesthetized with isoflurane (3% induction, 2% maintenance via inhalation) and euthanized by exsanguination. The tibial and femoral cartilages were dissected from 5-day-old rats as previously described (22). After washing in PBS, the cartilage pieces were digested with 3% (w/v) collagenase II (cat. no. BS164; Biosharp Life Sciences) twice for 45 min at 37°C each time. The primary chondrocytes (P0) were cultured in DMEM/F12 (cat. no. BL305A; Biosharp Life Sciences) containing 10% fetal bovine serum (cat. no. 11011-8611; Beijing Solarbio Science & Technology Co., Ltd.) in a humidified incubator at 37°C with 5% CO₂. Primary chondrocytes were cultured for 3 days until they reached 90% confluence prior to passage. Cells from the P0 and P1 generations in the logarithmic growth phase were selected for subsequent experiments. Cell contamination was routinely excluded by microscopic examination of cell morphology and culture medium condition.

Chondrocytes were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized using 0.1% Triton X-100 for 30 min at room temperature and subsequently blocked with 1% BSA for 15 min at room temperature. Cells were then incubated with anti-COL2A1 antibody (1:100; cat. no. AF0135; Affinity Biosciences) overnight at 4°C. After an extensive washing process in PBS, the cells were incubated with Cy3-labeled goat anti-rabbit IgG secondary antibody (1:200; cat. no. SA00009-2; Wuhan Sanying Biotechnology) for 1 h at room temperature. After staining the nuclei with DAPI at room temperature for 5 min and mounting with anti-fluorescence quencher, immunofluorescence images were captured under a fluorescence microscope at a magnification of ×400.

To assess the functional integrity of chondrocytes, the P0-P1 primary chondrocytes were treated with the pro-inflammatory cytokine IL-1β (10 ng/ml; cat. no. HY-P7097; MedChemExpress) with or without the HDAC inhibitor trichostatin A (TSA; 300 nM; cat. no. T129665; Shanghai Aladdin Biochemical Technology Co., Ltd.) for 24 h at room temperature (22,23).

For knockdown of DUSP26, two short hairpin RNAs (shRNAs) specifically targeting rat DUSP26 mRNA were inserted into the BglII/SalI sites of the pShuttle-CMV-H1 adenoviral vector (cat. no. BR009; Hunan Fenghui Biotechnology Co., Ltd.) (target sequence-1, 5'-ACTCGCCAGCCTTTGACATGA-3'; target sequence-2, 5'-CCTCATGCTGTACCACCACTT-3'). For knockdown of HDAC1/2/8, shRNAs targeting rat HDAC1/2/8 mRNA were inserted into the BglII/SalI sites of the pShuttle-CMV-H1 adenoviral vector. The specific target sequences were as follows: HDAC1, 5'-GCTGCTCAACTATGGTCTCTA-3'; HDAC2, 5'-CCCAATGAGTTGCCATATAAT-3'; and HDAC8, 5'-CTACAGTGTCAATGTGCCCAT-3'. A non-specific shRNA (target sequence, 5'-TTCTCCGAACGTGTCACGT-3') was inserted into the pShuttle-CMV-H1 adenoviral vector to generate the adenoviral negative control (Ad-shNC). For overexpression of DUSP26, the rat DUSP26 CDS fragment (NM_001012352) was inserted into the KpnI/NotI sites of the pShuttle-CMV adenoviral vector. An empty pShuttle-CMV adenoviral vector served as a control (Ad-EP). Once constructed, the shuttle vector containing shRNAs or the rat DUSP26 CDS fragment was linearized with FastDigest PacI (cat. no. FD2204; Thermo Fisher Scientific, Inc.) and co-transformed into Escherichia coli BJ5183 cells (Beijing Huayueyang Biological Technology Co., Ltd.) with pAdEasy-1 backbone plasmid (cat. no. 16400; Addgene, Inc.) following the manufacturer's recommended ratio (1:1, by mass) for homologous recombination. The resulting recombinant adenoviral plasmids (25 μg) were then transfected into 293A packaging cells (iCell Bioscience, Inc.) using a high-efficiency transfection reagent (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at room temperature. Cells were cultured for 10-14 days until the cytopathic effect reached >50%. Cells were then harvested by repeated freeze-thaw and filtered through a 0.45-μm filter to obtain the first-generation virus. Separate second-generation viral stocks were generated for each recombinant adenoviral vector via one additional round of amplification: Specifically, the two DUSP26 knockdown vectors (Ad-shDUSP26-1, Ad-shDUSP26-2), three HDAC knockdown vectors (Ad-shHDAC1, Ad-shHDAC2, Ad-shHDAC8), one DUSP26 overexpression vector (Ad-DUSP26), and two control vectors (Ad-shNC, Ad-EP) each had corresponding second-generation viral stocks. These second-generation viral stocks were used to infect chondrocytes at multiplicity of infection values of 100. After a 24-h incubation period, the chondrocytes were subjected to IL-1β stimulation.

Tissues were weighed, homogenized with normal saline (9X volume) on ice, centrifuged at 663 × g for 10 min at 4°C and the supernatants were collected. Cell supernatants from the primary chondrocytes were collected by centrifugation at 300 × g for 10 min at 4°C. Protein concentrations were measured using a BCA assay kit (cat. no. PK10026; Wuhan Sanying Biotechnology), and the levels of TNF-α and IL-6 in the tissue homogenates and cell supernatants were measured using the Rat TNF-α ELISA Kit (cat. no. EK382; Multi Sciences Biotech) and Rat IL-6 ELISA Kit (cat. no. EK306; Multi Sciences Biotech), according to the manufacturers' instructions.

Proteins were extracted from the primary chondrocytes on ice using a RIPA lysis buffer (cat. no. PR20001; Wuhan Sanying Biotechnology) containing 1% protease inhibitor (cat. no. PR20032; Wuhan Sanying Biotechnology) and phosphatase inhibitor (cat. no. PR20015; Wuhan Sanying Biotechnology) for 30 min. The supernatant fraction was collected by centrifugation at 10,000 × g for 3 min at 4°C, and the protein concentration was determined using a BCA assay kit. Subsequently, 15-30 μg proteins (loading volume of 15 μl per lane) were separated by SDS polyacrylamide gel electrophoresis on 10 and 12% gels and transferred onto a PVDF membrane. The membrane was then incubated in a blocking solution (cat. no. PR20011; Wuhan Sanying Biotechnology) for 2 h at room temperature, after which, it was probed with primary antibodies at 4°C overnight. These antibodies included anti-DUSP26 (1:1,000; cat. no. GTX109283; GeneTex, Inc.), anti-COL2A1 (1:500; cat. no. AF0135; Affinity Biosciences), anti-COL1A1 (1:500; cat. no. AF7001; Affinity Biosciences), anti-TNF-α (1:500; cat. no. AF7014; Affinity Biosciences), anti-HDAC1 (1:500; cat. no. AF6433; Affinity Biosciences), anti-p-HDAC1 (Ser421) (1:500; cat. no. PA5-36810; Thermo Fisher Scientific, Inc.), anti-HDAC2 (1:500; cat. no. AF6470; Affinity Biosciences), anti-p-HDAC2 (Ser394) (1:500; cat. no. bs-5389R; BIOSS), anti-HDAC8 (1:500; cat. no. AF6481; Affinity Biosciences), anti-p-HDAC8 (Ser39) (1:500; cat. no. AF3481; Affinity Biosciences), anti-β-catenin (1:1,000; cat. no. AF6266; Affinity Biosciences) and anti-β-actin (1:20,000; cat. no. 66009-1-Ig; Wuhan Sanying Biotechnology). Subsequently, the membrane was incubated with HRP-conjugated goat anti-rabbit (cat. no. SA00001-2; Wuhan Sanying Biotechnology) or anti-mouse (cat. no. SA00001-1; Wuhan Sanying Biotechnology) secondary antibodies at 1:10,000 dilution at 37°C for 40 min. The protein bands were visualized using an ultrasensitive enhanced chemiluminescent detection kit (cat. no. PK10003; Wuhan Sanying Biotechnology) according to the manufacturer's instructions and were analyzed by Tanon 5200 Image Analysis software (Tanon Science and Technology Co., Ltd.).

The primary chondrocytes were infected with a DUSP26-overexpressing adenovirus followed by treatment with IL-1β. The Co-IP assay was performed using a Pierce Co-IP Kit (cat. no. 26149; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, proteins were extracted from the primary chondrocytes on ice using an IP lysis buffer containing 1% protease inhibitor (cat. no. PR20032; Wuhan Sanying Biotechnology) and phosphatase inhibitor (cat. no. PR20015; Wuhan Sanying Biotechnology) for 30 min. The supernatant fraction of cell lysates was collected by centrifugation at 10,000 × g for 3 min at 4°C, and the protein concentration was determined using a BCA assay kit. An aliquot of the cell lysate supernatant (500 μg) was reserved as the Input group prior to IP. For each Co-IP reaction, 2 μl anti-DUSP26 antibody (cat. no. GTX109283; GeneTex, Inc.) or anti-rabbit IgG isotype control (cat. no. 30000-0-AP; Wuhan Sanying Biotechnology) was pre-conjugated to 20 μl aldehyde-activated agarose beads in 200 μl 1X coupling buffer and 3 μl sodium cyanoborohydride for 2 h at room temperature with gentle rotation. After washing twice with IP lysis buffer, the antibody-conjugated beads were incubated with 500 μg cell lysate for 2 h at room temperature with gentle rotation, and the bound protein complexes were eluted with elution buffer for 5 min at room temperature. The eluates were collected by centrifugation at 1,000 × g for 1 min at 4°C and subjected to western blot analysis.

For protein identification by MS analysis, protein lysates were obtained from primary chondrocytes infected with Ad-DUSP26and treated with IL-1β as aforementioned, and were reduced with 10 mM dithiothreitol at 37°C for 60 min, alkylated with 55 mM iodoacetamide at room temperature in the dark for 30 min, diluted with 50 mM ammonium bicarbonate for 10 min and subsequently digested with sequencing-grade trypsin overnight at 37°C. The resulting peptides were desalted using C18 columns, which were activated by 100% acetonitrile and equilibrated using 0.1% formic acid before loading samples. Subsequently, the columns were washed using 0.1% formic acid, and peptide fragments were eluted using 70% acetonitrile and lyophilized. Next, the lyophilized powder was dissolved in 10 μl mobile phase A (0.1% formic acid in water) and then centrifuged at 14,000 × g for 20 min at 4°C. Subsequently, the supernatants (4 μl) were injected into the UltiMate 3000 system (Thermo Fisher Scientific, Inc.) equipped with a column (inner diameter, 150 μm) packed with ReproSil-Pur C18-AQ 1.5 μm silica beads. Next, gradient elution was carried out using mobile phase B (0.1% formic acid in 80% acetonitrile) at a flow rate of 300 nl/min. The elution gradients were as follows: 8% B for 5 min, 12% B for 5 min, 30% B for 35 min, 40% B for 44 min, 95% B for 45 min and 95% B for 60 min. Following HPLC fractionation, the eluted peptide fractions were introduced into the Q Exactive HF-X mass spectrometer via a Nanospray Flex™ (NSI) ion source operating in positive ion mode, with a spray voltage of 2.2 kV and a capillary temperature of 320°C. For MS detection, a data-dependent acquisition mode was employed: The full-scan MS1 spectra (m/z range: 350-1,500) were acquired at a resolution of 120,000 (200 m/z), with an automatic gain control (AGC) target of 3×10⁶ and a maximum injection time of 80 msec. The top 40 most abundant precursor ions were subsequently selected for MS2 fragmentation via higher-energy collisional dissociation with a normalized collision energy of 27%. The MS2 spectra were acquired at a resolution of 15,000 (200 m/z), with an AGC target of 5×10⁴ and a maximum injection time of 45 msec. Data acquisition was performed by Beijing Qinglian Biotechnology Co., Ltd. and analyzed using the Proteome Discoverer 2.4 software (Thermo Fisher Scientific, Inc.), with searching against the Rattus_norvegicus_UP000002494_uniprot database (https://www.uniprot.org/proteomes/UP000002494). IgG was used to exclude non-specific interactors of DUSP26 in chondrocytes under IL-1β stimulation.

DUSP26-specific interactors were identified by IP/MS analysis. Non-differentially expressed genes were selected from mRNA-seq data using the following screening criteria: −1<log₂FC<1 and P≥0.05. GeneCards (https://www.genecards.org/) was used to retrieve genes associated with 'osteoarthritis', 'cartilage degradation' and 'inflammation'. The jvenn online tool (https://jvenn.toulouse.inrae.fr/app/index.html) was employed to visualize the intersection of gene sets using Venn diagrams. Finally, candidate genes related to 'osteoarthritis', 'chondrocytes' and 'phosphorylation' were identified through PubMed literature search engines (https://pubmed.ncbi.nlm.nih.gov/).

Analyses were performed using GraphPad Prism 8.0.2 (Dotmatics). The normal distribution of the data was determined using the Shapiro-Wilk test. One-way analysis of variance followed by Tukey test was employed for comparisons among multiple groups. Unpaired Student's t-test was used for comparisons between two groups. All data are presented as the mean ± SD. All quantitative experiments were conducted with at least three independent biological replicates, each including technical triplicates. P<0.05 was considered to indicate a statistically significant difference.

Neonatal rats were subjected to swaddling for 10 days. A total of 4 weeks later, the hip joints of the rats were macroscopically evaluated. Compared with the control rats, the rats that had undergone straight-leg swaddling exhibited shallower acetabular cavities, and their femoral heads were small and oval-shaped (Fig. 1Aa). Histologically, femoral head dislocation and acetabular labral tears occurred in the rats undergoing straight-leg swaddling (Fig. 1Ab), and the SO/FG staining intensity on the acetabular surface was weakened (Fig. 1Ac). Immunohistochemical staining of the acetabular cartilages from the 4-week-old rats showed a decreased expression of COL2A1, a major collagen synthesized by chondrocytes (Fig. 1B and E). Gene expression analysis further confirmed that the expression of COL2A1 was downregulated in the acetabular cartilage of the 4-week-old rats with DDH, whereas the expression levels of IL-1β and IL-6 were upregulated (Fig. 1C). Furthermore, ELISA showed a higher level of IL-6 in the acetabular cartilage of the 4-week-old rats with DDH (Fig. 1D). These results demonstrated that acetabular collagen degradation and inflammatory responses occurred in the rats with DDH induced by swaddling.

To identify the key factor in DDH induced by swaddling, mRNA-seq analysis of the acetabular cartilage was conducted. With the screening criteria of log₂FC >4 and P.adj<0.01, 117 markedly upregulated genes (subsequently referred to as 'Ups') in the acetabular cartilage of the rats with DDH were obtained (Fig. 1F). The expression trends of IL-1β and COL2A1 detected by RT-qPCR were fully consistent with the sequencing data (Fig. 1C), confirming the accuracy of the differentially expressed gene screening. Subsequently, the GO-biological process enrichment analysis showed that the Ups were mainly related to 'muscle cell differentiation' and 'muscle cell development'. The GO-cellular component enrichment analysis indicated that the Ups were mainly associated with 'myofibril' and 'sarcomere'. The GO-molecular function enrichment analysis revealed that the Ups were mainly involved in aspects such as 'structural constituent of muscle' and 'actin binding' (Fig. 1G).

DDH is a common abnormality leading to osteoarthritis (24). Therefore, the present study aimed to identify a novel gene that may be involved in osteoarthritis secondary to dysplasia of the hip. Genes related to cartilage degradation and osteoarthritis were searched using the GeneCards database. Venn interaction analysis revealed that 38 Ups were associated with cartilage degradation, with known osteoarthritic genes excluded (Fig. 2A). Among them, the DUSP26 gene, located on chromosome 8p12, has an active PTP-loop conformation (Fig. 2B and C). DUSP26 regulates intracellular signaling pathways by dephosphorylating the tyrosine and serine/threonine residues of proteins (9). Immunohistochemical staining showed higher DUSP26 expression in the acetabular cartilage of 4-week-old rats with DDH compared with that in the control group (Fig. 2D). Moreover, the mRNA levels of DUSP26 were upregulated in the cartilage of the acetabulum from 4-week-old rats with DDH (Fig. 2E).

To determine the effects of DUSP26 on chondrocyte degradation, primary rat chondrocytes were extracted and identified as chondrocytes by immunofluorescence staining of COL2A1 (Fig. S1). Subsequently, the chondrocytes were subjected to IL-1β stimulation with the intention of mimicking chondrocyte injury in vitro. Notably, the protein and mRNA expression levels of DUSP26 in chondrocytes induced by IL-1β were significantly increased (Fig. 3A). Subsequently, rat DUSP26 overexpression and knockdown adenoviruses were constructed and used to infect chondrocytes. After 24 h, the protein and mRNA expression levels of DUSP26 in the chondrocytes were effectively modulated (Fig. 3B and C).

An inflammatory environment in vitro can activate catabolic processes of chondrocytes and exacerbate cartilage erosion (4). As demonstrated in Fig. 4A, IL-1β induced chondrocyte degeneration, as manifested as enhanced COL1A1 expression and reduced COL2A1 expression. Overexpression of DUSP26 prevented upregulation of COL1A1 and downregulation of COL2A1 caused by IL-1β (Fig. 4A). Furthermore, overexpression of DUSP26 lowered TNF-α and IL-6 levels in the chondrocytes under IL-1β stimulation (Fig. 4B and C). In line with this, knockdown of DUSP26 aggravated IL-1β-induced chondrocyte damage, as evidenced by increased expression of catabolic markers (COL1A1, TNF-α and IL-6) and decreased expression of the anabolic marker COL2A1 (Fig. 4D-F).

Target proteins that bound to DUSP26 and whose activities were affected by the phosphatase DUSP26 were screened for. Firstly, 4,234 of proteins that specifically bound to DUSP26 in chondrocytes under IL-1β stimulation were harvested after the IP/MS analysis (Fig. 5A). Combining with the previous mRNA-seq results, 3,466 DUSP26-specific interactors with non-differential expression were obtained (Fig. 5B, two-set Venn diagram). Subsequently, the four-set Venn diagram revealed 538 DUSP26-specific interactors related to 'osteoarthritis', 'cartilage degradation' and 'inflammation' (Fig. 5B). Among them, activation of three Class I HDACs (HDAC1, HDAC2 and HDAC8) serves an important role in cartilage degeneration and osteoarthritis, as determined using search engines such as PubMed (Fig. 5B) (7,8). Therefore, the current study explored whether these three HDACs were involved in the progression of DUSP26-mediated osteoarthritis secondary to DDH. Increased HDAC1/2/8 total protein, p-HDAC1^Ser421, p-HDAC2^Ser394 and p-HDAC8^Ser39 levels were observed in the acetabular cartilage of the rats with DDH in vivo (Figs. 5C and S2). In chondrocytes treated with IL-1β, total expression and phosphorylation levels of HDAC1/2/8 were also enhanced (Fig. 5D). Co-IP experiments confirmed that DUSP26 bound to HDAC1/2/8 in IL-1β-stimulated chondrocytes (Fig. 5E).

Under IL-1β stimulation, overexpression of DUSP26 decreased the phosphorylation levels of HDAC1, HDAC2 and HDAC8 in chondrocytes, but did not affect the abundance of total proteins (Fig. 6A). By contrast, silencing of DUSP26 increased their phosphorylation levels. Adenovirus-mediated shRNAs against HDAC1, HDAC2 and HDAC8 were constructed and used to infect chondrocytes. After 24 h, the expression levels of HDAC1, HDAC2 and HDAC8 mRNA and protein in chondrocytes were effectively knocked down, respectively (Figs. 6B and S3). After the chondrocytes were co-infected with the HDAC1/2/8 interference adenoviruses and the DUSP26 interference adenovirus, the mRNA and protein expression levels of the catabolic marker COL1A1 and the inflammatory marker TNF-α were decreased relative to the Ad-shDUSP26 group (Fig. 6C and E). Furthermore, intervention with TSA, an inhibitor of HDAC activity, effectively reversed DUSP26 silencing-induced COL1A1 and TNF-α upregulation (Fig. 6D and F). These findings suggest that the DUSP26-HDAC1/2/8 phosphorylation regulatory axis may act as a key mechanism underlying the inhibition of cartilage degeneration. In addition, high β-catenin expression was observed in the acetabular cartilage of rats with DDH (Fig. S4). Therefore, DUSP26 may serve a potential synergistic protective role via the β-catenin pathway.